SIMPÓSIO INTERNACIONAL SÔBRE VENENOS ANIMAIS

INTERNATIONAL SYMPOSIUM ON ANIMAL VENOMS

INSTITUTO BUTANTAN

17 a 23 de julho de 1966

Sob os auspícios do GOVERNO DO ESTADO DE SÂO PAULO,

do CONSELHO ESTADUAL DE AUXÍLIOS E SUBVENÇÕES, da

SOCIEDADE DE BIOLOGIA DE SÃO PAULO, da FUNDAÇÃO DE

AMPARO À PESQUISA DO ESTADO DE SÃO PAULO e do

FUNDO DE PESQUISAS DO INSTITUTO BUTANTAN.

SUPLEMENTO COMEMORATIVO

CENTENÁRIO DE NASCIMENTO DE VITAL BRAZIL

COMISSÃO 1)E REDAÇÃO

Beçak, W. — Bücherl, W. -— Carvalho Lima, L. P. — Dessimoni v. Eickstedt, V.

— Emerson Belluomini, H. — Franco de Mello, R. -— Hoge, A. R. — Lavras,

A. A. C. — Leal Prado, J. — Lucas, S. M. — Mandelbaum, F. R. — Nahas, L.

— Ribeiro do Valle, L. A. ■— Saliba, F. — Schenberg, S. — Vallejo-Freire, A. —

Vital Brazil, O. — Zelnik, R.

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i SIMPOSIO INTERNACIONAL SOBRE VENENOS ANIMAIS

INTERNATIONAL SYMPOSIUM 0N ANIMAL VENOMS

1966 I

FASCÍCULO 3 1

COMUNICAÇÕES — COMMUNICATIONS 1

Animais venenosos — Venomous Animais I

Presidente .

L. D. BRONGERSMA (Holanda)

Vice-Presidente .

S. OHNO (E.U.A.)

Moderador .

P. SAWAYA (Brasil)

Secretário .

W. BEÇAK (Brasil)

Patologia do envenenamento

e prevenção de acidentes — Pathology of

envenomation and prevention

of accidents

Presidente .

A. OHSAKA (Japão)

Moderador .

G. ROSENFELD (Brasil) j

Secretário .

L. NAHAS (Brasil) 1

ui.

Imunologia — Immunology

Presidente .

P. A. CHRISTENSEN (África do Sul)

Vice-Presidente .

O. ZWISLER (Alemanha)

Moderador .

P. KRAG (Dinamarca) |

Secretário .

R. S. FURLANETTO (Brasil)

I IV.

Bioquímica — Biochemistry

Presidente .

J. MOURA-GONÇALVES (Brasil)

Vice-Presidente .

C. R. DINIZ (Brasil)

Moderador .

J. LEAL PRADO (Brasil)

Secretário .

R. ZELNIK (Brasil) |

1 v -

Farmacologia — Pharmacology

Presidente .

T. SUZUKI (Japão)

Vice-Presidente .

F. E. RUSSELL (E.U.A.)

Moderador .

J. H. WELSH (E.U.A.)

Secretário .

S. SCHENBERG (Brasil) !

SciELO

10 11 12 13 14 15 16

SIMPÓSIO INTERNACIONAL SÔBRE VENENOS ANIMAIS

INTERNATIONAL SYMPOSIUM ON ANIMAL VENOMS

INSTITUTO BUTANTAN

17 a 23 de julho de 1966

FASCÍCULO 3

COMUNICAÇÕES COMMUNICATI

ONS I

ÍNDICE — INDEX

1 I. An

1MAIS VENENOSOS — VeNOMOUS ANIMALS

Pág.

Venomous Coelenterates — M. Vannucci (Brasil) ...

659 |

Contribución al conocimiento de Theraphosa leblondi (La(reille)

1804 (ARANEA — THERAPHOSIDAE) — B. S. GeRSCHMAN

de Pikelin y R. D. Schiapelli (Argentina) .

667 i

Estúdio comparativo de Phoneutria fera Perty 1833 y Phoneu-

tria nigriventer (Keyserling) 1891 (ARANEA — CTENIDAE)

— R. D. Schiapelli y B. S. Gerschman de Pikelin (Ar-

gentina 1 .

675 |

Investigación ecológica sobre Loxosceles rufipes (Lucas) 1834

en la resión costera dei Perú — A. Delcado (Perni

683

Mi experiencia sobre Loxoscelismo — R. Gajardo-Tobar

(C hile) .

689

699 1

El Araneísmo en Bolivia — J. Vellard (Bolívia)

Ultraestrutura do aparelho venenífero de A pis (HY’MENOP-

TERA, APIDAE) — C. DA CrUZ LaNDIM e E. W. KlTAJIMA

(Brasil) .

701

Données histochimiques sur la glande à venin (glande chéli-

cérienne) des araignées dipneumones — L. Arvy (França)

711

Crotalus vegrandis Klauber, redescripción y distribución —

A. R. Lancini Villalaz (Venezuela) .

725 1

10.

The poisonous snakes of Irau — M. Latifi, A. R. Hoge and

M. Eliazan (Iran e Brasil) .

735 j

Die Sandotter — Vipera ammodytes amrrwdyles L. — M.

Stanic (Suíça) .

745 1

'LO 11 12 13 14 15 16

ÍNDICE INDEX

Pág. I

12.

Estudo citológico e ])oiuleral do testículo de Crotalus durissus

terrificus durante o ciclo reprodutivo anual — H. E. Bei.luomini,

R. Franco de Mello, A. M. Penha e G. Schreiber (Brasil)

761 1

13.

Probable significance of venom yield record studies — P. J.

Deoras (índia) .

767 |

14.

Constituição cromossômica e mecanismo de determinação do

sexo em ofídios sul-americanos. 11. Cromossomos sexuais e evo-

lução do cariótipo — W. Beçak (Brasil) .

775 J

II. Pato

LOG1A DO ENVENENAMENTO E PREVENÇÃO DE ACIDENTES -

1 PaTHOLOGY OF ENVENOMATION AND PREVENTION OF ACCIDENTS

15.

Acción dei veneno de Hapalopus limensis — N. C. de Espinqza

(Peru) .

799

, 16.

Casos de loxoscelismo grave occurridos en la ciudad de Buenos

Aires y alrededores en los últimos anos — A. Barrio y A.

Ibarra Grasso (Argentina) .

809

17.

Bee venom: glands, intoxication, accidents — E. de Lello

(Brasil) .

821 j

I 18.

Lepidopterismo y erucismo. Epidemiología y aspectos clínicos

en el Perú — H. Pesce y A. Delgado (Peru) .

829 |

19.

Toxic action of rove beetles (COLEOPTERA — STAPHYLINI-

DAE) — A. Fain (Bélgica) .

835 |

20.

Metronidazole in snake venom poisoning — F. E. Russell

( E.U.A.) .

845 1

21.

The release of biogenic amines from blood plalelets under lhe

influente of Crotalus d. terrificus venom — F. Markwardt

851 1

22.

Further observations on Coral snake bites in lhe United

States: symploms and lherapy -— J. G. Gennaro and N. C.

McCollough (E.U.A.) .

855

1 23.

Inyección in sita de antisuero en el tratamiento de Ia intoxi-

cación por mordedura de serpiente — J. M. Velasco (México)

857 I

1 III. Imunologia — Immunology

1 24.

Pharmacological effects of the venom of Hapalochlaena maculosa

861 i

E 25.

Diferencias inmunológicas entre entidades simpátridas de ara¬

rias dei género Latrodectus Walckenaer — A. Barrio

(Argentina) .

865

SciELO

10 11 12 13 14 15 16

ÍNDICE

INDEX

Pag.

26. Estúdio comparativo morfológico e inmunológico entre Ias di¬

ferentes entidades dei género Micr u r u s Wagler (OPHII)IA,

ELAPIDAE) de la Argentina — A. Barrio y M. E. Miranda

(Argentina) . 869

27. In vivo neutralization of Coral snake venom — P. Cohen

(E.U.A.) . 881

28. Las diferentes poblaciones de Bothrops alternata Duméril y

Bibrón (OPHIDIA, CROTALIDAE) de la Argentina, conside¬

radas desde el punto de vista morfológico y antigénieo -—- A.

Barrio y M. E. Miranda (Argentina) . 887

29. Antivenin production — M. Latifi and H. Manhouri (Iran) 893

IV. Bioquímica — Biochemistry

30. Separation of toxic eomponents from the Brazilian scorpion -

Tityus serrulatus - venom — M. V. Comez and C. R. Diniz

(B rasil) . 899

31. On the toxin of Trituras marmoratus Lalr. — H. Bachmayer

and H. Michl (Áustria) . 903

32. Biochemical investigations on ll elo d erma venom — D.

Mebs and H. W. Raudonat (Alemanha) . 907

33. Inhibition of electron transport chain by purified Phospholipase

A from Rothrops neuwiedi venom — J. C. Vidal, B. N. Ba¬

dano, A. O. M. Stoppani and A. Boveris (Argentina) .... 913

34. Preparation and properties of 1,1 1-labelled Croloxin — M.

Gomes Lomba, J. Kieffer. E. Waisbich and O. Vital Brazil

(Brasil) . 921

35. Action du venin de Naja nigricollis sur la coagulation san-

guine — J. Meaume, M. Jouannet, Y. Izard et P. Boquet

(F rança) . 929

36. Les venins de serpents, source dVnzymes anticancéreux —

I. Aspects biochimiques fondamentaux du problème — L.

Gili.o (Bélgica) . 933

37. Les venins de serpents, source dVnzymes anticancéreux -

II. Étude expérimentale — C. Wirtheimer el L. Gillo (Bél¬

gica) . 937

38. 5’-Nucleotidase Activity in Snake Venoms — Anima Devi,

S. S. Ashgar and N. K. Sarkar (índia) . 943

'LO 11 12 13 14 15 16

ÍNDICE

INDEX

V. Farmacologia — Pharmacology

39. Coiistituents of lhe venom of lhe Australian buli ant, Myrmecia

pyriformis — I. S. i»k la Lande and J. C. Lewis (Austrália) 951

40. Kecent advances on the mechanism of action of Brazilian

scorpion venom — T. serrulatus — A. P. Corrado, A. Anto-

nio and C. R. Diniz (Brasil) . 957

41. F.ffects of scorpion venom at neuromuscular junction — E. C.

Del Pozo, M. Salas and P. Pacheco (México) . 961

42. On the action of bufogenins and allied compounds on the in-

testine and other smoolh muscle organs — T. Suga (Japão) 965

43. Pharmacology of crystalline crotoxin. I. Toxicity — O. Vital

Brazil, J. P. Franceschi and E. Waisbich (Brasil) . 973

44. Pharmacology of crystalline crotoxin. II. Neuromuscular

blocking action -—- O. Vital Brazil (Brasil) . 981

45. Pharmacology of crystalline crotoxin. III. Cardiovascular and

respiratory effects of crotoxin and Crotalus durissus terrijicus

venom — O. Vital Brazil, R. Farina, L. Yoshida and V. A.

de Oliveira (Brasil) . 993

46. Pharmacology of crystalline crotoxin. IV. Nephrotoxicity —

W. A. Hadler and O. Vital Brazil (Brasil) . 1001

47. Pharmacological properties of cardiotoxin isolated from the ve¬

nom of Naja naja atra — C. Y. Lee, C. C. Chang, T. H.

Chiu, T. C. Tseng and S. Y. Lee (China) . 1009

48. The action of staphylococcal alpha toxin and the venom from

Bitis gabonica on rat stomach strip — K. Masek and H.

Rasicovà (Checoslováquia) . 1011

49. Nephrotoxic action of snake venoms — W. Raab and E. Kaiser

(Áustria) . 1017

ÍNDICE DOS AUTORES — AUTHOR INDEX . 1021

SciELO

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ANIMAIS VENENOSOS

VENOMOUS ANIMALS

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 659 - 666 , 1966

M. VANNUCCI

659

1. VENOMOUS COELENTERATES

M. VANNUCCI

Instituto Oceanogrúfico, Universidade de São Paulo, São Paulo, Brasil

An important aspect of the position held by COELENTERATES as poisonous

animais shoulcl lie emphasized immediately. All COELENTERATES, including

parasilic species, produce organelles with an elaborate structure filled with a

toxic substance. It is precisely this fact that makes them specially interesting

as ])oisonous animais since their tissues are not poisonous in lhemselves when

ingested by other animal species including man, there being a single doubtful

exception (Physalia physalis). They are venomous in the sense that, as a phylo-

genetic unit they have developed an unique and very specialized System by which

the venoms they produce are injected into lhe tissues of lhe prey or any offensive

hody that comes into contact with the coelenterate’s tissues, noticeably lhe epi-

dermis of the tentacles, mouth rim and of the acontia of anemones. This very

specialized system is represented by the organelles mentioned above which have a

complex genesis and structure. They are typical of the COELENTERATES

(CNIDARIA) and they are called nematocysts, cnidocysts or nettle cells.

They are ovoid or elongated capsules, filled with toxin. Before heing

discharged they contain the butt and the tube usually coiled spirally around the

butt. LIpon discharge, the hollow thread and butt are evaginated inside out

and function as a hypodermic syringe, emptying the contents of the capsule into

the foreign tissues which have been traversed by the tube upon explosion. Dis¬

charge is abrupt, usually explosive.

Nematocysts are usually contained in small groups of a few in an epidermal

cell provided with a cnidocil that plays a part in the discharge of lhe nemato¬

cysts lhemselves. The nematocyst carrying epidermal cell is frequently erro-

neously called a cnidoblast, but it does not usually produce the nettle cells, it

just houses them after they have been completely formed by the deeper laying

nematoblasts which derive direclly from interstitiaI undifferentiated cells. Dis¬

charge may be caused by either mechanical or chemical stimuli or both. Neither

agent can be said to be lhe universal discharge-causing factor. Nematocysts are

independent effectors(l) and their discharge is not controlled by the nervous

system nor is the stimulus for discharge transmitted by the nervous net. Structure

and ultrastructure of the nematocysts and of the cnidocil (2) have been described.

Nematocysts are very important systematic and phylogenetic characters to be

taken into account(3). The degree of intraspecific variation was shown by

Ito (4) and Vannucci (5) for a hydroid and a medusa respectively, their geo-

graph ic variation by Ito (6) and their importance to differentiate hydroids super-

ficially idêntica! belonging lo different species of different families was shown

by Brinckmann & Petersen(7).

SciELO

10 11 12 13 14 15

660

VENOMOUS COELENTERATES

Actually COELENTERATES are not the first animais in lhe animal kingdom

who have evolvetl organelles specialized in defense, offense and capture of prey.

The trichocysts of ciliates have certain similarities to nematocysts and the

polar capsules of CMDOSPOlilDIA are extraordinarily similar to them.

CN 11) OS P O lt 11) I A however, on the grounds of their morphology, life cycles

and other affinities must he placed among the SPOKOZOA. The polar

capsules also have a spirally coiled thread vvhich is evaginated upon discharge

and is used lo attach lhe spore to the inner surface of lhe host’s gut. As far

as I know, the polar capsule of the CNIDOSPORIDIA have no toxic action.

This leaves us wilh COELENTERATES as the only nematocyst producers in

the animal kingdom.

Summing up, the toxic effect of COELENTERATES is due lo their specia¬

lized organelles which inject toxic substances in other animais, when they come

in contact with them. Any animal producing nematocysts is a COELENTERATE

and inversely, all COELENTERATES have nematocysts As far as I know there

is a single exception to this ride which on the other hand is still open to dis-

cussion: this is the ctenophore Euchlora rubra which carries nematocysts

apparently produced by the animal itself and are not introduced as food(8).

A small diversion is pertinent at this stage. I have emphasized lhe fact that

COELENTERATES are animais that produce their own nematocysts, hecause

there actually are some gastropod molluscs, the NUDIRRANCHATA or sea-slugs

that feed on hydroids or siphonophores and are unable to digest undischarged

nematocysts, they rather store them away in chambers at the tip of papillae

the lumen of which is in continuity with the digestive tract of the slug. The

mechanism hy which lhe nematocysts are not digested, the migration from the

lumen of lhe gut along the diverticula and finally inlo the chamber, which is

provided with an externai pore, is yet unexplained.. Il is however true that the

nematocysts, stored in the apical chamber of the papilla are still functional and

they are used for defense hy the slugs that erect the papillae when pursued and

use the nematocysts effectively. They have been aptly called CLEPTOCN1DAE

since they are stolen cnidocysts.

As mentioned earlier, I ignore instances in which the tissues of COELENTE¬

RATES have been found to he poisonous when ingested. There are some large

species of jelly fishes which are normally eaten by man. We may refer to Kuragê

in the Ear East (Japan, also China) which indicates large SEMAEOSTOMEAE

and 1JHIZOSTOMEAE as well as the Te-Baitari of Gilbert Islands which indi¬

cates Carybdea alata (CARYBDEIDAE), all SCYRHOMEDUSAE. When consi-

dering the toxins injected hy the nematocysts, we must draw the difference

between their effect on man and on small aquatic animais. The toxins are

undoubtedly active on small animais, they have a paralyzing and finally a lethal

effect and on this property is based the normal feeding behaviour of COELEN¬

TERATES which are all carnivores and predators. Oídy a few species are lethal

to man or other large animais. About 30 cases of death hy stinging from jelly

fishes are known with certainty, almost all of them are from the Indo-Malayan

area between northern Auslralia and the Asian mainland (recent review by

Southcott (9).

We mentioned earlier that the toxin contained in the capsule of lhe nema¬

tocysts is injected in the prey as if hy a hypodermic syringe. The capsule is

usually about 10-20 p long or less, it is exceplionally large in SCYPHONOPHORA

as for instanec in Ag alma where they may reach a length of some 200 g

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 659 - 666 , 1966

M. VANNUCCI

661

or more. Their numbers however are so great in the epidermis of the COELEN-

TERATES, mainly in the tentacles and moutli rim or lips, that is has been esti-

mated that a large medusa may inject some 3 ce of toxin in a single discharge.

The nature of the toxin or toxins is not known with certainty. The principal

difficulties involved are two: 1) — il is difficult to isolate nematocysts in

sufficient quantity uncontaminated by proteins and indols from other lissues;

2) — the presence of different nematocysts in the same and in different species

each with a different function and their different effect on lhe prey suggests

that they probably contain different toxins. Lane & Dodge(lO) have elaborated

a method by vvhich nematocysts may be isolated and lhe toxin extracted and

tested separately from other tissue extracts.

The venom always has a paralyzing effect on other animais and when i(

is sufficiently strong to act on man specially when contact has occurred at

places where the skin is finer or at mucous memhranes, it causes a strong pain

that feels like stinging fire. The paralyzing effect may be due to lhe toxiiTs

action on the respiratory System. This is attributed to a glycerin soluble fraction

called congestin, which would also cause a congestion of the gut and respiratory

mucose. The alcoholic extract called thalassin causes itching, erythema and

general irritation of lhe affected area allied to nausea, prostration and death in

guinea-pigs. A fraction called hypnotoxin has also been descrihed which is

water soluble and was extracted from Siphonophores; il causes anesthesia, somno-

lence and death. All lhese toxins, whether they have been isolated in a pure

State or not, are proteins, either mucoproteins with or wilhout an indol absorbed

on them, or compounds of tetramethylammonia associaled to indols or to 5-hydro-

xytryptamine. Solutions of tetramine chemically pure when injected, cause res¬

ponses similar lo lhose of lhe extract of tentacles of different Coelenterates

such as to reduce the tendency to autotomy of the fiddler-crab, Uca mordax,

and to stimulate the excised moluscan heart. According to some authors the

intense pain is caused by tetramine and paralysis to quarternary bases associaled

lo it. Lane & Dodge(lO) obtained similar results by injecting the extract of

nematocysts freed and isolated from other tissues by a method they devised.

The lethal dose of isolated toxin in mice is of 0.037 ml/kg of a solution containing

0.201% of total N.

As mentioned above, nematocysts are independent effectors uneontrolled by

lhe nervous net, thus discharge may be highly localized, as for instance from

a fragment of a tentaele or a small portion of the lips and also occurs in anes-

thetized animais. Peterson & Vannucci (11) while working out of the cycle of

KoéUikerina jasciculata observed that this species starves to death when kept at

temperatures lower than 11°C because at such temperatures the nematocysts fail

to discharge while all other functions appear to be maintained normally. It is

interesting to note that nowhere, even at greater depths and in winter, the tem-

perature in the Mediterranean Sea falis below 13.5 or 13.8°C at most. We

doiTt know why low temperatures inhibit nematocysts discharge in this case and

the matter requires further attention since preliminary observations suggest that

an enzymatic mechanism may be at play.

Southcott(9) gives a detailed list of species of medicai importance. Based

on my own observations and experience I may add lhe following one for Bra-

zilian waters:

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10 11 12 13 14 15

662

VENOMOUS COELENTERATES

HYDBOZOA

Macrorhynchia philippina, PLUMULARIIDAE, colonial hydroid lacking a

medusa free-living stage. It looks likc inoffensive extremely delicale pens grow-

ing on algae and rocks below low tide mark. They produce an intense itching

on human skin, irritation with erylhema and the formation of little papulae,

lasting several days. Southcott records the same efíécts from lliis species in the

Indo-Pacific arca; he ralis it Lytocarpus philippinus.

OUndias mmbaquiensis, LIMNOMEDUSAE, is called “relojinho” or little watch

by local fishermen. It has a definite nettling action, lhe skin is reddened and

later becomes vesiculated; stings may cause dizziness, muscle pain and dyspnoea.

It is especially disagreahle when hands soiled hy the contact are taken lo lhe

eyes, a serious conjuntivitis may ensue.

The mosl disagreahle and painfuI species in Brazil is PhysáUa physális,

popularly knovvn as “Caravela” (Porluguese man-of-war).

There are records in the older literature of lethal cases in animais who were

fed on P h y s a lia tissues, toxicity in this case is presumably due to the

stinging of the gut hy the Siphonophore’s nematocysts. Any contact with P h y -

sal ia tentacles, even the slightest are extremely painful, produce erythema,

papulae similar to urticaria, [>ain and swelling of lymphatic system. dizziness,

nausea and muscle pains lasting several days. The reaction lo the stinging of

P hy s alia is instant and violent, it diminishes gradually with time while the

effect of lhe stinging of Hydroids is slower, growing gradually < luring the first

hours.

SCVPHOZO A

Chiropsalmus quadrumanus (CARYBDEIDAE) from Coastal water cause an

intense dermatitis, erythema, oedema and muscular pains along with swelling

of lmyphatic ganglia. Arcas stung hy the jelly-fish develop local inflammation

and ulcerations, in severe cases this is followèd hy fever, dizziness and cold

tremors.

In comparison, Chiropsalmus quadrigatus a closely related species from the

Indo-Pacific, may he lethal through pulmonar oedema, weakening of lhe pulse,

respiratory difficulties, cardial blocking and death accompanied hy slrong pains

shortly after contact with the jelly-fish.

Accidents caused hy jelly-fishes are frequently accompanied hy nervous and

psychic phenomena such as screaming, exaltation, depression, suicidai tendencies,

dyspnoea and cardiovascular disorders.

It is interesting to point out that all the dangerous species mentioned from

Brazil have closely related representatives in the Indo-Pacific area belonging to

lhe same genus or to a closely related genus. They are all mentioned by South¬

cott (1. c.). It appears that all the Atlantic counterparts are much milder than

their Indo-Pacific relatives.

Summing up we may say that lhe numher of species dangerous to man is

small in the Atlantic Ocean, while their numbers are large in the Orient, espe¬

cially in the Malayan region, in Japan and in the Philippines. Jelly-fishes are

specially dangerous because they frequently occur in very dense and sometimes

large patches, they are frequently transparent and rather difficult to see in lhe

water and further lheir long tentacles drift about with the current, sometimes

quite far away from the medusa and are quite invisible.

SciELO

10 11 12 13 14 15

Mcm. Inst. Butantan

Simp. Internac.

33 ( 3 ): 659 - 666 , 1966

M. VANNUCCI

663

Among the sea-anemones or ANTHOZOA, allhough many will produce

stinging sensible to men. lhere are no really dangerous species. We inay con-

clude by saying that among the HYDROZOA, the MILLEPORINA and other

hydroids may affect men hui not as acutely as the HYDROMEDUSAE; among

the SCYPHOZOA, the SCYPHOMEDl SAE include the species most dan¬

gerous and some may have a lelhal effect. The ANTHOZOA are not really

dangerous and the disagreable effect produced by corais is primarily due to

lacerations caused by lheir skeleton and the breakage of small fragments which

become inerusted in lhe victirns wound. lheir action through nematocysts is

only secondary.

Il is knovvn that in relation to certain species a certain degree of immunity

may be developed with time, as may be observed from fishermen that are fre-

quently exposed to certain abundant species when handling the nets soiled by

the jelly-fishes. This has been shown in relation to C y an e a a species of the

order SEMAEOSTOMEAE abundant in lhe norlhern seas. Prohably lhe same

happens here when there are large patches of Olindias sambaquiensis.

Acquired immunity may be an explanation of certain curious cases of sym-

biosis, difficult to explain otherwise, except perhaps by natural immunity or

selection. The association between different species of PAGURIDAE, the hermit

crabs, with certain sea-anemones has been famous for centuries, specially that

between Eupagurus bernhardus and Adamsia palliatu. Also the fact that young

fishes, mainly cod find refuge between the hanging tentacles of large medusae,

as for instance Cyanea, is well known and equally not fully understood.

The symbiosis between certain coral fishes and

STOICHACTIDAE is less widely known but has

experimentally. In European aquaria, noticeably

aquaria, specially at Marineland of the Pacific,

Amphiprion percula some 5-10 cm long that live

three in the tentacular crown of large sea-anemones of the genus Stoichactis.

They never swim very far from their host, they defend their territory most

actively and over and over again return to lhe sea-anemones’ tentacles, lying

and rubbing against them. Neither the fish appears to be stung by lhe coelen-

terale’s nematocysts nor does the sea-anemone show any attempt to feed on

the fishes. A feeding behaviour would immedialely be induced by any other

animal behaving as that particular fish. Davenport (12) studied this behaviour

experimentally. It is well ascertained that both the sea-anemone may live in

symbiosis with other fishes of closely related species, and viceversa, the fish

may live in symbiosis with closely related sea-anemones, either may be found

isolated. Amphiprion which have been kept in isolation for months require

several days to acclimatize lo the anemone, during the process they began

eontact slowly and gradually with the deadly tentacles. Il is believed that this

is the means by which the bonds between the two species are established as

well as the physiological barrier that protects the fish from the nematocysts and

from being swallowed by the COELENTERATE. DavenporPs experiments suggest

that the mucus secreted by the fish contains an active principie that raises the

threshold required for nematocyst discharge in this species of sea-anemone. This

active principie is inactive at the threshold of eleclrically induced discharge,

it acts fast, is specific and thermolabile. The same principie is not contained

in lhe fish’s muscles.

Since a Stoichactis acclimated to Amphiprion does not exhibit

the. feeding reactions it shows to other fishes or to a non acclimated A m p h i -

p r i o n , it may be presumed that the active principie contained in lhe epi-

sea-anemones of the family

in recent years been studied

at Monaco and in American

one may observe fishes like

in small groups of two or

SciELO

10 11 12 13 14 15

664

VENOMOUS COELENTERATES

(Icrrnal

well

mucus of Amphiprion acts on

reactions.

the

anemone s nervous lissue

inhibiting its feecling

dischargel

Pantin (1) studied the mechanism hy which nematocysts are

He showed among other points that: a) nematocysts are independent effecto

írecently Ross, unpuhlished, has expressed the view that he has evidences

show that in one case at least the nervous system may interfere with nematoeyl

discharge, see Hand (13), b) discharge of nematocysts may he stimulated

mechanical and hy Chemical means, among the latter specially hy surface acti

substances, c) Solutions of usual foods make nematocysts more sensitive

discharge, hy mechanical means.

Thus, if the discharge threshold of nematocysts is lowered hy means

adequate food stuffs, inversely one must admit that lhe threshold for dischar

must he raised hy disagreahle substances. If the mucus secreted hy Amphipric

percula is disagreahle lo Stoichactis, its nematocysts will not he dischargel

and further the sea-anemone will have no feeding reactions, similar lo wh

happens with Anemonia sulca ta that does not reaet to and therefore does n

ingest and does not feed upon fragments of flesh of the same species. As f,

as I can see it, this may be a tentative explanation of the behaviour of S t o

chactis and Amphiprion when acclimated one to lhe other. Furthi

Parker & Van Alstyne (14) showed that extract of the epidermis of the fid

Fundulus causes the discharge of a eertain nurnber of nematocysts of ll

sea-anemone Metridiu m and of P h y s a l i a and Davenport has shown th,

wounded Amphiprion or fishes from which even a single scale is missi

are no longer immune to the stinging hy Stoichactis. Analysing all the:

facts the following tentative hypothesis may be drawn: Stoichactis’ nem

tocysts normally react only feebly to the mucus of lhe epidermis of A m p h

prion, the anemone does not react to this fish with its normal feeding beh

vionr, probably due to inhibition of the feeding behaviour hy lhe mucus. Fu

ther, Davenport has shown that fishes not acclimated to the sea-anemone on

gradually develop more intimate contacts with the sea-anemone. This suggesl

that the fish acquire immunity gradually and that the stinging and intoxicatioi

which it receives at first are not very effective due to lhe small amount

discharged nematocysts. It seems to me that experiments should be carried o

along these lines that may take to the explanation of one of the most interestir

and obscure cases of symbiosis yet described. Information available suggests

acquired immunity of the fish to the nematocysts and a feeding behaviour

the sea-anemone inhibited hy the fish’s mucus.

Before closing I wish to rernind the audience that the present extreme

modest communication was purposely just a short reminder of the venomo

aspect of beautiful COELENTERATES, in a symposium that is mainly concerni

with such terrific animais as serpents, spiders and scorpions.

References

1. PANTIN, C. F. A., J. exp. Biol., 19 (3), 294-310, 1942.

2. BOUILLON, G., CASTIAUX, P., and VANDERMEERSSCHE, G., Buli. MU |

apyl., 8 (3), 61-63, 1958.

3. WERNER, B., Helgoland wiss. Meeresunters., 12 (1-2), 1965.

4. ITO, T., Rep. Tohoku Univ., Ser. 4, 18 (2), 211-215, 1948.

SciELO

10 11 12 13 14 15

Mem. Tnst. Butantan

Simp. Internac.

:«< 3 ): 659 - 666 , 1966

M. VANNUCCI

665

5. VANNUCCI, M., Pubbl. Staz. Zool. (Napoli), 31 (3), 393-420, 1960.

6. ITO, T., Mem. Ehime Univ., Sec. II, 1 (2), 69-80, 1951.

7. BRINCKMANN, A., and PETERSEN, K. W., Pubbl. Staz, Zool, (Napoli), 31

(3), 386-392, 1960.

8. PICARD, J„ Rec. Trav. Stat. Mar. Fac. Marseille, 15, 99-103, 1955.

9. SOUTHCOTT, R. V., in H. L. KEEGAN, and W. V. MAC FARLANE (Editors),

Venomous and Poisonous Animais and Noxious Plants of the Pacific Region,

1963.

10. LANE. C. E„ and DODGE, E„ Biol. Buli,, 115 (2), 216-219, 1958.

11. PETERSEN, K. W., and VANNUCCI, M„ Pubbl. Staz. Zool. (Napoli), 31, 473-

492, 1960.

12. DAVENPORT, D., and NORRIS, K. S., Biol, Buli, 115 (3), 397-410, 1958.

13. HAND, C., Biology of Hydra, 1961, pp. 187-202.

14. PARKER, G. H„ and VAN ALSTYNE, M. A., J. exy. Zool., 63, 329, 1932.

SciELO

10 11 12 13 14 15

Riem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 667 - 67 - 1 , 1966

BERTA S. GERSCHMAN DE PIKELIN y

RITA D. SCHIAPELLI

667

2. CONTRIBUCIÓN AL CONOCIMIENTO DE THERAPHOSA LEBL0ND1

(LATREILLE), 1804 ( ARANEA: THERA PHOSI DAE)

BERTA S. GERSCHMAN DE PIKELIN y RITA D. SCHIAPELLI

Museo Argentino de Ciências NaHirales “Bernardino Rivadavia’’,

Buenos Aires, Argentina

En nuestra visita al Natural History Museuni of New York en el ano 1960,

tuvimos oportunidad de ver entre el material no determinado de la colección

de ararias de ese Museo, un ejemplar macho de MVGALOMORPHAE,

que a pesar de estar partido por la mitad nos llamó la atención por su enor¬

me talla.

Aunque en nuestros estúdios sohre géneros de THERAPHOSIDAE, como

X ene st is Simon, 1891; Acanthoscurria Ausserer, 1871 y Grani¬

mos tola Simon, 1892, cuyas especies pueden tener ejemplares que llegan

a los 80 mm, estáhamos acostumhrados a ver aranas gigantes, el ejemplar dei

Museo de Nueva York nos asombró, no solo por su enorme cuerpo, sino por

el grosor de sus patas, cuyos fémures anteriores tienen un diâmetro ántero-poste-

rior de más de 8 mm, casi el doble dei de las otras aranas de gran tamano.

Por gentileza dei Dr. Gertsch, Curator of ARACHNIDA, pudimos traer al

Museo Argentino de Ciências Naturales “B. Rivadavia”, en préstamo y para

su estúdio, vários ejemplares de MYGALOMORPHAE que necesitábamos para

comparación, e incluímos esa araria gigantesca que estaba sin determinar y que

no conocíamos; procedente de Venezuela, Auyan-te|)ui, a 1.100 m. de altura,

recolectada en el ano 1937.

Con las claves de Simon (1892) llegamos fácilmente al género Thera-

phosa Walck., 1805 y a su especie-tipo Th. leblondi (Latreille), 1804.

Hicimos la determinación no exentas de emoción, porque es una especie muy

rara en las colecciones, y a pesar de que está muy citada en la literatura araneo-

lógica, nada se sabe de su veneno y ecologia. Este se debe a que la literatura

está llena de datos contradictorios porque muy pocos aracnólogos la han visto.

La mayor parte de ellos repite lo dicho por otros y mucbos la han confundido

con aranas de gran tamano pertenecientes a otros géneros. Por cuya razón

Simon dice (1892, p. 159): “Es la única especie conocida descripta por Latreille

en Ilisl. nat. Crust. Ins., 7, 159, 1804. El nombre de Mygale leblondi o blondii

ha sido aplicado a muchas especies de gran talla que habitan América dei Sud.

El tipo de Latreille, traído de la Guayana por Leblond existe todavia en el

Museo” (Se refiere al Museo de Paris). “Esta rara especie parece propia de

la región dei Maroni, de donde yo la he recibido; es por error que ha sido

citada para las Antillas, Brasil y aun para Java! (Por C. L. Koch)”. Mas

adelante dice Simon que el género se encuentra también en el Orinoco.

Así se explica que algunos autores la hayan visto trepando por los árboles

en busca de nidos de colibríes de cuyos huevos y pichones se alimentarían; otros.

SciELO

10 11 12 13 14 15

dentro de cuevas en el suelo, sin tapizar, y en cuya entrada se encuentran ai

anochecer al acecho de sus presas.

Para algunos son ararias tímidas que tratan de huir al ser hostigadas, no

siendo peligrosas. Para otros son ararias temidas en los lugares en donde viven.

Por esa razón, si bien la tarea de determinaria fué fácil, se complico mu-

ehísimo su estúdio al consultar la bibliografia, por la confusión, no solo en su

ecologia y distribución, sino también en los caracteres considerados para su

determinación.

Finalizado este trabajo y después de haber sido presentado nuestro “Estú¬

dio sistemático comparativo de los géneros Theraphosa Walck., 1805,

Lasiodora C. L. Koch, 1851 y Sericópelma Ausserer, 1875” al

Simpósio Sôbre a Biota Amazônica, Belém, Pará, Brasil, junio 6-11, 1966,

tuvimos oportunidad de ver en el Museo Gõldi de aquella ciudad, ejemplares

macho y hembra de Theraphosa leblondi (Latr.), 1804, recolectados por el

seííor P. Waldir en Benevidez y Utinga. Por lo que dejamos estableeido en

contra de la opinión de Simon, que esta especie se encuentra también en el

Estado de Pará, Brasil ecuatorial.

En el afio 1965, el Sr. Pablo San Martin, dei Uruguay, nos trajo para su

estúdio, varias ararias MYGALOMOBPHAE procedentes de Rio Caura, Departa¬

mento Bolívar, Venezuela, las que resultaron ser hembras de Theraphosa leblondi

iLatr.), 1804, coincidentes en Iodos los caracteres con el ejemplar macho que

leníamos en nuestro poder. Pudimos así observar las espermalecas e ilustrarias,

las que basta ahora no se habían estudiado (carácter muy importante en la

determinación de las MYGALOMOBPHAE — Schiapelli & Gerschman de Pike-

lin, 1962).

El Sr. San Martin, que luvo una de esas ararias viva en su laboratorio

durante un ano, alimentándola con insectos, nos dijo: “Fueron bailadas bajo

bases de hojas de palmera en la selva, en la proximidad dei rio. Esas bases de

hojas, al desprenderse y precipitarse al suelo con la parte externa bacia arriba,

eonstituyen un notable refugio, en forma de media caiia. El lugar en donde se

establece la arana está alisado. A su captura presenta una fuerte defensa y gran

agresividad, Ia que también se observo en la araria mantenida viva en el labo¬

ratorio.”

Es curioso, y lo hacemos notar aqui, que el ejemplar traído de Nueva York

fué partido por la mitad con un machete por el que la recolectó. Latreille dice

que el ejemplar típico de la especie que denomino leblondi fué nruerto con la

espada por el Dr. Leblond. Según parece la vista de esa arana despierta el

espíritu guerrero de sus colectores.

El aporte de San Martin fué de gran valor para nuestra investigación. En

las ararias MYGALOMORPHAE es muy difícil la determinación de los ejem¬

plares hembras por falta de genitalia externa, epigino, que se encuentra en las

ararias ARANEOMORPHA. habiendo sido rnuchas veces confundidos los distintos

géneros y aun determinados como holotipos hembras de espécies nuevas, ejem¬

plares machos jóvenes. En los machos adultos el problema no es tan difícil,

porque su genitalia externa se ve fácilmente en los tarsos de los palpos y permite

su identifieación, lo que está facilitado por los caracteres sexuales secundários

que se encuentran presentes.

Con el material a nuestra disposición, 1 macho y 5 hembras, que pudimos

comparar con los tipos macho y hembra. remitidos por el Prof. Dr. Max Vachon,

dei Muséum National d’Histoire Naturelle de Paris, en donde se encuentran

2 3

5 6

10 11 12 13 14 15

SImp. Internac.

Mem. Inst. Butantan

33 ( 3 ): 667 - 674 , 1966

BERTA S. GERSCHMAN DE PIKELXN y

RITA D. SCHIAPELLI

669

depositados, damos en este trabajo la diagtiosis de la especie y de acuerdo a

nuestro sistema, precedida por la bibliografia temática; (fí: bibliografia; Cal:

catálogo; Cl: clave; Distr: distribución geográfica; L: lista; Morf : morfologia;

Ref: referencia; Syn: sinonimia; Ven: veneno.) e ilustramos los caracteres espe

cíficos diferenciales como ser ojos, órgano estridulatorio, bulbo, palpo y esper-

matecas. La escala en los dibujos no indica medida sino orientación. Las

medidas están dadas en milímetros.

Theraphosa leblondi (Latreille), 1804.

1804 Mygale Blondii: Latreille P. A., Hist. nat. gen. part. Crustac. Insec., Paris.

an XII, 7:159 (D c? sp. n.).

1805 Mygale blondii: Walckenaer C. A„ Tab. Aran. trib. gen. famil., Paris, XII: 4

(D ecol.).

1805 Mygale blondii: Palisot de Beauvois, A.M.F.J., Insec. rec. Afrique et Ameri-

que 1786-1797, Paris, p. 4.

1806 Mygale Blondii: Latreille P. A., Genera Crast. et Insect., Paris, 1:83.

1818 Mygale Blondii: Latreille P. A., Encycl. méth. Hist. nat. /ws.. Paris, p. 9.

1818 Mygale Blondii: Latreille P. A., N. Dic. hist. nat., Paris, 22:109.

1820 Mygale Blondina: Hahn C. W., Monographia Aranearum, Nürnberg, 8.

1825 Mygale Blondii: Jarocki F. L., Zool. swierz. ogol, podl., Warszawie, 5:332.

1830 Mygale Blondii: Martius F., in Delect. Anim. Art., Monachii, p. 36 (Ven).

1831 Mygale de Leblond: Latreille P. A., Conrs d’Entom., Paris, p. 506.

1832 Mygale de Le Blond: Latreille P. A., N. Ann. Mus. Hist. nat., Paris, 1:69.

1833 Mygale blondii: Perty M., in Delect. Anim. Art., Monachii, p. 271.

1837 Mygale Blondii: Koch C. L., Uebers. Arachn., Nürnberg, 1:35.

1837 Mygale Blondii: Walckenaer C. A„ Hist. nat. Ins. Apt., Paris, 1:210 (D ecol.).

1837 Mygale Leblondii: Lucas H., Dic. pit. hist. nat., Guerin, Paris, 5:542.

1840 Mygale Blondii: Lucas H„ Hist. nat. Anim. art., Paris, p. 335 (D d 1 Distr.).

1842 Mygale Blondii: Koch C. L., Die Arach., Nürnberg, 9:91.

1849 Mygale Blondii: Menzel A., N. jahrsbl. naturf. Ge s. Zurich, 51:6.

1851 Mygale Blondii: Koch C. L., Uebers. Arachn., 5:72.

1851 Mygale Leblondii: Lucas H., Ann. Soc. ent. Fr., 9(2):LXLI (Ecol.).

1851 Mygale Blondii: Blanchard E., C. R. Acad. Sei., 34:402 (Morf.).

1857 Mygale Blondii: Lucas H., C. R. Acad. Sei., 45:1103.

1857 Mygale Blondii: Lucas H., Arachn., in F. Castelnau, Exp. Amerique Sud.

3:14 (Ref.).

1857 Mygale Blondii: Carus J. V., Icon. Zootec. Arachnida, I, Lam. XIII.

1858 Mygale de Leblond: Lucas H., Ann. Soc. ent. Fr., 6(3) :CCXXXIII (Morf.).

1863 Mygale Blondii: Bates H. W., The Naturalist on the River Amazons. Lon-

don, p. 53, p. 194 (Ecol.).

1864 Mygale de Leblond: Simon E., Hist. nat. Ar., Paris, p. 70.

1871 Theraphosa Blondii: Ausserer A., Verh. bot. zool. Ges. Wiener, 21:216 (D

gen Syn).

1873 Mygale Blondii: Taczanowski L., Horae Soc. ent. Ross., 10:101 (Ref.).

1874 Theraphosa blondii: Moggridge J. T„ Trap.-doors Spiders, London. pp. 188,

234.

SciELO

10 11 12 13 14 15

C70 CONTRIBUCIÓN AL CONOCIMIENTO DE THERAPHOSA LEBLONDI

(LATREILLE), 1804 (.ARANEA: THERAPHOS1DAEI

1880 Theraphosa Blondii: Karsch. F., Zeits. gesam. Naturw., 53:844 (D t? Ecol.).

1882 Mygale blondii: Künckel d’Herculais J., in A. E. Brehm, Les Insectes, etc.,

Paris, 3:719.

1890 Mygale blondii: MacCook H. C., American Spiders, 2:142, 189 (Ecol.).

1890 Mygale Blondii: Trouessart E. L., La Geographie zoologique, Paris, p. 125

(Ref.).

1892 Mygale Blondii: Wood J. G., Homes without hands, etc., London, p. 114.

1892 Theraphosa Leblondi: Simon E., Hist. Nat. Ar., 1:156 (Morf.), 159 (Dá 1 y 9 ).

1895 Mygale Blondii: Wagner J., Ann. Mag. Nat. Hist., 15(6) :308 (Anat).

1902 Theraphosa Blondii: Kobert R., Beitr. Kennt. Giftspinnen, Sluttgart, 26:678.

1903 Theraphosa Leblondi: Simon E., Hist. Nat. Ar., 11:940 (Distr.).

1907 Theraphosa Leblondi: Strand E., Jahresh. Ver. Naturk. Wurt., 63:62 (D J

y 9 Ecol.).

1909 Theraphosa leblondi: Warburton C. in Cambridge Natural History, London,

4:336.

1911 Theraphosa blondi: Petrunkevitch A., Cat. Spiders, Buli Amer. Mus. Nat.

Hist., 29:91 (Cat).

1912 Theraphosa leblondi: Strand E., Jahrb. nassau. Ver. Naturk., 65:176.

1913 Theraphosa leblondi: Merian P., Rev., Mus. La Plata, 20:55 (Ref.).

1922 Mygala blondii: Phisalix M„ Animaux venimeux et venins, 1:262.

1927 Theraphosa blondi: Pawlowsky E. N., Giftiere und ihre Giftigkeit, Jena,

p. 153.

1928 Theraphosa leblondi: Petrunkevitch A., Trans. Connect. Acad. Sc., 29:81 <L).

1931 Theraphosa leblondii: Savory T. H., Arachnida, in W. P. Pycraft, The

Standard Nat. Hist. from Amoeba to Man, London, p. 397.

1932 Theraphosa leblondi: Berland L.. Encyclop. entom.. Paris, 16:92 (Morf.), 333

(Distr. Morf.).

1933 Theraphosa leblondi: Ewing H. E., Nation. Geogr. Mag., 64:166.

1936 Theraphosa leblondi: Vellard J., Le Venin des Araignées, Paris, p. 137 (Ref.).

1939 Theraphosa blondii: Petrunkevitch A., Trans. Connect. Acad. Sc., 33:247 (Cat.).

1949 Theraphosa leblondi: J. Millot, in Traité Zool. Grassé, Paris, VI:723.

1959 Theraphosa leblondi: Bonnet P., Bibliographia Araneorum, 11(5) :4425(B).

1961 Theraphosa leblondi: Loc. cit., 111:231 (Ref.).

Diagnosis.

cT Ejemplar de la colección dei Natural History Museum of New York.

/•* rocedencia: Venezuela (Auyan-tepui) a 1.100 m. de altura.

Col. Phelps exp., 6.XII. 1937 (El colector la ha dividido por la mitad con

un machete).

Largo total: 80. Cefalotórax: 40 X 40. Lábio: 14 de ancho por 7 de alto.

Pata

Fémur

Patela mds tibia

Metatarso

Tarso

Total

109

102

III

28,5

100,5

113

SciELO

10 11 12 13 14 15

Simp. Internac.

Mem. Inst. Butantan

33(3) : 667-674, 1966

BERTA S. GERSCHMAN DE PIKELIN y

RITA D. SCHIAPELLI

671

Cefalotórax igual de ancho que de largo, muy redondeado, su mayor an¬

chura a la altura de la coxa II. Foseta apenas recurva, muy profunda, más

ancha que el túmulo ocular (6).

Túmulo ocular muy elevado, casi redondo (5 de ancho por 4 de alio).

Fila anterior muy procurva, ojos iguales, casi equidistantes; más grandes

que los ojos de la fila posterior. Fila posterior recurva, los MP casi dei mismo

tamano que los LP. (Lam. 1:6).

Lábio más ancho que alio, con una estrecha banda de espínulas próxima

al borde.

Coxas de la pata maxila con el ângulo súpero-inlerno muy prolongado.

Patas IV, 1, II y III muy espinosas. Las anteriores más gruesas que las

posteriores. Llaman la atención por el grosor de sus artejos basales. En la

pata 1 el fémur tiene un diâmetro dorso-ventral de 9 mm; el II. 8,5; el III, 9

v el fémur IV, 8. Patela más tihia I es un poco mas larga que patela más

tibia IV. El fémur IV tiene en su cara posterior una densa escópula de pelos

no plumosos. El melatarso IV no tiene escópula.

Órgario estridulatorio. La coxa I en su cara anterior y parte superior, por

encima de la sutura, tiene 3 cerdas plumosas de gran tamano (Lam. I: 5) dis-

puestas en línea, las que están acompanadas por otras cerdas mucho menores y

pelos plumosos que recubren todo el resto de la coxa, trocánter y mas de la

mitad hasal dei fémur. En la coxa de la pata maxila no se observan las cerdas

gruesas en su cara externa, pero presenta en su mitad apical espinas negras muy

recostadas sobre el artejo, que aumentan de tamano a medida que se acercan

a su extremidad apical. Los pelos plumosos se encuentran en el borde inferior

de la cara externa de la coxa, recubren el trocánter y mas de la mitad hasal

dei fémur de la pata maxila.

Pata maxila (Lam. 1: 3) Bulbo: es casi cilíndrico en toda su extensión

siendo excavado en su extremidad apical en forma de cuchara cuyo borde es

dentado. (Lam. I: 1 y 2).

9 de la colección dei Museo de Historia Natural de Montevideo.

Procedência. Venezuela, Dep. Bolívar, Rio Curá.

Col. Pahlo San Martin, V. 1957.

Largo

total : 84.

Cejalotórax: 32,5 X

32,5. Labio:

8 de ancho

X 5 de

Pata

Fémur

Patela más tibia

Metatarso

Tarso

Total

III

24,5

27,5

SciELO

10 11 12 13 14 15

672

CONTRIBUCIÓN AL CONOCIMIENTO DE THERAPHOSA LEBLOND1

(LATREILLE), 1804 (ARANEA: THERAPHOSIDAE)

Ejemplar $ N.° 2.

Largo total : 76. Cefalotórax : 31 X 31.

Pata

Fémur

Patela más

tíbia Metatarso

Tarso

Total

III

Ejemplar

9 N.°

Largo total : 81.

Cefalotórax:

34 X 34.

Pata

Fémur

Patela más

tíbia Metatarso

Tarso

Total

31,5

12,5

III

28,5

85,5

27,5

101,5

La hembra es en todo igual al macho. El cefalotórax es también igual de

ancho que largo en los 5 ejcmplares estudiados; tampoco hay diferencias en el

órgano estridulatório. La foseta torácica muy profunda, transversa, puede ser

más o menos recurva.

Espermateca : Lam. 1: 7 y 8. No presenta diferencias en los 5 ejemplares.

Distribución geográfica. Este género tiene una sola especie, la especie-tipo

Theraphosa leblondi (Latr., 1804) procedente de las Guayanas (región dei Ma-

roni) y de Venezuela. Y a pesar de que Simon niega su presencia en Brasil,

dejamos estahlecido que se encuentra en el Estado de Pará.

Observaciones. Tanto el ejemplar macho como las hemhras sobre los que

se ha hecho la diagnosis arriba consignada, fueron comparados con los ejemplares

típicos de la colección dei Muséum National d’Histoire Naturelle de Paris. Dos

de las hemhras colectadas por P. San Martin quedan depositadas en la colección

dei Museo Argentino de Ciências Naturales “B. Rivadavia”, como Homeotypus

con los números 5874 y 5875.

SUMMARY

With one mate specimen from Venezuela belonging to the collection of the

American Natural History Museum of New York, and five females collected also

in Venezuela by P. San Martin, this species, rare in the museums, has been studied.

Much has been written about it in the last century and at the beginning of this

one; but since 1936 it has not been mentioned in the spider literature, only in

catalogues and lists. In this paper the diagnosis is given pointing out the valid

characters to separate it from the other THERAPHOSIDAE. These characters

are iliustrated in one table with 8 figures. The studied specimens were compared

with the Types kept in the Muséum National d’Histoire Naturelle de Paris. For

the first time the spermathecae are iliustrated. Up to now they were not been

described.

Agradecimientos — Agradecemos al Dr. W. H. Gertsch, al Prof. Dr. Max Vachon

y a los Senores Pablo San Martin, Roger Arlé y P. Waldir, la gentileza que han

tenido al poner el material estudiado a nuestra disposición.

SciELO

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Mem. Inst. Butantan

Slmp. Internac.

33(3):675-682, 1966

RITA D. SCHIAPELLI y BERTA S. GERSCHMAN

DE PIKELIN

675

3. ESTÚDIO COMPARATIVO DE PHONEUTRIA FERA PERTY, 1833 Y

PHONEUTRIA NIGRIVENTER (KEYSERLING), 1891

(AR AN EA: CTENIDAE)

RITA D. SCHIAPELLI y BERTA S. GERSCHMAN DE PIKELIN

Museo Argentino de Ciências Naturales “Bernardino Rivadavia’’,

Buenos Aires, Argentina

Durante largos anos el género Phoncutria Perty, 1833 flguró en la

literatura araneológica como sinónimo de Ctenus Walckenaer, 1805.

En 1936, Mello Leitão, en su revisión de la familia CTENIDAE da una

clava de géneros y separa a los dos mencionados, principalmente, por la presen¬

cia en Phoneutria de una densa escópula en la cara interna de las tildas

y tarsos de las patas maxilas, tanto en machos como en hembras.

Como género, los ejemplares de Phoneutria se determinan fácilmente:

pero sus especies están muy confundidas. Son en total unas 12 especies, casi

todas dei Brasil, de las cuales Ph. fera Perty, 1833 y Ph. nigriventer (Keys.),

1891 se han citado también para la Argentina. Esas dos especies, de las cuales

nos ocupamos eu este trabajo, han sido sinonimizadas últimamente por algunos

autores; sinonímia que no está bien fundamentada.

Phoneutria fera Perty, 1833 a pesar de haber sido citada para la Argentina

por Holmberg y Mello Leitão, no es autóclona; esta especie recolectada por J. B.

de Spix en Rio Negro, Brasil ecuatorial, se encuentra en Rio de Janeiro y en

litoral de B rasil. A la Argentina llega solamente con los cachos de banana im¬

portados de Brasil.

El tipo de Phoneutria nigriventer (Keys.), 1891 procede do Rio Grande do

Sul, en el Estado dei mismo nombre, fué recolectado por von lhering; esta especie

es común en São Paulo y en el Estado dei mismo nombre, llegando a la pro¬

víncia de Misiones, Argentina, en donde se la encuentra con bastante frecuencia.

Para nuestro estúdio hemos contado con material de distintas colecciones.

Agradecemos al Dr. Max Birabén por habernos facilitado el material de la co-

lección dei Museo de La Plata y el de su propia colección; al Dr. Avelino Barrio

por habernos entregado para su estúdio el material dei Instituto Nacional de

Microbiología; al Dr. Tomsic dei Instituto Miguel Li 11o de Tucumán por habernos

facilitado algunos ejemplares que poseían en la colección; al Dr. J. de Araújo

feio y a la Dra. Anna Timotheo da Costa por la gentileza de enviamos en

préstamo material dei Museo Nacional de Rio de Janeiro.

Además contamos con el material depositado en la colección dei Museo Ar¬

gentino de Ciências Naturales “B. Rivadavia” en donde trabajamos.

Ph. fera y Ph. nigriventer son muy semejantes; pero su estúdio comparativo

nos ha permitido establecer dos caracteres que se correspondeu y permiten su

SciELO

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676

ESTÚDIO COMPARATIVO DE PHONEUTRIA FERA PERTY, 1833 Y PHONEUTRIA

N1URIVENTER (KEYSERLING), 1891 ( ARANEA-CTENIDAE )

diferenciación; esos caracteres son: la proporción cie ancho y largo dei epiginio

en ia hemhra y la proporción dei ancho y largo de la tihia dei palpo en el

macho, y el colorido dei ejemplar.

Perty, en 1833 hace una descripción muy somera de Ph. fera senalando

su colorido uniforme.

Keyserling, en 1891 al describir su especie nigriventer, que él llama Ctenus

nigriventer, hace notar que es muy semejante a fera dc la que se diferencia

por el abdômen con manchas dorsales y el vientre con un campo negro; considera

que los epiginos de ambas son iguales, crec que nigriventer podría ser una sub-

especie de fera ; no puntualiza que en su especie el epígino, es más o menos igual

de ancho que alto. Que es lo que hace Pickard-Cambridge en 1897 en su “Revi-

sión de la familia CTENIDAE", trabajo que ilustra con dibujos de los epiginos

de ambas espeeies; dibujos que muestran claramente la diferente proporción

en los mismos. Esta diferencia Ia hemos constatado en el material estudiado.

(Lam. I: 1 y 2; 5 y 6).

En cuanto al colorido, fera se ha descrito siempre como de abdômen uni¬

color; en cambio nigriventer además de la mancha ventral que le da su nomhre.

presenta un folio dorsal más claro. Si bien en los machos la mancha ventrai

negra está a veces muy disminuída, siempre se puede observar una banda obscura

en el borde posterior dei pliegue epigástrico siendo constante su dibujo dorsal.

Mello Leitão (1936) ilustra los palpos de ambas espeeies y hace notar la

diferencia de proporción de la tibia en ambas; siendo más delgada en nigriventer

y más gruesa en fera.

Vellard (1936) al estudiar el veneno de esas ararias, senala que su acción es

muy semejante, no sólo en fera y nigriventer sino también en Ph. rufibarbis,

1833 cuyo epigino es completamente distinto. El veneno de fera actua más inten¬

samente sobre las fibras musculares, produciendo convulsiones más violentas y

una parálisis más rápida. El veneno de nigriventer tiene una acción hipotensa

más marcada. I'h. rufibarbis excita menos las secreciones.

La correspondecia de los dos caracteres: proporción dei epigino en las hembras

y de la tibia dei palpo dei macho con el colorido general: epigino corto y tibia larga

con abdômen de dibujo dorsal; epigino largo y tibia corta con abdômen de colo¬

rido uniforme; caracteres constantes en el material estudiado (15 ejemplares de

fera y 50 de nigriventer ) y como se pueden apreciar en los dibujos que ilustrau

este trabajo, nos permiten establecer que las dos espeeies, Phoneutria fera Perty,

1833 y Phoneutria nigriventer (Keys.), 1891 son válidas.

Los dibujos están realizados por las autoras con câmara clara y pasados en

tinta por la dibunante Sra. Sara Kahanoff con un subsidio otorgado por el Con-

sejo Nacional de Investigaciones Científicas y Técnicas de Buenos Aires.

Se agrega un mapa con la distribución geográfica de las espeeies basadas

en las localidades dei material estudiado, y una lista bibliográfica de los trabajos

mas útiles en su estúdio.

Summary

Phoneutria fera Perty, 1833 and Phoneutria nigriventer (Keyserling), 1891 are

carefully studied, pointing out the differences and similarities, that are illustrated

with drawings with camera lúcida made by the authors. The study was made on

the material of the collection of the Museo Argentino de Ciências Naturales “B.

Rivadavia”, Museo de La Piata, Museo Nacional de Rio de Janeiro, Dr. Max Bira-

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33<3):675-682, 1966

RITA D. SCHIAPELLI y BERTA S. GERSCHMAN

DE PIKELIN

677

ben’s private collection and other private collections. The diagnosis preceded by

its bibliography is given, and also a map showing their distribution based on the

material studied;

Bibliografia

1. BONNET, P., Bibliographia Araneorum, Vol. II, Douiadoure, Toulouse, 1958,

p. 3619.

2. BÜCHERL, W., Mon. Inst. Butantan, 1, 22, 40, 53, 67, 1951.

3. BÜCHERL, W., Mem. Inst. Butantan, 24 (2), 127-156, 1952.

4. BÜCHERL, W., Mem. Inst. Butantan, 25 (1), 153-174, 1953.

5. BÜCHERL, W., Mem. Inst. Butantan, 31, 31-85, 1964.

6. HOLMBERG, E. L., Arácnidos Argentinos, Coni, P. E., Buenos Aires, 1876,

p. 26.

7. KEYSERLING, E., Die Spinnen Amerikas (Brasilianische Spinnen), Bauer und

Raspe, Nürnberg, 1891, pp. 143-145, tab. 4, fig. 98.

8. MELLO LEITÃO, C. DE, Festschr. Strand, 1, 1-18, 1936.

9. MELLO LEITÃO, C. DE, IV Congr. int. Entomol., Madrid, Vol. 9, Notes sur

la famille Ctenidae Keys. et sa distribution geographique, 1940, p. 104.

10. MELLO LEITÃO, C. DE, Arq. Inst. Biol. (S. Paulo), 11, 242, 1940.

11. MELLO LEITÃO, C. DE, Arq. Mus. Nac., 37, 196, 1943.

12. PERTY, M., Delectus Animalium Articulatorum, Monachii, 1833, p. 197, tab. 39,

fig. 3.

13. PICKARD-CAMBRIDGE, F. O., Ann. Mag. Nat. Hist., Ser. 6, 19, 52-105, 1897.

14. STRAND, E., Z. Naturw., 79, 425, 1907.

15. VITAL BRAZIL e VELLARD, J., Mem. Inst. Butantan, 2, 5-78, 1925.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Intcrnac.

33(3):675-682, 1966

RITA D. SCHIAPELLI y BERTA S. GERSCHMAN «70

DE PIKELIN

0) , „,.

o o

'Õ

o o

Limm

O o

Lam. I: Phoneutria nigriventer (Keys.), 1891. FIg. 1 — epigino, cara ventral. Fig. 2

— epigino, cara dorsal (clarificado). Fig. 3 — grupo ocular dei macho.

Fig. 4 — grupo ocular de la hembra.

Phoneutria fera Perty, 1833. Fig. 5 — epigino, cara ventral. Fig. 6 — epi¬

gino, cara dorsal (clarificado). Fig. 7 — grupo ocular dei macho. Fig. 8

grupo ocular de la hembra.

SciELO

10 11 12 13 14 15

Lam. II: Phoneutria nigriventer (Keys.). 1891. Fig. 9 — palpo dereeho dei macho, cara

interna. Fig. 10 — palpo dereeho dei macho, cara externa.

Phoneutria fera Perty, 1833. Fig. 11 — palpo dereeho dei macho, cara in¬

terna. Fig. 12 — palpo dereeho dei macho, cara externa.

Mem. Inst. Bulantan

■Simp. Internar.

33(3): 675-682, 1966

RITA D. SCHIAPELLI y BERTA S.

DE PIKELIN

Lam. III: Bulbo distendido de Phoneutria nigriventer (Keys.). 1891: Fig. 13 — cara

interna, Fig. 14 — cara externa. Bulbo distendido de Phoneutria fera Perty-

1833: Fig. 15 — cara interna, Fig. 16 — cara externa.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3): 683-688, 1966

ÁLVARO D*

4. INVESTIGACIÓN ECOLÓGICA SOBRE LOXOSCELES RU FIE ES

(LUCAS), 1834, EN LA REGIÓN COSTERA DEL PERÚ

ÁLVARO DELGADO

Facultad de Medicina, Universidad San Marcos, Lima, Pení

Introducíon

El objeto principal de este trahajo es el análisis de las formas de vida de

la araria antropotóxica, Loxosceles rujipcs en la Província Andina Central, situada

en la parte Occidental de Sud-América. De esta gran area geográfica que ecoló¬

gica y fitogeográficamente comprende 3 subprovíncias — Costa, Cordillera y

Selva — para el presente estúdio elegimos la subprovíncia costera que se extiende

desde 30° S (Norte de Chile) hasta 8 o S (Norte dei Perú), pues es más satis-

factorio cubrir una area natural que una polílica desde que las fronleras de los

países son independentes de Ias harreras naturales que limitam la Flora y Fauna.

Senalamos también que todavia este estúdio es incompleto y que precisamos

realizar investigaciones más acuradas.

Material y método

La parte principal de este estúdio fué realizada durante investigaciones hio-

cenóticas en los principales valles de la costa peruana, donde comprohamos cl

ecoclima y microelima de este loxoscelineo, tanto en el campo vital rural cuanto

en el urbano. Colectamos material particularmente de los valles de Lima e Ica

para su estúdio en el laboratorio.

Habitat y distribución

Loxosceles rufipes se distribuye ampliamente a lodo lo largo de los valles de

la Costa. Vive preferentemente en el campo vital rural comportándose lapidícola

con máxima presencia de alta constância, pues aqui, de 7.963 araneidos captura¬

dos en los nichos ecológicos que deja el pedregal seco y sombreado 69% resul-

taron identificados como Loxosceles rufipes (Lucas), 1834. Con casi igual pre¬

valência la encontramos debajo de las grandes piedras, terrones de los campos

eriáceos, adobes de las minas precolomhinas, aprovecha también los espacios tu¬

bulares de los tallos de la caíía brava, Gynerium sagitatum de que están cons¬

truídas las casas rurales. Invade los espacios que dejan los basurales vegetales

de gramíneas, cannaceas y otras plantas industriales. No puede sobrevivir dentro

dei área húmeda de cultivo ni en los biotopos caldeados o mojados dei monte

ribereno, lomas, zona desértica y zona cultivada. En la zona marina vive cn las

oquedades que dejan las rocas y grandes piedras de las islas guaneras. A la

zona dei litoral Ilega por las playas arenosas con vida vegetal activa, a la desem-

SciELO

10 11 12 13 14 15

(LUCAS), 1834 EN LA REGIÓN COSTERA DEL PERO

bocadura <le los rios en el Océano y a las zonas <k‘ fauna cadavérica marina en

busca de la gran canlidad de insectos especialmenle larvas de coleópteros y dípte

ros. Menos frecuentemente, en la zona de los desiertos busca albergue en el

pedregal protegido por la vegetación xerofítica a donde también llega la entomo-

fauna que utiliza como alimento.

En el campo vital urbano, capturamos 3.646 araneidos y sólo 15% fucron

especímenes de Loxosceles rufipes. Sin embargo, es oicofílica sinantrópica, pues

al no ineomodarle la influencia humana invade los edifieios modernos. En las

habilaciones prefiere los lugares oscuros, abrigados, de poeo movimiento y aseo

que le garantizan sustraccíon a Ia vista dei hombre durante el dia. La búsqueda

realizada en el interior de 720 casas tomadas con la técnica dei muestreo casual

simple en 32 distritos de Lima demonstro que los nichos ecológicos preferenciales

son: las azoleas (57% ), los rincones oscuros de los sótanos (48%), de las

eocinas (42%), de las bibliotecas (12%). Los espaeios angulares coreanos al

lecho o al piso que dejan las molduras de los grandes muebles y cuadros col-

gados (7%). En los elosets, alacenas, cajones y hendidura de las paredes

(4 a 5%). En fin esta conducta las lleva también a ocultarse en los espaeios

interiores que dejan los vestidos colgados en las paredes o en los roperos (3%).

La autoconservación'

Alimento — A los biotopos referidos urbanos y rurales acuden tam¬

bién para refugiarse infinidad de ARTHROPÒDA y en los nichos ocupados

por este loxoscelineo algunos de entre i lios caen mortalmente por la acción pre-

datcra. La constitución dei aparato queliceral está admirablemente apropriado

para esta actividad eoadyuvante de las funciones de nutrición lo cual permite a

la araria aproveehar la entomofauna a11 í existente. Además su tela algodonosa

y viscosa sirve como trampa donde quedan selectivamente prendidos artrópodos

pequenos particularmente larvas y adultos de dípteros y coleérpleros (Phaleria

koepcki Pic.) y con cierta freeuencia pequenos lepidópteros y ortópteros.

Son voraces; parecen no saeiarse succionando las vísceras lisadas de su

víetima, pero también experimentalmente los especímenes adultos pueden soportar

ayuno hasta en media 16 semanas notándose entoces gran reducción dei volumen

abdominal.

Durante la luz crepuscular así como en la noche las hembras grávidas se

muestran más activas y voraces. Cualquier movimiento las estimula; en cambio

las hembras juveniles y los machos continuan al aceeho en su madriguera.

Examinando el contenido intestinal microscopicamente a fresco y con colora-

ción de Lugol y Grarn encontramos detritos indeterminables y una que otra vez

músculos estriados. Casi siempre pudimos detectar la presencia de PHOTO-

ZOA (rizopodos y flagelados), bactérias (cocos y bacilos), hongos (levaduras).

La locomoción dentro de su hábital o para ampliar su campo vital es enor¬

memente facilitada por la plasticidad de sus patas que se adaptan a los laberin-

tos dei pedregal y basural ocupando mínimo espacio. Al avisorar el peligro son

veloces, adquieren impresión vivaz. Se empinan para correr sobre superfícies

horizontales y parece que amplian la base de sustentación arrastrando casi la

superfície ventral corporal cuando trepan superfieies verlicales. Evitan caminar

contra la gravedad pues no puden utilizar la substancia serígena de sus hileras

como paracaídas.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

simp. Internac.

33(3):683-688, 1966

ÁLVARO DELGADO

685

Resistência: medio físico — Este loxoscelineo es muy sensible a la excesiva

humedad; el exoesqueleto parece fallar contra la acción lítica dei agua. El

abdômen es el más vulnerable pues se moja con gran facilidad debido a que

es pubescente y guarda mayor liempo el agua permitiendo el desarrollo de

hongos; por esto muere cuando se riega el biotopo que ocupa.

Huye penosarnente y a veces sin êxito de los pedregales que quedan enlo-

dados por el polvo y llovizna. Logra salir de los muy ealdeados por el sol

estival; es entoces que suelen correr de día procurando otro biotopo sombreado

cercano a la vegetación y es así que pasan de las azoteas bacia las habitaciones

dei hombre. Cuando huye se desplazan a 0,95 m/seg. en me<lia.

El biotopo sombreado puede calentarse por encima de 60°C en las horas

de fuerte insolación o enfriarse bruscamente a 4°C al declinar el sol. Sin em¬

bargo parece que su microhabitat amaina estas transiciones creando un micro-

clima favorable; además Loxosceles rujipes es euriterma con lo cual logra condi¬

ciones óptimas en el biotopo sombreado durante el verano. Aqui permanece in-

móvil aún con una humedad atmosférica relativa de 40 a 70% en el aire que

está en contacto con Ia superfície dei piso.

Experimentalmente soporta 6 horas en media a la sequedad de la estufa a

37°C. Vive en condiciones óptimas a 17 y 21 Ü C. Las temperaturas de 26 a

•H0"C la matan en 12 horas. Puede vivir sin recibir agua durante 12 semanas

lo que acontece cuando está en cautividad. El riego de sua biotopo es un factor

desfavorable aun durante el verano.

Resistência: medio biológico — Las hormigas y los véspidos (Polistes sp.)

son los principales enemigos específicos. Los animales de pastoreo rnueven el

pedregal y el hombre limpiando y quemando los abrojos alteran el biotopo.

Los Protozoários, hongos y bactérias encontrados en el aparato digestivo

evidentemente son parásitos, cuya araneidopatogenicidad e identificación taxonó-

mica ignoramos.

En la competência con otros araneidos ejerce acción dominadora ganando

el biotopo a ararias de su misma talla o menores, siendo por esto especie repre¬

sentativa dei basural vegetal y pedregal.

Algunas especies de FORMICIDAE suelen invadir momentaneamente en horas

dei día el biotopo de Loxosceles rufipes para apoderarse de las ootecas. En

breves instantes se apoderan de los huevos en presencia de la propia madre, que

no efectúa resistência alguna contra esta agresión.

Los indivíduos adultos tienen coloración simpática con el terreno polvoriento de

color marrón con todos sus matices. Los indivíduos jóvenes tienen de modo ge¬

neral coloración que vira dei amarillo claro citrino al castano oscuro ferruginoso.

Así, los estádios juveniles parecen protegidos por el color contra la predación de

véspidos ( Polistes, E um enes), sólo si permaneceu inmóviles, pues basta

que realicen la fuga para que invariablemente caigan víctimas dei hymenóptero.

Raras veces pueden escapar cuando se las coge por los apêndices, de los euales

se desprendeu por autotomía. Los apendices desprendidos continúan rápido movi-

miento, distrayendo de este modo al predator, que puede ser un pájaro de pequeno

tamano o una lagartija (Tropidurus peruvianus) .

En el mismo biotopo gana la competência alimentaria a miiltiples

ARACHNIDA; sin embargo la pierde cuando existen en abundancia nidos

de HYMENOPTERA y/o SCORPIONIDA ( Hadruroides lunatus, Brachistosternus

ehrembergi) , en sus vecindades, pues estos son enemigos ocasionales.

SciELO

10 11 12 13 14 15

686

INVESTIGACIÓN ECOLÓGICA SOBRE LOXOSCELEÜ RUFIPES

(LUCAS), 1834 EN LA REGIÓN COSTERA DEL PERO

Resistência: medio social — Los indivíduos que no han aleanzado madu¬

rez sexual, eu presencia de abundante alimento comparten el nicho ecológico;

así pudimos encontrar ocupando espacios contíguos intercomunicados y bajo una

misma piedra hasta 19 indivíduos próximos a la muda que les confirió adullez.

Los adultos son antisociales, sin embargo, pueden tolerar la presencia mo¬

mentânea de estádios juveniles de su misma especie tanto en su medio natural

como cuando están en cautividad.

Los individuos dei mismo estádio son siempre canibales; el indivíduo rnejor

conslituido mata a su rival mediante el aparato queliceral.

Cuando los individuos han logrado madurez, su coloración aumenta de tona-

lidad, se tornan absolutamente antisociales iniciando entonces una nueva búsqueda

de nichos ecológicos.Entran en competência con otros ARTHlíOPODA también

lapidícolas a los euales no solo les quita campo vital sino que los usa como ali¬

mento. El indivíduo adulto que ha logrado total desarrollo se encuentra rnejor

protegido por su corpulência y por la acción mortífera de su aparato quelice-

ral. Prohablemente por esta razón los pequenos pájaros que se alimentan de

artrópodos y la lagartija (T. peruvianus) que son enemigos ocasionales de Loxos-

celes rujipes, prefieren dar eaza a los estádios juveniles.

La conservación de la especie

Las hemhras a su cuarta muda, excluyendo la que se efeetúa intraooteeal-

mente, devienen aptas para las funciones de reproduceión, en tanto que los

machos se muestran sexualmente aptos a la tercera. Los especímenes adultos

con easi 51 semanas de edad muestran marcado dimorfismo sexual. El macho

es de cuerpo gracil, de patas más largas, palpos plegahles de morfologia caracte¬

rística especialmente en el bulbo y embolo adaptables a los receptáculos seminales

de la hembra que también exhiben morfologia de valor taxonómico específico.

La estructura morfológica de los palpos sirve para el transporte de los esperma-

tóforos e inoculación durante cl acoplamiento que generalmente es nocturno.

La hembra grávida tórnase voraz e irritable, llegando al máximo de agre-

sividad y canibalismo. No permite la presencia de sus semejantes de la misma

cronologia o de otros individuos en su biotopo, sin embargo muéstrase inerme

para impedir la visita de las hormigas que Ilegan para examinar las ootecas, que

las invaden si uo están bien construídas.

Du rante la oviposición, que sospechamos se deba efectuar a los 2-3 meses

dei apareamiento, la hembra se queda dentro dei saco ovígero, que es laxo y

que previamente fué tejido por ella. El saco ovígero que suele contener de 40

a 150 huevos embrionados es revestido, por una capa de tela algodonosa y

laxamente asegurado a los accidentes de su madriguera. La oviposicion puede

durar de una a dos semanas y por lo general construyen una sola ooteca, habiendo

casos que llega a fabricar hasta 3, otros casos en que la ooteca no tiene huevos

o estos no son viables.

La eclosión de los huevos depende de la temperatura, por esto es más fre-

cuente en la estación cálida y dentro de las habitaciones abrigadas. Durante

lodo el ano en los biotopos naturales referidos se puede encontrar todos los

estádios dei ciclo evolutivo en correlaeión positiva a la variabilidade climática

que se da en Ia Costa. Además dentro de las casas tienen microclima indepen-

diente de la estación dei afio dentro de un margen relativo.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3):683-688, 1966

ÁLVARO DELGADO

687

En su medio rural así corno en cautividad la hemlrra puérpera no pródiga

cuidados a las crias; sin embargo en ausência de alimenlo demuestra su instinto

maternal absteniéndose dei canibalismo con su prole. El macho tampoco tiene

participación alguna en el cuidado de los vástagos.

Las aranitas recién eclosionadas al cabo de 4 o 5 dias realizan su primcra

muda y abandonan la ooteca. Desde este momento empiezan a cumplir las fun¬

ciones biológicas básicas de la autoconservaeión.

En su medio natural se dispersan en pos de alimento en los espacios que

dejan el pedregal, basural y espacios protegidos de las habitaciones donde en-

euentran larvas de coleópleros y dípteros que utilizan como alimenlo. En cautividad

reciben Drosophila sp., ninfas de OKTHOPTERA (Blata y I 1 eriplaneta),

larvas de MUSC1DAE ( M u seu. De s t r u s , S t o mo x is) y larvas de

COEEOPTEIÍA (Phaleria sp.) a las cuales atacan para luego succionar las xis-

ceras licuadas por los jugos digestivos que inyectan.

Hiocenosis

Las formas de vida que hemos senalado sou adaptaciones de este loxosee-

lineo a los espacios protegidos y oscuros que le ofrece el campo de vida rural y

urbano y a las comunidades vitales lapidícolas.

En fin, el comportamiento y complejos estructurales de esta arana antropo-

tóxica, la senalan como muy apropiada para vivir en las viviendas dei hombre

de la misma maneia que en los biotopos dei pedregal y basural vegetal secos

próximos a terrenos con vegelación dei medio ambiente biótico rural.

SciELO

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33(31:689-698, 1966

R. GAJARDO-TOBAR

689

5. Ml EXPERIENCIA SOBRE LOXOSCELISMO

R. GAJARDO-TOBAR

Chile

EI objetivo de esle trabajo es analizar el cuadro clínico producido por la

pioadura de la araria de los rincones, Loxosceles lacta *. a través de 200 casos

de nuestra casuística, tomados al azar. ocurridos en Valparaíso y pueblos vecinos

(Chile), entre 1956 y 1966.

Los accidentes causados por la introducción de ponzonas de ararias en el

liombre, siguiendo el camino natural de picadura a través de la piei, están con¬

dicionados por el tipo dei veneno, la época dei ano. el estado de la araria, la

cantidad de ponzona disponible, el lugar por donde es introducido, el camino

seguido por el tóxico y la resistência dei paciente (5).

El accidente ocurre cuando las ararias son martirizadas inadvertidamente.

De ordinário no acometeu, sólo se defienden. En nuestra casuística la picadura

se produjo cuando el afectado se vestia o mientras estaba en cama durmiendo.

La época dei ano en que sucede el accidente tiene importância porque du¬

rante el verano las ararias están más vivaces y el calor, alcalinizando las pon¬

zonas, las liace más activas (6). De nuestros 200 casos, 98 acaecieron durante

el verano (49%). En cambio, en invierno se registraron solamente 31 casos

(15.5%). En primavera hubo 42 casos (21%) y en otono 29 (14.5%).

El lugar de la piei por donde es introducido el tóxico es también impor¬

tante. Las zonas de piei fina o las regiones muy irrigadas son las que deter-

minan los cuadros clínicos más sérios.

En cuanto a resistência humana, es faetor que dehe considerarse. Los niiios

son los que hacen más graves casos de aracnidismo.

El veneno dei Loxosceles laeta es dermotóxico y en algunos casos dermo-

viscerotóxico.

La picadura de la araria de los rincones es capaz de engendrar, enlonces,

dos cuadros clínicos diferentes: — Lhro localizado, benigno o de mediana gra-

vedad, llamado loxoscelismo cutâneo o “mancha gangrenosa” y, otro generalizado,

muy grave y mortal con frecuencia, el loxoscelismo cutaneovisceral. — En el

primero, el veneno actúa sólo localmente. En el segundo, difundido, obra sobre

todo el organismo y afecta en especial la sangre, el hígado y los rinones.

Cualquiera está expuesto a ser picado por esta araria que es común en las

viviendas, aún en las muy aseadas y cuidadas. En nuestra casuística hubo 97

casos de mujeres (48.5%) y 103 de hombres (51.5%).

Continuamos usando el término “laeta” y no “rnfipes” hasta que los especialistas en

sistemática se pongan de acuerdo acerca de cual debe perdurar (1, 2, 3, 4).

SciELO

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690

MI EXPERIENCIA SOBRE LOXOSCELISMO

La zona afoitada dei cuerpo pucde ser cnalquiera. Con todo, el mayor

número de casos se refiere a las extremidades, el cuello y la cara. De nuestros

200 casos, 139 fueron de las extremidades (09.5%), con predominio en los ante-

lirazos, 20 casos (14%) y en los muslos, 21 casos (10.5%), en el cuello y

nuca 18 (9%) y en la cara 21 (10.5%). En cl tórax tuvimos 15 casos (7.5%),

en el abdômen 4 (2%) y en los genitales 3 (1.5%).

En matéria de edad, el mayor número de afeitados lenia entre 16 y 50

anos, cs decir 135 casos, o sea, el 67.5%. El menor tenía un mes y el mayor

sobre 70 anos.

Producido el empozonamiento, en las primeras veinlicuatro horas no sc pucde

predecir lo qnc va a sobrevenir. El comienzo cs igual en cnalquiera de las dos

formas de este aracnidismo.

Se inicia con el dolor de un pinchazo. Sin embargo, en algunas oportuni¬

dades, los afeitados no recuerdan el incidente inicial. De nuestros 200 pacientes,

158 sintieron cl pinchazo (79%). La pieadura sirve para que el afeclado

busque la causa y, muchas veces, encuentre la araria y la dcstruya. De nuestros

enfermos no la vieron en 48 casos (24%). la vieron 149 (74.5%), la destruyeron

77 (38.5%), se pudo identificar en 36 casos (18%). En todos ellos era Loxos-

celes laetu.

Al lancetazo sigue un |)eríodo asintomátieo o de latcncia, de ordinário corto,

de 5 a 10 minutos, poro que a veces puede llegar basta 8, 10 y 24 horas,

Estalla, entonces, un dolor local creciente, con scnsación de quemadura, y

unas pocas veces con prurito, dolor que puede adquirir caracteres impresionantes

y ciiva pertinácia e intensidad, en cualquier caso, mortifica mucho. En nues¬

tros casos, el dolor local se hizo presente en 193 (96.5%). acompanado de scn¬

sación de quemadura en 180 (90%), con prurito en sólo 22 casos (11%).

La piei, en el sitio afectado, enrojece por congestión de los capilares, se

edematiza y se pone tensa y dura.

En este momento cl cuadro clínico se individualiza en una de las dos for¬

mas antes senaladas. Va al proceso cutâneo o se generaliza y se hace visceral.

Por lo común esto sucede dentro de las primeras veinlicuatro horas.

LOXOSCELISMO CUTÂNEO O MANCHA GANGRENOSA

En la forma cutânea no hay o es limitado el eompromiso dei estado general,

en cambio se produce una lesión local de variables proporciones, muchas veces de

alarmante extensión y de difícil reparación.

El dolor local va aumentando y, se hien es cierto en algunos casos es tole-

rable, en otros puede llegar a adquirir tal violência y pertinácia que más de

algún paciente ha deseado que le amputasen la extremidad afeitada para lihrarse

de él o en su desesperación ha pensado en suieidarse. El dolor es permanente,

con exacerbaciones periódicas. Limita los movimientos, crea impotência funcional

transitória, causa insomnio y desasosiego. Tiene la ya citada característica tan

uesagradahle y típica de la scnsación de quemadura desesperante.

Desde las primeras horas aparece, en el sitio de la pieadura, una mancha

de tamano variable, de color rojo vinoso, en cuyo centro es, a veces, posible dis¬

tinguir los puntos por donde penelraron los qnelíceros de la araria. Alrededor de

las veinticuatro horas el centro de Ia lesión se pone violáceo y después negro.

En su contorno se disenan zonas pálidas, blancas y otras moradas, como vetas

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R. GAJARDO-TOBAR

691

irradiadas irregularmente, todo lo cual recuerda el color dei mármol jaspeado,

de allí el nombre que le damos de “placa marmórea”. Depende de una isque-

mia local y de hemorragia con induración. Como im halo, en torno de ella, la

piei no directamente afectada, desde un comienzo roja, acentua su color, con

grau vasodilatación.

Típica placa marmórea vimos en 173 de nuestros casos (86.5%), algunas

pequenas y otras de tamano impresionante.

Surgen sobre esta placa y, a veces antes que ella se forme o sin que se

forme, en tiempo variable de pocas horas, flictenas, a veces gigantescas, de con-

tenido seroso o sero-hemático, aséptieas por cierto. En algunas circunstancias

la vesícula es única y en oportunidades constituye la exclusiva manifestación de

la picadura de la araria. Tuvimos flictenas en 80 casos (40%).

El edema que se insinua desde el principio, se va extendiendo a vastas

superfícies dei cuerpo, sobre todo en las zonas declives. Es consistente, regular.

Bajo la placa marmórea se hace más duro, sitio donde se produce gran infiltra-

ción. En 162 de nuestros casos fue notorio (81%).

En la placa marmórea y sobre todo en la parte negra de ella desapareceu

la sensibilidad al dolor y térmica. Mientras tanto que en la región roja peri¬

férica la sensibilidade es exquisita.

El tamano de la placa marmórea es variable. Nosotros tuvimos casos en que

media desde uno hasta treinla y más centímetros de diâmetro.

Entre el 5.° y el 6." día después de la picadura, han disminuído el edema

y el rubor, mientras que la placa marmórea se pone negra. Se deslinda una

escara apergaminada y seca, dura y algo brillante. cuyo tamano guarda relación

con las dimensiones de la placa, escara que delimitándose bien. se va soltando por

los bordes hasta caer a la tercera semana (5, 7, 8).

El aspecto de gangrena seca que presenta la lesión y su color negro le han

significado de denominación, desde hace más de un siglo, de “mancha gangre-

nosa” (9, 10, 11). En nuestra casuística hubo necrosis de importância en 78

casos (39%).

Cuando se desprende la escara queda una úlcera de superfície rosada, ma-

melonada, irregular, anfractuosa, resumando un líquido amarillento y sangrando

con mucha facilidad.

En los casos en que la piei está directamente aplicada sobre las aponeurosis

musculares, éstas se ven en el fondo de la úlcera.

Es interesante senalar que con el deslinde de la escara y la disminución dei

edema, también menguan los dolores y por fin cesan dei lodo. El desprendi-

miento de la escara es indoloro, lo que acontece, como lo hemos dicho. haeia la

tercera semana (7, 8, 12).

Es menester hacer hincapié en que lo que en el 96,5% de nuestros casos deses-

peraba al paciente era el dolor local, la sensación de quemadura y el insomnio

que, naluralmente, resultaba su consecuencia.

La adenopatía que se registra, en oportunidades, se debe a infecciones pio-

genas sobreagregadas. La presentaron 27 de nuestros casos (13.5%).

La reparación de los tejidos es muy lenta. En lesiones extensas es menester

bacer injertos cutâneos. El tiempo que ha demorado la curación total ha sido,

en general de 20 a 30 dias, pero tenemos casos de 5 a 8 dias y oiros hasta de

116 dias.

SciELO

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692

MI EXPERIENCIA SOBRE LOXOICELISMO

El proeeso deja una cicatriz irregular, parda o azuleja. a veces queloídea

y retráctil, excepcionalmente dolorosa.

Loxoscelismo

El edema.

EI curso de esta forma de loxoscelismo es favorable siempre. Los problemas

surgen de las incapacidades para el trabajo, por suerte de tipo transitório o en

casos rnuy raros de carácter permanente, derivadas de la larga evolución hasta

la mejoría, de la ubicación de la lesión. de la cicatriz y de la retracción de los

tejidos que puedan limitar los movimientos de las extremidades o dei cuello, o

bien de la secuela de cicatrices dcforrnantes de la cara.

En el loxoscelismo cutâneo hay nulo, escaso o mediano compromiso dei estado

general dei paciente, completamente independiente dei carácter, tamano e impor¬

tância de la lesión local.

Consideramos en el loxoscelismo, clínicamente baldando, cuatro grupos de

pacientes: l.° — Sólo con lesiones locales, sin ningún compromiso dei estado

SciELO

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R. GAJARDOTOBAR

693

La mancha gangrenosa.

Flictcnas.

La úlcera.

Con mediano compro-

general. 2.° — Aquellos con ligero compromiso. 3.°

miso. 4.° — El grave proceso cutáneo-visceral.

Los tres primeros grupos correspondeu ai loxoscelismo cutâneo. EI cuarto

fornia la entidade cutáneo-visceral.

Del primero, con sólo lesiones locales, sin ningún compromiso dei estado

general, de los 200 casos, tuvimos 108 (54%).

SciELO

694

MI EXPERIENCIA SOBRE LOXOSCELISMO

LIamamos con ligero compromiso a los que además de las lesiones localcs

presentaron cefaleas y fiebre. Contamos con 69 casos (19.5%).

Contemplamos como de mediano compromiso a aquellos que presentaron,

fuera de las lesiones locales, fiebre, cefaleas, vertigos, nauseas, vómitos, diarrea,

sudores y crisis nerviosas. Tuvimos 31 casos, en este grupo (15.5%). Excepcio-

nalmente hay prurito o erupción cutânea morbiliforme. Ello sucedió en 19

casos (9.5%).

En cualesquiera de los citados casos el pulso se acelera en relación con la

temperatura; las presiones se mantienen; la uremia y la glicemia no se modi-

íican; las pruebas de función hepática, los tiempos de sangria y de coagulación

y la resistência globular son normales. Igualmente el hemograma, sedimentación,

orina y electrocardiograma no cambian.

Finalmente agrupamos en el último conjunto los casos graves, es decir la

entidad clínica que lamamos loxoscelismo cutáneo-visceral, de impresionante vio¬

lência, con una lesión local mínima y iin compromiso dei estado general fulmi¬

nante con anemia hemolítica, necrosis hepática y nefrosis hemoglobinúrica. De

estos tuvimos 22 casos, es decir el 11% dei total, com 7 muerles (3.5% de los

200 casos y 31.81% dei loxoscelismo cutáneo-visceral).

LOXOSCELISMO CUTÁNEO-VISCERAL

Clínica — En la forma clínica cutáneo-visceral dei loxoscelismo la gravedad

cs mucha y el pronóstico sombrio.

La lesión local, las más de las vezes, pasa a segundo término, porque la

violência y la rapidez dei proceso casi no le dan tiempo para formarse y, en

aquellos pacientes que mueren rápidamente, apenas hay una mancha rojo-cianó-

tica, edema y un esbozo de placa marmórea. El dolor quemante, en cambio, no

falta nunca.

Contrastando con esta condición el compromiso dei estado general es im¬

presionante. Hay intenso malestar general, desasosiego, cefaleas y fiebre. Del

grupo de 22 casos cutáneo-viscerales que tuvimos dentro de los 200 de loxos¬

celismo, los 22 presentaron malestar general, 15 desasosiego, 16 cefaleas y 20

fiebre. En muchos de ellos sobrevinieron vértigos, taquicardia, hipotensión y en

un fuerte número, 13 de los citados, hubo náuseas, vómitos con frecuencia con

sangre. Alrededor de las 12 a 24 horas surgen los sintomas más alarmantes:

Equímosis, icterícia y hematúria y hemoglohinuria. En nueslros 22 casos 5

hicieron equímosis en diferentes partes dei cuerpo; 15 icterícia y 19 hematúria.

Horas más tarde aumentan las cefaleas, vienen calofríos y decaimiento, desapa¬

rece el sueno y mientras la taquicardia acrece la hipertermia alcanza a 40° centí¬

grado, las presiones descienden y la piei se pone terrosa.

Como se vaya agravando el cuadro clínico irrumpen acentuadísimas dispnea

y cianosis y se marcan más la icterícia y las hemorragias imponiéndose la ane¬

mia. En muchos pacientes aparecen signos de congestión y edema pulmonar.

For fin la respiración se hace estertorosa; tos y desgarro muco-espumoso y

sanguinolento ahogan al enfermo; el pulso se pone filiforme, las presiones des¬

cienden más aún, se suprime la diuresis, el desasosiego se extrema y el enfermo

entra en coma basta que muere.

Pruebas de Laboratorio — Las pruebas de laboratorio nos ofrecen una

serie de hechos muy importantes.

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R. GAJARDO-TOBAR

695

En Ia sangre ensenan una anemia de lipo hemolítico. Hay formidable des-

trucción de los hematies. En uno de nuestros casos se contaron sólo 840 000

por milímetro cúbico. Lc aeompanan trombocitopenia, y reducción dei hemato-

crito (en uno de nuestros pacientes no alcanzaba a 7%). En la serie blanca

irrumpe una fuerte leucoeitosis, verdadera reacción leucemoíde con 30, 40 y más

de 50 000 leucócitos por milímetro cúbico, con desviación a la izquierda de Ia

fórmula, en algunos casos com 31 segmentados y 47 baciliformes. La sedimen-

tación, que en el loxoscelismo cutâneo es normal, sube en la forma visceral a

más de 50 mm cn la primera bora. La uremia sube también. En uno de

nuestros casos llegó a 5 gramos por mil. La glicemia al principio alza transi¬

toriamente y luego queda en hipoglicemia. Hay hiperbilirrubinemia y hemo-

globinemia.

En la orina, la densidade baja (1009 y 1010), aparecen albuminúria, hema¬

túria, hemoglobinuria y cilindruria.

Los casos más graves terminan en la muerte, entre las 30 y las 48 horas

después de la picadura de la arana. En nuestra casuística, de los 22 casos de

loxoscelismo cutáneo-visceral, luvimos 7 mortales, es decir, 31.81% de los 22 y

3.5% de los 200.

Anatomía 1’ATOLÓgica — Los hallazgos coinciden con lo que otros médicos

y nosotros mismos liemos observado en los experimentos de laboratorio, con

animales sometidos a la acción de la ponzona de esta arana (5, 7, 8, 12, 13, 14).

Localmente se produce edema sero-sanguinolento; los tegumentos se infiltran;

el proceso puede exlenderse muchísimo por vecindad, comprometiendo hasta los

músculos regionales. En el dermis bay fuerte dilatación capilar con perivascula-

rilis. Las lesiones típicas de la placa necrótica rara vez se ven en las autopsias

porque la muerte sobreviene antes de que puedan formarse. El resto de la piei

aparece pálida y con icterícia.

En la sangre, la ponzona determina hemolisis intravascular, con las corres-

pondientes hemoglobinemia y hemoglobinuria, con lesión renal ulterior, la ne-

frosis tóxica aguda o hemoglobinúrica. Con la liberación excesiva de hemo¬

globina aumenta la concentración sérica de bilirrubina y aparece Ia icterícia.

Como consecuencia se produce una hemosiderosis generalizada. El revestimiento

endotelial de los vasos sanguíneos se va tinendo de hemoglobina. La excreción

de los pigmentos de ella termina por produzir la ya citada nefrosis hemoglobi¬

núrica.

En los casos más graves sobrevienen câmbios grasos en el corazón, rinón

e hígado. Pero, no debemos olvidar que los agentes tóxicos no sólo actúan

sobre la sangre sino que también causan simultáneamente lesiones graves en otros

órganos que a veces llegan a predominar.

En muchos casos bay trombocitopenia. Enlonces el liempo de coagulación

resta normal, pero se alarga el tiempo de sangria. En algunos enfermos hemos

tenido lendencia a las hemorragias de las mucosas y en la piei, con grandes

equímosis. Esto refleja una acción dei tóxico sobre la médula ósea.

Siendo el hígado uno de los principales centros de desintoxicación que

posee el organismo, los venenos actúan sobre él. En los casos benignos, sólo bay

tumefacción turhia de las células hepáticas. En lesiones más graves hay infil-

tración grasa y en las peores circunstancias destrucción o necrosis en focos o

SciELO

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696

MI EXPERIENCIA SOBRE LOXOSCELISMO

difusa, además de hemosiderosis. En nuestros easos falales hahía focos de ne-

crosis, moderada disociación travecular. difusa infiltracióu grasa v hemosiderosis.

(Las células de Kupfer tumefactas y cl citoplasma hemogloliínico, además de nó¬

dulos dc hierro cu los espacios intertravecularcs I.

Los rinones cxperimentan con violência la acción dc la ponzona. La parte

más vulncrahle dc ellos sou los luhos contorncados, cu su parte proximal. que

sufren tumefacción, metamorfosis grasa y aún nccrosis. segím la gravedad dei

caso. Los glomérulos y tubos eolectores resisten más.

Las alteraciones rcnales comienzan antes de las 21 horas. En la orina

rápidamente aparecen cilindros granulosos y pigmentados con hemoglobina. Los

luhos se llenan dc cilindros y sobreviene necrosis y degeneración de los e])itelios.

El cuadro, en lodos nuestros casos de loxoscelismo cutaneo-visceral fuc de nefrosis

hcmcglohinúrica. En ellos hahía edema, hemorragias en los rinones, túbulos

llenos de cilindros hemáticos y paredes tumefactas. Tanto la pared de los túbulos

como la de la cápsula de Bowman aparecían cargadas dc hemoglobina y la cápsula

llena de glóbulos rojos y hemoglobina. En la vejiga encontramos orina con

critrocitos y hemoglobina y sus paredes embebidas de hemoglobina.

En la medula ósea bailamos predomínio de elementos primitivos, mielocitos,

granulocitos macrófagos, eosinófilos y escasos plasmocitos.

En los pulmones encontramos acentuado edema, hiperemia y hemorragias.

Igualmente se compromete la pleura y termina ésto en hemotórax. Las arteriolas

están llenas de sangre hemolizada.

En el corazón, en algunos casos encontramos degeneración grasosa y lume-

facción turbia dc las fibras musculares, además de hcmopericardio.

En cl cerebro, la reacción a la ponzona se parece a lo que ocurre con algu¬

nos otros tóxicos, encontramos edema, hiperemia, perivascularitis y piqueteado

hemorrágico.

En cl resto de los órganos hahía hiperemia, edema, foquitos

(suprarenales y bazo) o bien edema y erosiones (tubo digestivo).

morraaicos

Diagnóstico — Clínicamente es posible hacer el diagnóstico. Ni la placa

marmórea, ni la escara pueden eonfundirse. Con relativa frecuencia el paciente

no ha visto la arana y no se sabe cómo se ha iniciado su enfermedad, pero el

dolor quemante, la placa marmórea, las flictenas, la mancha gangrenosa, sin

compromiso o muy eseaso dei estado general, la mayor [tarte de las veces sin

gânglios afectados, llevan al diagnóstico de loxoscelismo cutâneo.

En la forma cutáneo-visceral, la lesión local es poco ostensible, pero existe

y. acompafiándose de fulminante agravamiento dei estado general, con fiebre,

ictericia, hematúria y hemoglobinuria no es un problema llegar al diagnóstico.

Naturalmente, en Ia forma cutânea hay que distinguir de carbunclo, forún-

culos y picaduras de insectos. En la forma visceral, dc aquellas enfermedades

con ictericia v hemólisis.

PronÓSTICO — Antes tle las primeras 24 horas no es posible predecir la

evolución. Pasadas éstas, en el caso dei loxoscelismo cutâneo no hay peligro

para la vida pero, la curación puede demorar meses y dejar cicalrices dolorosas

y aún impotência funcional de los miembros si la lesión se produjo en las extre¬

midades. Tenemos un caso en que a pesar de los injertos de piei que se lc

hicieron, por la extensión de la pérdida de piei que sufrió en el muslò izquierdo,

demeró 116 dias en curar.

SciELO

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Mem. Inst. Butantan

Slmp. Internac.

33 ( 3 ) : 689 - 698 , 1966

R. GAJARDO-TOBAR

697

La fiebre, cl grave compromiso dei estado general, la hematúria, Ia hemoglo-

binuria y la icterícia precoces marcan la gravidad dei caso. Si aparecen antes

de las 24 horas signifiean extrema gravedad. Si

ninguno de estos sintomas y

signos se presentan en las primeras 18 horas sólo habrá loxoscelismo cutâneo.

En el loxoscelismo visceral que va a la curación, las manifestaciones pato¬

lógicas comiezan a atenuarse entre las 70 y las 80 horas. Ya dijimos que en

nuestros 22 casos de loxoscelismo eutaneo-visceral luvimos 7 muertos, es deeir

81.81% de ellos.

Thatamiento — Tiene grau importância que sea precoz. El ideal es usar

suero específico. Desgraciadamente, entre iiosotros se eonsigue dificilmente.

Guando se dispone de él debe emplearse en dosis suficiente y no atenerse a que

en las instrucciones se dice que una ampolleta, por via intramuscular es lo indi¬

cado, sino que poner dos, Ires y muchas más si el caso lo exige. En el excelente

suero que se prepara en el Instituto Butantan 5 ml. neutralizan 50 dosis míni¬

mas necrosanles para la oreja de un conejo(15).

En su defeelo, se ban empleado desensibilizantes, antialérgicos, corticoides,

analgésicos y tratamienlo sintomático.

En los emponzonamientos produeidos por picaduras de aranas es preferible

no emplear la via digestiva porque el tubo digestivo se afecla y la absorción

es mala.

No habiendo suero específico, y aún con él, lo más útil ha sido el empleo

de corticosteroides, cuanto más luego mejor, eu dosis altas, por via parenteral,

cortisona 800 milígramos al dia los 4 primeros dias y después 100 milígramos

diários hasta cuando sea uecesario. El depomedrol (methyl prednisolone) 80 mg.

intramusculares prontamente repitiendo cada 12, 24 o 48 horas según necesidad,

disminuyendo despúes las dosis cada 24 horas (16, 17).

Hemos usado tamhién toda la gama de antialérgicos y desensibilizantes, siem-

pre de acción muy inferior a los corticoides.

La ligadura, la ineisión, la cauterización, Ias inyecciones perifocales de per-

manganato de potasio, el amoníaco, etc., son ineficaces.

Como es natural, hay que usar también tratamienlo sintomático, mas que

nada para combatir el dolor, la sensación de quemadura, el insomnio y la an¬

gustia, así como estimulantes cardíacos y generales, sueros glucosados y transfu-

siones cuando la situación lo requiera.

El tralamiento local deberá ser cuidadoso y vigilante. Si la úlcera es muy

grande y la cicatrización tórpida habrá que recurrir a injerlos de piei.

SUMMARY

Loxoscelism is clinically studied through 200 cases in Chile. The venom of

Loxosceles laeta acts on the skin, determining a cutaneous form or “Grangenous

spot”, or on the skin and vísceras, causing a very serious poisoning with a high

mortality. Clinicai differences between the two forms are described. In the visceral

form, death occurs in 31.81% of these cases, 30 to 48 hours after the spider’s bite.

There is toxic hemolytic anemia, hepatic focal necrosis and hemoglobinuric nephrosis,

with a series of tissue and general organic Iesions. The differential diagnosis,

prognosis and the results of treatment with specific sera and other medicaments

are analysed.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 31 : 699 - 700 , 1966

J. VELLARD

699

6. EL ARANEÍSMO EN BOLÍVIA

J. VELLARD

Bolívia

Existen niuy pocos clatos concretos sobre el araneísmo en Bolivia.

La más antigua es dei ano 1886 (E. Simon, Ann. Soc. Ent. Belgique).

Un químico establecido en Cochahamba, el Dr. Sacc, envió a E. Simon

diversas ararias designadas como mico-mico, sindicadas de producir accidenles

graves: la picadora es igual al dolor producido por un hierro al rojo, seguido

de violenta inflamación; la sangre pasa en las orinas y la muerle puede sobre-

venir en algunas horas. Las especies recibidas por E. Simon fueron determina¬

rias como Dendryph antes y D. sacei, de la familia de las SALTICIDAE,

pero la atribución de accidentes a estas ararias carecia totalmente de bases. El

mico-mico es en realidad el Latrodectus mactans.

Cuatro regiones biogeográficas principales deben ser consideradas para

Bolivia:

En las altas regiones andinas, arriba de los 3.000 m. no existen ararias

peligrosas. EI veneno de las mayores especies dei Altiplano Lycosa rujimanoides

y L. guaquiensis es muy poco activo para el hombre.

En los valles secos, al oriente de los Andes, entre 1.500 y 2.800 m, prin¬

cipalmente, se observan dos formas de araneísmo: un araneísmo dei campo pro¬

ducido por Latrodectus mactans, el mico colorado; y un araneísmo domiciliário

cuyo responsable es un Loxosceles que no he podido determinar por haber

recibido únicamente bendirás.

No existe ningún dato estadístico al respecto, pero los médicos de Cocha-

bamba y de Sucre conocen bien estos accidentes, que a veces son mortales.

La sintomatologia no difiere de la observada en otros paises.

Solo notaré después de la picadura de L. mactans, un pequeno edema local

duro alrededor dei punto de inoculación dei veneno. Los otros sintomas son clá-

sicos: dolor irradiante violento, cefáleas, raquialgia, dolores abdominales, con¬

tracturas musculares, espasmo de la musculatura lisa. El restablecimiento es la

regia, pero se ba observado algunos casos mortales a 2 o 3 dias, por anuria.

Durante muchos aiíos todos los casos de araneísmo de estas regiones fueron

atribuídos a L. mactans.

Con la colaboración de diversos clínicos de Cochabamba, entre ellos el

Dr. Hermógenes Sejas, lie podido identificar a otra especie, la araria negra, un

Loxosceles sp, que produce los accidentes más graves, con frecuencia mortales,

con el cuadro habitual de loxoscelismo, edema, flictenas, necrosis local, ictericia,

hematúria.

SciELO

10 11 12 13 14 15

700

EL ARANE1SMO EN BOLÍVIA

En Ia región dei Chaco Ias THERAPHOSIDAE conocidas localmente por su

nomhre guarani de nandú-guassú son muy temidas, así como la anarimba, Pho-

ncutria rufibarbis, pero no se han publicado relaciones clínicas de accidentes.

En la región tropical de tipo amazônico, dei Oriente Boliviano faltan también

datos positivos. EI pueblo teme diversas especies de araria; las THERAPHOSIDAE,

los grandes Ctenídeos son frecuentes. Es muy probable que los Phormicto-

pas, y Acanthoscurria sean responsables de accidentes. Es un campo

de investigaciones todavia abierto.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 701 - 710 , 1966

C. DA CRUZ LANDIM e E. W. KITAJIMA

701

7. ULT R AESTR UTUR A DO APARELHO VENENÍFERO DE A PIS

(. HYMENOPTERA , APIDAE)

C. DA CRUZ LANDIM e E. W. KITAJIMA

Faculdade de Filosofia, Ciências e Letras, Rio Claro e Instituto Agronômico,

Campinas, São Paulo, Brasil

Introdução

Os acessórios do ferrão são conhecidos nas abelhas com detalhes, desde o

trabalho de Hordas (1), que fêz um estudo comparativo dêste órgão em várias

espécies de himenópteros. Em A p i s , como nas demais abelhas providas de

ferrão, existem duas glândulas associadas à base dêsse órgão. A principal delas

consiste de túbulo longo, fino e convoluto, localizado na parte posterior do

abdômen. O túbulo termina apicalmente por uma bifurcação sob a forma de

duas pequenas dilatações secretoras. Posteriormente, o túbulo se abre num re¬

servatório, o saco de veneno (2). Esta glândula denomina-se ácida devido ao

pH de sua secreção, e é aquela que produz o veneno.

A segunda glândula não se acha ligada ao ferrão, de maneira que a sua

secreção possa ser injetada durante a picada, tendo sido denominada glândula

básica.

Kerr e Lello (3) sugeriram a possibilidade da glândula ácida em Bom bus

ser secretora em tôda a sua extensão, estando porém a atividade mais intensa

restrita à parte apical bifurcada. Sugeriram também que o saco de veneno

poderia exercer função secretora. Por outro lado, Autrum e Kneitz (4), verifi¬

caram em A pis , que a secreção do veneno iniciava-se no final da pupação,

pouco antes da emergência, atingindo seu máximo entre 10 a 16 dias de vida da

abelha adulta.

No presente trabalho, pretende-se descrever a morfologia e ultraestrutura da

parte apical bifurcada (glândula propriamente dita), e mediana da glândula

ácida e do saco de veneno de pupas prestes a emergir e de abelhas adultas,

campeiras.

Material e métodos

a. Abelhas utilizadas — 1 . Pupas prestes a emergir, de operárias de abe¬

lhas híbridas ( Apis mellifera ligustica e A. mellifera adansonii) ; 2. Operárias

campeiras (aproximadamente 30 dias de idade) dos mesmos híbridos.

Pesquisa realizada sob os auspícios da Fundação de Amparo à Pesquisa do Estado de

São Paulo, Conselho Nacional de Pesquisas e Organização dos Estados Americanos.

SciELO

10 11 12 13 14 15

702

ULTRAESTRUTURA DO APARELHO VENENIFERO DE A PIS

( HYMENOPTERA, APIDAE)

1). Técnicas histológicas para microscopia eletrônica — As abelhas foram

dissecadas em solução lisiológica (NaCl a 0,7 ' c /c) lamponada para pH 7,1 com

tampão fosfato, tendo sido estas abelhas prèviamente anestesiadas com resfria¬

mento a 0“C durante 15 minutos. As glândulas e o saco de veneno, após sua

extração, foram fixados em solução de tetróxido de ósmio(5) ou numa combi¬

nação de tetróxido de ósmio e permanganato de potássio (6). A desidratação

foi efetuada em acetona e o material fixado, foi incluído em Epon 812(7).

Após serem seccionadas em ultramicrótomo Porter Blum, MT-2, as secções ultra-

finas foram contrastadas em acetato de uranila, durante 1 hora, e a seguir em

hidróxido de chumbo (8), e examinadas em microscópio eletrônico Elmiskop I

da Siemens.

Resultados

A. Glândula ácida — A fim de facilitar a exposição dos resulta¬

dos, esta glândula foi dividida em duas porções arbitrárias, respectivamente, a

parte apical, bifurcada e a porção mediana. Não se estudou a porção basal, em

contacto com o saco de veneno, pois esta não difere hislològicamente da região

mediana.

1. Pupas — Nas pupas, a luz da glândula ácida, na extremidade apical

dêste órgão, tem secção de forma estrelada e sua parede é formada de uma

espessa cutícula (ca. 5 p .), de material provavelmente não esclerolizado, disposto

em camadas concêntricas.Em tôrno desta cutícula achavam-se dispostas as células

secretoras. Estas apresentavam-se com grandes vacúolos, dando à parte secretora

um aspecto rendilhado. Não se observou uma membrana conspícua, limitando

lais vacúolos (Fig. la). A semelhança do que ocorre em células vegetais, devido

à presença dos enormes vacúolos, os núcleos destas células encontravam-se em

geral comprimidos em pequenas faixas do citoplasma.

A porção citoplasmática destas células era rica em ribossomas, embora pobre

em elementos do retículo endoplasmático (Fig. 1). Nas junções celulares, as mem¬

branas justapostas se apresentavam sinuosas nas secções, e em alguns pontos

formavam alças. O espaço entre estas membranas justapostas achava-se preen¬

chido por um substância densa, e em certas regiões elas se espessavam, o que

talvez pudesse representar desmossomas, muito embora não tivessem um aspecto

característico destas estruturas celulares. Nesta fase, os mitocôndrios ocorriam

em pequeno número, uniformemente distribuídos nas células e se caracterizavam

por abundantes cristas. Zonas de Golgi foram raramente observadas e em ne¬

nhum caso pôde-se identificar as vesículas de secreção, usualmente a elas asso¬

ciadas.

Dois tipos de núcleos foram constatados nestas células. Um grande, apro¬

ximadamente esférico, que representaria o núcleo da célula secretora de localiza¬

ção mais periférica e outro, menor, localizado próximo aos canalículos intracelu¬

lares ou à cutícula que reveste a luz da glândula. Nos núcleos maiores, nota¬

vam-se vários nucléolos bem desenvolvidos e um retículo de material cromáti¬

co (Fig. 1). Disperso no nucleoplasma, apresentava-se grande número de grâ¬

nulos densos, semelhantes aos ribossomas. Os núcleos pequenos não apresentavam

nenhuma característica marcante. Aparentemente, os núcleos pequenos e os maio¬

res estavam contidos nas mesmas células, isto é, nesta fase não se percebiam

membranas divisórias compartimentando o citoplasma pertencente a cada tipo de

núcleo. As células desta porção, como as da parte mediana e as do saco de

veneno, apresentavam uma série de canalículos intracelulares. Tais canalículos

1, | SciELO

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Flg. la - Micrografias <la porção apical da f.‘ 1 ( ^' a , 0 (lese nvolvido (r) mas nume-

vsvrüolos (Va) sem membrana limltante, u “\ . . células (mp) têm espessamen-

rf-sos rlbossomas (rb). As membranas ^P^ratodas . ( . unuiu . u ,„-, intercelulares (O

tos que podem ser tomados como ^mossomas^d). fixado em Palade.

apresentam microvilosidades imv) a

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 701 - 710 , 1966

C. DA CRUZ LANDIM e E. W. KITAJIMA

705

possuíam uma parede densa, possivelmente de quitina esclerotizada, disposta em

ramadas concêntricas e em sua luz ocorria uma substância granular de densidade

média. Dois tipos de canalículos puderam ser distinguidos: um de diâmetro

maior e de paredes mais espessas, localizado preferencialmente próximo à luz

da glândula (Fig. 2a); e outro mais fino, de paredes mais delgadas. Êstes

últimos possivelmente representavam ramificações menores dos primeiros e se

caracterizavam por serem de menor calibre e se acharem envolvidos por microvi-

losidades da célula (Fig. f e Fig. 2a). Estas microvilosidades mediam cêrca de

1 n de comprimento e 20-25 m/j. de diâmetro e entre elas não se pôde notar

nenhum material denso.

A região mediana da glândula ácida da pupa apresentava um aspecto muito

semelhante ao descrito para a zona apical. As únicas diferenças encontradas

foram quanto à presença de pequenas vesículas de cêrca de 30 m/r de diâmetro,

contendo material relativamente denso, aparentemente associadas às microvilo¬

sidades em tórno dos canalículos intracelulares. Os vacúolos eram bem menores

do que os da região apical.

2. Abelhas campeiras — A porção apical da glândula destes indivíduos

apresentava-se semelhante à da pupa, mas diferenciava-se pelo falo do citoplas¬

ma apresentar-se extremamente vesiculado e pela ausência dos vacúolos (Fig. 2a).

Tais vesículas provavelmente representariam elementos do retículo endoplasmático

agranular alargados, mas com um conteúdo de baixa densidade.A zona citoplas-

mática, adjacente às microvilosidades apresentava-se menos densa e livre de orgâ-

nulos celulares, talvez refletindo um acúmulo de secreção, não organizada nesta

região (Fig. 2a). Freqiientemente observon-se um alargamento do espaço entre

as microvilosidades adjacentes, na porção basal, formando-se pequenas bolsas.

Todavia, a diferença mais marcante, quando comparada esta região das abe¬

lhas adultas com a das pupas, era a ausência quase total dos enormes vacúolos,

ocorrendo êstes, em tamanho e número bem reduzido e próximos à luz da glân¬

dula, condição semelhante à verificada na região mediana da glândula da pupa.

Nesta fase é possível perceber uma membrana plásmica divisória, próxima aos

canalículos maiores.

Em abelhas campeiras, as células da região mediana da glândula apresen-

tavam-se com sinais evidentes de degeneração, pràticamente sem orgânulos celu¬

lares distintos, restando somente identificáveis o núcleo e as vilosidades em tôrno

dos canalículos. O resto do espaço intracelular achava-se preenchido por vesí¬

culas de forma e dimensões irregulares. Estruturas em forma de lamelas con¬

cêntricas aparecem freqiientemente nestas células e provàvelmente correspondem

aos parassomas (p), que surgem caracteristicamente em células em vias de

degeneração (Fig. 2b).

B. Saco de veneno — Distinguem-se duas regiões no saco de vene¬

no: a porção apical, onde desemboca a glândula ácida, formada de células rela¬

tivamente altas e a zona basal, próxima ao dueto excretor, constituída de células

bem baixas. A cutícula, revestindo êste saco, era muito espessa, principalmente

na parte basal, onde mostrava organização típica do exoesqueleto de insetos.

Na parte apical, esta cutícula assemelhava-se à da glândula ácida. As células do

saco de veneno ostentavam também canalículos intracelulares e pequenos núcleos

próximos à cutícula e aos canalículos.

Na pupa, as células do saco de veneno diferenciavam-se das da glândula

ácida por apresentarem invaginações da membrana plásmica associadas a mito-

côndrios, na zona cortical. Nesta célula, pequenos bastonetes ou grânulos densos,

SciELO

10 11 12 13 14 15

T E ' í T Glanrlula aclda tIe abe| ha campeira, a) Vista geral cie um corte transversal

cia glândula, vendo-se a luz (lu) contendo material pouco denso, a cutícula (c) disposta

em camadas concêntricas e o aspecto bastante deteriorado da célula. Notar que existem

dois tipos de eanalirulos intercelulares (ci), sendo que os mais próximos à cutícula não

tem rnicrovilosidaa.es ao redor. Os pontos marcados com « correspondem a depósitos

de secreção, b) Detalhe da célula mostrando estruturas parecidas com os lisossomas d>

e estruturas lamelares concêntricas < p).

Mem. Inst. Butantan

Sirnp. Internar.

33 ( 3 ): 701 - 710 , 196(5

C. DA CRUZ LANDIM e E. W. KITAJIMA

707

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Fig. 3 — Saco de veneno, a) Vesículas contendo mitoeôndrios em degeneração no inte¬

rior. b» Origem de secreção a partir do núcleo. Bastões densos (bd) vêem-se primei¬

ro entre as membranas do envoltório nuclear (en) e depois no interior de vesículas

(v ) no citoplasma.

708

ULTRAESTRUTURA DO APARELHO VENENÍFERO DE A PIS

(IIYMENOPTERA , A PIDAE )

de dimensões variadas (75-100 m/x X 100-400 m/x), foram encontrados (Fig. 3I>).

Êstes ocorriam livres no citoplasma, agrupados ou individualmente no interior

de vesículas e também freqüentemente no espaço pcrinuclear (Fig. 3b). Milo-

côndrios, de forma atípica, foram encontrados em células de abelhas adultas.

Êstes mitocôndrios apresentavam-se com poucas cristas, e um substrato muito denso

e embora alguns ocorressem normalmente no citoplasma, outros apareciam fre¬

quentemente em diversas fases de degeneração, encerrados em vesículas (Fig. 3a).

Além destas vesículas contendo mitocôndrios, numerosas outras, contendo um

material de baixa densidade ocorrem nestas células. Tais vesículas parecem au¬

mentar à medida que envelhece a abelha e, presumivelmente, originam-se do retí¬

culo endoplasmático. Na base das microvilosidades que circundam os canalí-

culos intracelulares, frequentemente observam-se pequenas e numerosas vesículas

e também gotículas densas, provàvelmente de natureza lipídica. As paredes dos

canalículos, por outro lado, ocasionalmente mostravam interrupções semelhantes

àquelas descritas na glândula hipofaríngea de Apis(9).

Estruturas consideradas como lisossomas foram freqüentemente encontradas

nestas células do saco de veneno. Tais orgânulos eram limitados por uma mem¬

brana simples e continham grânulos densos e lamelas concêntricas cm seu inte¬

rior. Nas células da região mediana do saco de veneno foram encontradas dife¬

rentes fases de evolução destas estruturas.

A musculatura que envolve o saco de veneno, bastante desenvolvida, dispu-

rdia-se tanto circular como longitudinalmente. As fibras desta musculatura eram

do tipo estriado, embora não apresentassem tôdas as características de uma mus¬

culatura estriada típica. As células musculares eram alongadas tendo miofibrilas

apenas na porção central. Tais fibrilas mostravam, a espaços proximados de

3 jx, linhas densas que corresponderiam à linha Z (10). Lateralmente às miofi¬

brilas. elementos do retículo endoplasmático e mitocôndrios foram observados.

Tal tipo de musculatura é encontrado comumente em vísceras de insetos.

Traquéias, com armação helicoidal de quitina, foram freqüentemente encon¬

tradas nas células da glândula ácida e do saco de veneno. Membrana basal,

espessa, de densidade mediana estava invariàvelmente presente nas células glan¬

dulares e do saco de veneno, bem como nas musculares.

Discussão

Tôda a glândula ácida, da pupa de A p i s , parece ter atividade secretora,

confirmando e até reforçando as observações de Autrum e Kneitz (4). Porém,

em abelhas de 30 ou mais dias de idade, a glândula ácida acha-se degenerada,

restando parcialmenle intacta apenas a porção apical. Estas observações suge¬

rem que o processo degenerativo destas glândulas progride da parte basal para a

apical. O veneno produzido pela abelha durante a fase final da pupação e pri¬

meiros dias da vida adulta é armazenado no saco de veneno até o momento do

uso. Geralmente as operárias de A p i s picam uma vez só porque, ao fazê-lo,

perdem o ferrão, essa talvez seja a causa da degeneração precoce da glândula

ácida. Em Bombas( 3) mesmo nas abelhas campeiras, tôda a glândula é

secretora, mas esta abelha não deixa o ferrão ao picar. Fenômeno similar ocorre

no saco de veneno, onde principalmente as células apicais passam por processos

degenerativos.

Particularmente notável é a ausência pràticamente total do complexo de

Golgi. neste sistema venenífero, pois sua importância nos fenômenos de secreção

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 701 - 710 , 1966

C. DA CRUZ LANDIM e E. W. KITAJIMA

709

é bastante conhecida (5).Esta ausência talvez seja explicável pelo fato de a secre¬

ção ser armazenada, intracelularmente, em enormes vacúolos na fase pupal, pois

o complexo de Colgi teria mais função como concentrador da secreção.

Nas abelhas adultas, aparentemente haveria a secreção de substâncias dife¬

rentes daquelas produzidas durante a pupação e eliminadas nos primeiros dias

da vida adulta.

Os basloneles ou grânulos no saco de veneno, que migrariam do espaço

perinuclear para o citoplasma, poderiam representar uma destas substâncias. O

envolvimento do núcleo em atividades secretoras já foi relatado em hipotálamo

de rato associado à neurcsecreção (11). Também as vesículas associadas ou não

a mitocôndrios, poderiam conter outro tipo de secreção, embora mais pareçam

um resultado de degeneração celular.

O conteúdo dos enormes vacúolos encontrados em células das glândulas ácidas

de pupa representaria uma secreção ainda não eliminada. Possivelmente nas

fases subseqüentes da existência da abelha, tal secreção seria lançada para a luz

da glândula, sendo armazenada aí ou no saco de veneno, pois em células de

abelhas adultas, em vias de degeneração, não há sinal de acúmulo desta secreção.

É provável que a glândula ácida produza uma parte do veneno, ao qual

são adicionadas posteriormente outras substâncias, secretadas pelo saco de vene¬

no. Estas últimas, poderiam determinar a especificidade do veneno de cada

espécie de abelha, uma vez que se originam em íntima relação com o núcleo.

Evidentemente os canalículos intracelulares devem funcionar como um siste¬

ma coletor da secreção. Tais canalículos facilitariam a eliminação da secreção

aumentando a superfície polarizada (12, 9). As dobras da superfície celular

em forma de microvilosidades que envolvem os canalículos, principalmente nas

suas ramificações menores, proporcionariam uma superfície maior para a elimi¬

nação da secreção. É provável ainda que, principalmente na glândula ácida, em

que a secreção parece ser mais fluida, parte do solvente possa ser reabsorvido

pelas microvilosidades antes da secreção passar para a luz do canalículo, o que

representaria uma economia de água para a célula.

A origem dos canalículos intracelulares parece ser diferente da das células

secretoras. Frequentemente, adjacentes à cutícula e aos canalículos, estão células

achatadas e de núcleo pequeno. As membranas separatórias entre estas células

e as secretoras não são visíveis nas abelhas jovens, mas tornam-se evidentes nas

abelhas campeiras. Isto reforça a teoria (4) da origem independente das célu¬

las secretoras e formadoras da cutícula e canalículo. Neste caso a cutícula seria

uma verdadeira íntima.

As presentes observações confirmam também o ponto de vista de que os

canalículos intercelulares estão, na realidade, topològicamente fora das células se¬

cretoras e portanto sua denominação é incorreta (12). Tais canalículos apre¬

sentam-se tipicamente separados da célula secretora por uma membrana plásmica,

normal ou dobrada em forma de microvilosidades.

O aparecimento de estruturas consideradas como parassomas e lisossomas,

além da extensa vacuolização do citoplasma, anomalias no núcleo, etc., devem

representar inegavelmente, sinais flagrantes da degeneração das células glandu¬

lares e do saco de veneno nos indivíduos adidtos.

A musculatura em volta do saco de veneno deve estar associada à injeção

do veneno na vítima, durante o ato da picada.

SciELO

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ULTRAESTRUTURA DO APARELHO VENEN1FERO DE APUS

( HYMENOPTERA, AP1DAE )

SuMMARY

Electron microscope studies were made on acid gland and venom sac of

A pis workers. Workers ot A pis are sLudied in lhe pupal phase and adull

foragers (30 days older). Jn pupa all lenght of acid gland and the apical

portion of venom sac was secretory and probably was in greal activity. In

adull workers the cells of acid glands and the venom sac degenerate. The dege-

neration was less intense in the apical portion of the acid gland.

In pupa acid gland cells, appear enormous vacuoles that possibly contai n

lhe secretion of this phase. In adults this vacuoles practically desappear. It was

suggest that the adults secrete another type of substances. In lhe venom sac dense

material in form of rods or small granules free or within vesicles seem to be the

secretion. These materiais appear in lhe cell always associated wilh the nucleus,

first located in the perinuclear space.

The venom sac is provided with a powerful striated musculature, that pro¬

bably add in the venom injection.

Bibliografia

1. BORDAS, M. L„ Ann. Sei. Nat. Zool., 19, 1, 1895.

2. SOTELO, J. R., and PORTER, K. R„ J. biophys. biochem. Cytol., 5, 327-342,

1959.

3. KERR, W. E„ and LELLO, E., J. N.Y. enthomol. Soc., 70, 190-214, 1962.

4. AUTRUM, H. VON, und KNEITZ, H„ Biol. Zbl., 593-602, 1959.

5. PALAY, L. S., in L. S. PALAY (Editor), Frontiers in Cytology, New Haven,

Yale, Univ. Press., 1958, pp. 305-342.

6. MILLONIG, G., J. Cell Biol., 11 , 736-739, 1961.

7. McALEAR, J. H., Ann. Histochem., Suppl. 2, 261-276, 1962.

8. MILLONIG, G., in BUFFA (Editor), From Cell to Molecules, Symposium on

Electron Microscopy, Roma, 1964, pp. 347-362.

9. BEAMS, H. W„ and KING, R. L„ J. Morphol., 53, 223-241, 1932.

10. FORBES, A. R., Mem. entomol. Soc. Canada, 36, 1-75, 1964.

11. KAWABATA, I„ J. Cell. Biol., 26, 101-113, 1965.

12. BEAMS, H. W., and ANDERSON, E„ J. Morphol, 109, 159-171, 1961.

1, | SciELO

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Simp. Internai .

33 ( 31 : 711 - 724 , 1066

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DONNÉES HIST0CHIM1QUES SUH LA GLANDE À VENIN

(GLANDE CHÉLIGÉRIENNE) DES ARAIGNÉES DIPNEUMONES

L. ARVY

Station de Physiologie animale, C.N.R.Z., Jouy-en-Josas, France

“That such minute glands should secrete

enough venom to kill human beings. . . is hard

to believe” (1).

“Une piqüre dont ma loupe ne peut trou-

ver les traces. . . a suffi. . . pour tuer ia vigou-

reuse bête. Toute proportion gardée, le Crotaie,

le Céraste, le Trigonocephale, et autres serpents

d’odieux renom, n'obtiennent pas, sur leurs

victimes, des effects aussi foudroyants” (2).

Cest à une telle estimation qu’était coniiuil le célèbre eniomologlsle en

observant l’effet clu venin des Epeires, faseiées ou soyeuses, sur la grande libèllule

Aeschna grandis L. et un observateur contemporain a pu dire, sans exagération

semble-t-il, que les araignées venimeuses (en particulier, Latrodectus et

Loxosceles ) tuent autant dlmmains que les guêpes, aussi, leur petite taille.

oomparée à ceile de 1'être humain, en fait-elle “de grands tueurs’ . II est donc

parfaitement étonnant qu’une sécrétion aussi puissamment dévastatrice n ait pas

encore fait 1’objet de recherches comparées méthodiques, quant à sa nature réelle

et aux particularités de la structure des cellules qui 1’élaborent, chez des repré-

sentants des divers genres d Araebnides. Les quelques données dont nous dis-

posons sont si rudimentaires qu’en 1964, encore, Bücherl admettait que de

nouvelles recherches étaient nécessaires.

Cependant, des Iravaux récents ont précisé la nature, le plus souvent fonda-

menlalement protidique du venin des araignées, ainsi que 1’existence d importantes

variations spécifiques; s’est ainsi que le venin de Phoneutria )era est einq fois

moins riche en lysine et trois fois mcins riche en histamine que le venin de

Lycosa erythrognatha (3). Le venin sec de cette lycose est remarquablement

riche en histamine quand on le compare à celui de Phoneutria fera-, il contient.

en effect, de 14 à 19 mg/g d’histamine quand celui de Phoneutria n’en

contient que de 0,6 à 0,8 mg/g (4). Par contre, le venin de Phoneutria

est environ deux fois plus riche en glutamine (3) que celui de Lycosa', c’esl

grâee à cette nature essentiellement protidique qu il est possible de préparer des

antivenins efficaces.

Nous savons aussi que le venin de certaines araignées est riche en séro-

tonine et en ]>olypeptide de type bradykinine; le venin de Lycosa contient

environ trois fois plus de sérotonine que celui de Phoneutria (soit 1.5 à

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DONNÈES HISTOCIIIMIQUES SUR LA GLANDE A VENIN (GLANDE

CHÈLICÉRIENNE) DES ARAIGNlíES DIPNEUMONES

1.9 mg/g); par contre, il est infiniment plus pauvre cn polypeptide que celui

de I‘honeutria, quand il a une aclivité de 1.5 à 5 unités de hradykinine,

le venin sec de Lycos a a une aclivité de 360 à 420 unités de liradykinine (4).

Cependant, la plus large part du venin de certaines araignées n’est pas

protidique; chez Atrax robnstus, par exemple, le venin cst décomposable, par

électrophorèse, en Irois fractions toxiques, de mobilités bien différentes, donl

1’une csl dépourvue d’acides aminés; c'csl à eelle particularité que Wiener (5)

attribue 1’impossibilité d’obtcnir un antivenin vraiment efficacc contre les morsu-

rcs de cette araignée.

Lebez (6) a trouvé des lipides dans le venin de Latrodectus Iredecimgultatus ;

cependant, étudiant la même espèce, Cantore cl Bettini (7) n'ont pas trouvé de

lipides soudanophiles. A ccl égard, il convienl de rappeler que certains hislo-

logisles (8, 9) ont observé des inclusions osmiophiles dans les cellides glandu-

laires et le venin des ARACHN1DES; ils ont attribué cette osmiophilie, sans

autre preuve d’ailleurs, à des lipides.

Fischer et Bohn (3) ont trouvé un penlose dans le venin de Phoneutria

fera et de Lycosa erythrognatha ; Gabe (10) a mis en évidenee une glycoprotéine

bislocbimiquement décelable dans le venin de 9 espèces d’Aranéides (appartenant

à 7 genres) et Wiener (5) a décelé un glueide dans le venin (VAtrax robuslus.

Nos connaissances sur les enzymes des venins des Araignées sont três succin-

tes. Dès la fin du siècle dernier, Gaubert (11) admettait que la sécrétion des

glandes venimeuses “doit renfermer des ferments qui modifient les liquides que

1’animal absorbe par succion (p. 56); cependant, la plupart des auteurs ivont

('nvisagé pour le venin qu’un róle de défense ou d’attaque, utile pour supprimer

un adversaire ou pour immobiliser les proies. Comme le suggérait Millot (12)

pour lever tout dout, quant au rôle du venin dans 1’alimentation, ‘41 faudrait

rechereher in vitro, 1’action digestive dYxtraits des glandes”, ce qui à ma

connaissance n’a pas encore été fait. Quoi qu’il en soit sur ce point, Duran-

Reynals (13) a vu que les extraits d’araignées contenaient un remarquable

“íacteur de diffusion” absent des èxtraits de sauterelle, de libéllule ou de four-

mi... et ce íacteur a été identifié depuis à 1’hyaluronidase. En outro, le venin

de la plupart des araignées qui ont été examinées à ce point de vue, a des

effects protéolytiques et anti-acétylcholinestérasique (7).

II semble que nos connaissances doivent désormais évoluer rapidement; en

effect, 1'électrophorèse, en particulier, est d'une aide précieuse; elle fait parfaite-

ment a|>paraítre la complcxité du venin des araignées; suivant 1’éspèce étudiée,

suivant la technique d’électrophorése mise en oeuvre, six fractions (14), cinq

fractions (15, 16, 7, 17, 18), voire huit fractions (19) ont pu être dissociées.

Ainsi, à 1’heure actuelle, nous sommes relativement mieux documentés sur

les caracteres du venin des araignées que sur Ia morphologie des glandes qui

1’élaborent. Les mémoires classiques, dont un relevé soigneux figure dans le

travail de Millot (12) ont, certes, abouti à des précisions intéressantes sur le

développement embryonnaire de ces organes et leur anatomie chez 1’adulte, mais

les bistologistes semblent sêtre contentés d appliquer les seules méthodes de l ana-

toinie microscopique, d'oü une carence évidente de nos informations relatives

aux caracteres cytologiques et histochimiques des cellules glandulaires, ainsi qu’à

leur mode de sécrétion. Parmi les articles de Traité portant sur des ARANÉIDES,

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L. ARVY

713

eelui de Gerhardt et Kãstner (20) reprend Ies notions essenlielles du travail

de Millot (12), sans leur ajouter d’élément nouveau. Millot (21), à son tour,

a exposé, sous une fcrme plus concise, les mêmes donnces. Les quelques publi-

eations orientées vers la morphologie parues depuis cetle date ne représentent

pas mi progrès, si liien que la parcimonie des documents disponibles, apparait

clairement à la lecture de la monographie de Savory(22). II devait donc pa-

raítre op])ortun d’appliquer quelques techniques cytologiques et surtout histochi¬

miques à ces glandes à venin, puisque seuls les composants glucidiques semlilent

avoir élé explorés avec les techniques histochimiques récentes (10).

Materiel et techniques

Les caracteres morphologiques généraux et certaines particularités cylolo-

giques des glandes à venin ont pu être étudiées eliez les espèces suivantes:

Dysdera crocata C. Koch.

Pholcus phalangioidcs Füssly.

Teutana grossa C. Koch.

Araneus diadematus Clerck.

Clubiona ter r es/ris Westr.

T egenaria parietina Fourcroy.

Tegenaria derhami Scopoli.

Le plus souvent le matériel a été fixé par le liquide de Bouin, inclus à la

paraffine et débité eu coupes sériées de 5 p. Parmi les méthodes histologiques

générales, 1’azan de Heidenhain, le Irichrome eu un tenips sans différencialion,

le triehrome de Masson-Goldner et la coloration de Mann-Dominici nous ont

fourni les images les plus significatives. Les variations anatomiques des glandes

sont importantes d Tm genre à 1’autre et cependant aucune de ces techniques

n a fait apparaitre de différences dans la structure histologique chez les sept

espèces examinées (Tableau I).

TABLEAU I — AFFINITÉS TINCTORIALES DES PRODUITS DE SÉCRETION

DE LA GLANDE CHELICÉRIENNE

Colorations

Flaques

Grains

Triehrome en un temps

vertes

rouges

Triehrome de Masson-Goldner

vertes

rouges

Azan

bleues

rouges

Mann-Dominici sans oxydation

roses

rouges

Mann-Dominici avec oxydation

roses

bleues

Mann biacide

bleues

rouges

La plupart des constatations histochimiques ont été oblenues sur des coupes

de glande chélicérienne de Tegenaria derhami et de 'Tegenaria pcrActina ; elles

sont résumées dans le Tableau II.

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DONNÉES HISTOCHIMIQUES SUR LA GLANDE A VENIN (GLANDE

CHELICERIENNE) DES ARAIGNÉES DIPNEUMONES

TABLEAU II —

CARACTERES HISTOCHIMIQUES DES PRODUITS DE

SÉCRÉTION DE LA GLANDE CHÉLICÈRIENNE

Méthode

Flaques

Grains

APS

APS après diastase du malt

Réaction métachromatique

Coloration par la fuchsine-paraldéhyde

idem, après oxydation permanganique

Coloration au bleu alcian

Alloxane-Schiff

Tétrazoréaction de Danielli

Réaction au ferricyanure ferrique

idem, après blocage au sublimé

Azoréaction

Réaction argentaffine

Réaction du rosindole

Resultats

LA GLANDE

Rappel anatomique — Les glandes chélicériennes des Aranéides dipneu-

mones sont piriformes, siluées dans la région proximale et dorsale du prosoma.

La partie renflée de ces glandes, sacciforme et plus ou moins longue, occupe

un emplacement superfície!, très proche de la paroi dorsale du corps (Fig. 1

à 3) ; sa face inférieure est plus ou moins adjacente, suivant les espèces, aux

diverticules thoracentériques et à Ia masse nerveuse sus-oesophagienne; les parois

latérales des glandes sont au contact des muscles du prosoma. La partie anté-

rieure effilée de la glande se continue par un canal qui parcourt toute 1’étendue

de la chélicère, pour déboucher dans 1'article terminal de celte pièce, non loin

de 1’extrémité du crochet. L’exploration d’un matériel ahondant (23, 24, 25.

26. 9, 27, 28, 12, 29, 1. 8, 30), a révélé des différences de taille enormes suivant

les genres; c’est ainsi que Ia glande chélicérienne est petite chez Amaurobius

erberi, moyenne chez Helecnemus pluchei et grande chez Scytodes delicalula (271:

elle est entièrement contenue dans la chélicère. chez Ia Mygale , elle s’étend

dans 1’article ])roximaI de la chélicère chez Clubiona palUcula et elle est entiè¬

rement dans le céphalothorax chez E p e i r a , A g e l e n u et T e g e n a riu (23 I ;

elle est relativement énorme chez les Pholcides et la Filistale (24). La taille

de la glande est sans rapport avec la toxicité du venin. ou le comportement de

1’araignée; les eas extremes soul représentés [)ar la Filistale et les Ulohorides; en

effect, la Filistale, qui possède une énorme glande à venin, est apparemment

incapable de mordre, car les deux chélicères sont soudées à leur hase et sur une

partie de leur étendue (il en est de même chez les Pholcides). Au contraire,

chez les LIloborides, il n’existe pas de glande à venin. mais la chélicère est

fonctionnelle et les crochets peuvent mordre.

La glande à venin est composée d'une musculeuse, d une membrane basale

et d’un épitbélium sécréteur; le canal excréteur de la glande est dépourvu de

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L. ARVY

715

musculeuse; sa paroi est très mince, elle lie comporte qu un basale tapissée d'un

épithélium dont les caracteres cytologiques diffèrent de ceux de la partie pro-

prement glandulaire.

Caracteres histologiques — La musculeuse qui engaíne le sac glandulaire

est composée de fibres striées dont la structure est semblahle à celle des muscles

du prosoma, mais qui sont remarquables par leur disposition en spires régulières

autour de la glande (Fig. 1 à 3).

Les auteurs classiques ne mentionnent pas 1’existence d une basale, sur la-

quelle repose 1’épithélium glandulaire; Legendre (1935) signale une basale mince

chez T e g e n a ria ; en fait, celte basale est assez épaisse, homogène et pourvue

des affinités tinctoriales habituelles aux basales glandulaires: elle est fortemenl

cyanophile, elle relient le vert solide du trichrome en un temps, le vert lumière

du trichrome de Masson-Goldner, le bleu d’auiline de Fazan. La réaction à

1’ acide periodique-Schiff lui confere Fhabituelle teinte rouge intense, signalétique

de composés glucidiques; elle ne contient apparemment pas de mucopolysaccha-

rides acides.

L’épithéliutn glandulaire est fait de cellules prismatiques, très hautes et

étroites, à parois minces et fragiles; le plus grand diamètre peut atteindre 100 /i.

la largeur est voisine de 10 /x. Chez certaines espèces, il existe des inégalités

de la hauteur de l’épithélium glandulaire; signalées dès 1880 par Mac Leod.

chez Epeira diademata, clles ont été décrites par Bordas (26), chez Latrodeetus

tredecimguttatus ; ces inégalités peuvent aller jusqu’à la diposition en éventail

des cellules, et un véritable cloisonnement de la lumière glandulaire par des

replis épithéliaux existe chez Filistata insidiatrix 1121 ; une telle disposition des

cellules glandulaires n "existe pas chez les espèces éludiées ici (Fig. 1 à 3).

Les noyaux des cellules glandulaires sont de petite taille et basaux; ils appa-

raissent régulièrement arrondis sur les coupes de la glande; ils mesurenl en

moyenne 8 g de diamètre; leur chromatine apparait en mottes irrégulières. uni-

formément réparties dans le cytoplasme; il existe un petit nucléole, fortemenl

basophile. Les mitoses dans l’épithélium des glandes chélicériennes des araignées

adultes sont extrêmement rares.

Le cytoplasme basal, périnucléaire, tranche sur le reste du corps cellulaire,

dès 1'examen des préparations colorées par les techniques histologiques géné-

rales (Fig. 4); il est, en effet, pourvu d’une forte affinité pour les colorants

basiques et la disparition de cette basophilie après mise en oeuvre de la ribo-

nucléase, démontre que la basophilie est due à des ribonucléines (Fig. 5).

La partie supré-nucléaire du cytoplasme constilue, en raison de la position

Irès basale des noyaux, la majeure partie du corps cellulaire; sou aspect varie

avec les stades du cycle sécrétoire; dans certaines cellules, elle apparait entière-

ment vidée (Fig. 8). alors que dans daulres. elle contient une quanlité plus ou

moins considérable de sécrétion (Fig. 6 el 7).

Le produit de sécrétion de la glande chélicérienne n’est pas unique: la seule

morj)hologie suffit pour distinguer deux substances. En effet. Fun des produits

sécrétés forme des flaques plus ou moins étendues et des trainées, qui parfois

remplissent toute la partie supra-nucléaire des cellules, tandis que 1'autre se

présente sous forme de granules et de gouttelettes, aux conlours bien définis,

dont la taille ne dépasse généralement pas 1.5 /j.; ces deux produits coexistenl

.souvent (Fig. 4, 5 et 91. dans une même cellule épithéliale.

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DONNÉES HISTOCHIMIQUES SUR LA GLANDE A VENTN (GLANDE

ClIÈLICÉRIENNE) DES ARAIGNEES DIPNEUMONES

Les affinités tinctoriales des deux produits de sécrétion soul fondamenlalemenl

différentes. En effet, la sécrétion en flaques est faiblement éosinophile après

mise en oeuvre de la technique de Mann-Dominici et elle est cyonaphile avec les

colorations Irichromes nlilisées ici; le produit retient fortement le vert Inmière

du trichrome de Masson-Goldner, le vert solide du trichrome en un lemps de

Gabe et Martoja, le bleu d’aniline de 1’azan de Heidenhain. Au contraire, le

produit de sécrétion en granules ou en gouttelettes esl fortement éosinophile après

eoloration suivant Mann-Dominici, lorsque cetle méthode esl pratiquée sans oxy-

dation préalahle des coupes; après oxydation, il devient au contraire, fortement

liasophile; érythrophile avec les colorations Irichromes (Fig. 4 et 5), ce produit

retient Ia fuchsine acide, 1’azoruhine S et 1’azocarmin (Tableau I).

Caracteres histochimiques — Certains caracteres histochimiques sont com-

muns aux deux produits de sécrétion de la glande ehélicérienne (Tableau II).

G’est ainsi que les flaques aussi hien que grains sont APS-positifs et dépourvus

de glycogène, aussi hien que de mucopolysaccharides acides; il semble (pie leur

réactivité à 1’acide périodique-Schiff traduise, soil la présence de muco poly-

saccharides neutres, soit de glycoprotéines, comme l a admis Gabe (10).

Les colorations par 1’acide periodique-Schiff et par la fuchsine paraldé-

hvde (Fig. 6 à 8) permettent d’apprécier parfaitement la plus ou moins grande

abondance du produit de sécrétion. Certains caracteres histochimiques révèlent

la dualité de la sécrétion de la glande ehélicérienne; c’est ainsi que la recherche

des protides fail apparaitre des différences nettes entre deux constituants du

venin; en effet, les flaques cyanophiles réagissent faiblement à l’alloxane-Schiff

et à la tétrazoréaction de Danielli. alors que les grains érythrophiles réagissent très

fortement. Inversement, la recherche des protides sulphydrilés fait apparaitre

nettement les flaques cyanophiles, alors que les grains restent inapparents et ne

se colorenl que par le colorant de fond, après mise en oeuvre de la technique

au ferricyanure ferrique. La recherche de dérivés indoliques n’est positive que

dans les grains. La recherche des polyphenols est restée totalement négative

(Fig. 9 à 12).

Les différences histochimiques notées au niveau des cellules épithéliales du

sac glandulaire, restent les mêmes dans la lumière du sac, oü s’accumulent les

deux produits après leur extrusion, si hien que leur distinction reste, là, aussi

facile qu’au sein des cellules; elles persistent encore dans la sécrétion du canal

excréteur.

LE CANAL EXCRÉTEUR

La limite entre le sac glandulaire et le canal excréteur est

des modifications anatomiques très nettes; d’une part, la lumière se

(1’autre part la tunique musculeuse de la glande disparait. En oulre,

des cellules épithéliales diminue hrusquement pour n’être plus que

environ. La hasale glandulaire s’amincit fortement. Fnfin, seule la

tiale du canal excréteur est tapissée par un épithélium prismatique

indiquée par

rétrécit et

la hauteur

de 25 n

parlie ini-

dont les

cellules ont toujours leurs noyaux hasaux, mais

ces cellules s’aplatissent rapidemenl, si hien

10 fx, dans Ia partie du canal intra-chélicérienne;

du canal excréteur, les

plates; il est dont peu

joue quelque rôle, autrt

gla ndulaire.

elles

que leur

n’ont plus d’ergastoplasme;

hauteur atteint à peine

dans la partie loute terminale

cellules de 1’épithélium canaliculaire sont

vraisemhlahle que le canal de la glande

que celui de vecteur du venin élahoré

extremement

ehélicérienne

par

sac

SciELO

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Mem. Inst. Butantan

Slmp. Internac.

33 ( 3 )-. 711 - 724 , 1966

L. ARVY

717

Discission

En somme, les caractères histologiques de la glande chélicérienne sonl ceux

qui ont élé décrits par les classiques; senis deux points sonl à discuter, à savoir

les caractères histochimiques du produit élahoré par la glande et le mode de

fonctionnement des cellules glandidaires.

Les techniques qne nous avons mises en oeuvre concourent pour faire

admettre la dualité des produits figures, élaborés par les cellules glandulaires. La

morphologie

des produits de sécrétion esl très significalive à cet égard.

Certaines des affinités tinctoriales observées n’ont pas obligatoirement une signi-

fication chimique précise; on sail, en effet, que l’érythrophilie ou la cyanophilie

d'une structure peut dépendre de son état physique autant que de sa constitution

chimique, mais 1’histochimie fournit des indications formelles à cet égard: si les

deux produits de sécrétion contiennent des glycoproléines ou de mucopolysaecha-

rides neutres (puisqu’ils sont colorables par 1’acide periodique-Schiff et qu ils

perdent leur pouvoir de recolorer la fuchsine de Schiff par acétylation pour re-

trouver ce pouvoir par saponification ), ils ne sont pas métachromatiques au bleu

de toluidine, ils ne sont pas alcianophiles et sont bien colorables par la fuchsine

paraldéhyde après oxydation permanganique), leur constitution protidique est

cependant essentiellement différente; seul le produit érythropbile est riche en

acides aminés aromatiques, alors que seul le produit cyanophile est riche en

protides sulfhydrilés. En outre, seul le produit de sécrétion érytbrophile contient

des composés indoliques en conCentration supérieure au seuil de sensibilité de la

réaction au rosmdole de Glenner.

11 est évidemment difficile de relier ces constatations histochimiques aux

résultats des nombreuses recherches toxicologiques concernant le venin des Ara-

néides; en effet, les méthodes mises en oeuvre dans ce Iravail pourraient déceler

des précurseurs, ou des substrats des principes actifs, et il n’est pas certain

quVlles montrent ces principes actifs eux-mêmes. D autre pari. les principes toxi¬

ques ne sonl, à 1’heure actuelle, definis que par certains caractères physico-chi-

miques et certains effect pharmacodynamiques. Les premiers chercheurs (31, 32)

admettaient 1’existence de deux principes distincts dans le venin (au moins ehez

les Latrodectidés) , un príncipe hypertenseur et un principe neurotoxique; mais

les recherches recentes, menées à 1'aide de 1’electrophorèse, permettent de révéler

jusqu’à huit fractions (19). 11 est cependant admis que ces fractions sont essen¬

tiellement de tnême nature et ne présentent que des différences quantitalives

spécifiques; néanmoins, il convient de souligner que celte appréciation a été

éniise après une seule coloration des fractions électrophorétiques de nature pro¬

tidique: celle qu’on obtient par 1’amido-black acétique; il n’esl pas invraisem-

lilable d’amettre qu’une autre (ou d’autres colorations) ferait apparaíte d’autres

composés.

Quoiqu il en soit sur ce point, la pluralité des actions pharmacodynamiques

du venin des AI1ANÉIDES est sans doute, pour une part, liée à la dualité du

produit, décelable grâce à 1’histochimie.

Quant au mode de fonctionnement des cellules glandulaires, la plupart des

auteurs classiques (26, 33) ont cru à une fonte holoerine des cellules glandulaires,

mais toute une série d arguments s’oppose à cette manière de voir et avec

Millot (12) il faut admettre que la sécrétion est du type mérocrine. En effet.

on ne rencontre jamais de noyaux dans la lumière glandulaire et la recherche

de débris cytoplasmiques y reste négative, Contrairement à ce qu’on voit dans

toutes les glandes à fonctionnement holoerine, la glande chélicérienne des AKA-

SciELO

10 11 12 13 14 15

718

DONNÉES HISTOCHIMIQUES SUR LA GLANDE À VENIN (GLANDE

CHÉLICÉRIENNEJ DES ARAIGNÉES DIPNEUMONES

NÉIDES ne comporte pas de zone “germinative”, faite <lc cellules indifférenciées r

riche en mitoses et destinée à prendre la relève des cellules épuisées. Le renou-

vellement cellulaire ne semble pas particulièrement rapide, de sorte qu’il est vrai-

semblable que chaque eellule parcourt un certain nombre de fois le cycle séeré-

loire, allant depuis le prélèvement. dans le milieu intérieur, des matériaux né-

cessaires à la synthèse du venin, jusqu’à 1’extrusion de ce dernier.

A côte des arguments négatifs mentionnés, il convient de signaler que les

images dVxtrusion des produits de sécrétion, sur les coupes de glandes fixées

avec soin, ne sont pas rares; cependant les méthodes de la cytologie infra-

structurale sont nécessaires pour décider du mode, mérocrine ou apocrine, de

rextrusion des produits élaborés |)ar répithélium de la glande chélicérienne.

Conclusion

La coloration par 1'azan de Heidenbain révèle dans le venin des araignées

la coexistence de deux produits de sécrétion d'affinités tinctoriales différentes,

l’un est en flaques cyanophiles, 1’autre est en granules érythrophiles, de lailles

variées. La sécrétion semble unique après coloration suivant Mann-Dominici,

comme après coloration par 1'APS ou par la fuchsine paraldéhyde; la dualité de

la sécrétion apparait après oxydation permanganique et coloration suivant Mann-

Dominici. Les flaques cyanophiles sont riches en groupements sulfhydrilés. Les

grains érythrophiles sont riches en acides aminés aromatiques.

SUMMARY

In seven species of DIPNEUMONES ARANEIDS the sécrétion of lhe che-

liceriau glands is complex; histochemically two substances can be distinguished:

one is granular, it is rich in aromatic amino-acids and in indolic compounds for

il gives the alloxan-Schiff reaction, the Danielli tetrazoreaction and the rosindole

reaction of Glenner; the other, whieh is nct granular, contains sulfhydrils groups.

lhe two substances are APS + and deprived of glycogen or acid mucopoly-

saceharids, they contain, probahly, nêutrons mucopolysaccharids for they lost their

power to stain the Schiff-fuchsin after acetylation and recover that power after

sapcnification; moreover, they are not metachromatie with toluidin hlue, they

are not alcianophilic and they are fairly well stained hy paraldehydfuchsin after

permanganie oxidation.

SciELO

10 11 12 13 14 15 16

I Bouin-trlchrome cie Masson-Gclldne ^"'90 'riiamTtT™) 3 ? &lS <,e Tcfl,e,,oria Varietina

partie antérieure ciu prosoma, à ras cie ■ „ wl ^ ' 1°: . = coupe Passant par la

rhelicériennes, en position clorsale séoarées <ilr es chéllceres - Remarquer les glandes

culaire, la grande hauteur des eelluJes énithéii-, i«"' , ' gne ! T,e<ilane P ar u n raphé mus-

dulaire. Fig. 2 = les glandes chélicérienneT 1 f 1 ^gularité de la lumière glan-

F !°- 3 - la partie aborale des glandes est »'? ° mb ^ nt un divertieule thoracentérique.

nques et Ia masse nerveuse sus-oesoDhaeienne . Pport avec les diverticules thoracenté-

la photographie. Fig. J, _ Coudc^ nhiim . ’ qU1 apparait dans la partie inférieure de

de Tegenaria derhami (Bouin-coloration suivant^M glal J' lulaire de la Klande chélicérienne

375 diamètres, écran oraneei R prnnr f nt Mann -Dominici, sans oxydation préalable,

cellules et les deux produUs sécréUon" r,„ ““ régÍ °" S des

laire, chez Tegenaria derlmmi ,r„„í„ „ V- 0 ' 5 ~ Coupe transversale du sae glandu-

permanganique, 375 diamètres cer in °° '” a 1<>n EUivant Mann-Dominici après oxydation

<*■ cellules et ia bawpwfle du produit de^si' * emar, » uer «'«gaatoplasme du pôle basa,

P ime ou produit de sécrétion en grains, alors que les flaques ont

ccnservé leur acidophllie.

SciELO

10 11 12 13 14 15

Fig. à 12 — Coupes adjacentes d’une glande chéllcérienne de Tegenaria parietina

(Bouin — 200 diamétres). Fig. 9 — coloratton par 1'azan de Heidenhuin, écran vert:

les flaques du prodult cyanophile apparaissent en gris, les grains érythrophiles en nolr.

Fig. 10 — APS, écran vert: les deux produits de sécrétion sont APS +. Fig. 11 — tétra-

zorcaction de Danielli, écran vert: la concentration des protides est beaucoup plus forte

dans les grains érythrophiles que dans les flaques cyanophiles. Fig. 12 — ferricyanure

ferrique, écran orange: seules les flaques cyanophiles sont riches en groupements sulphy-

drilés hlstochlmiquement décelables, les grains érythrophiles apparaissent en négatif.

700 DONNÉES HISTOCHIMIQUES SUR LA GLANDE A VENIN (GLANDE

‘~ Z CHÊLICÉRIENNE) DES ARAIGNÉES DIPNEUMONES

Bibliographie

1. REESE, A. M., Trans. amer. micr. Soc., 63, 170-174, 1944.

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206 et 1951, p. 155.

3. FISCHER, F. G., und BOHN, H., Z. yhysiol. Chem., 306, 265-268, 1957.

4. DINIZ, C. R., Acta yhysiol. lat.-amer., 12, 211, 1962.

5. WIENER, S., Med. J. Aust., 48, 693-699, 1961.

6. LEBEZ, D., Z. phys. Chem., 298, 73-76, 1954.

7. CANTORE, G. P., et BETTINI, S., R. C. Ist. sup. Sanità, 21, 794, 1958.

8. LEGENDRE, R., Ann. Univ. saraviensis, 4, 527-528, 1953.

9. BARTH, R., Mem. Inst. Osw. Cruz, 60, 275-292, 1962.

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11. GAUBERT, P., Buli. Soc. Philom., 3, 82, 1891.

12. MILLOT, J., Ann. Sei. Nat., 14, 113-147, 1931.

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70-74, 1956.

15. NEUMANN, W., und HABERMANN, E., Naturvrissenschaften, 39, 286-287, 1952.

16. BARRIO, A., Acta physiol. lat.-amer., 5, 132-143, 1955.

17. BETTINI, S., and TOSCHI-FRONTALI, N. XI Congr. int. Entomol. (Vienne),

3, 115-121, 1960.

18. FRONTALI, N„ et GRASSO, A., Arch. Biochem., 106, 213-218, 1964.

19. McCRONE, J. D., and NETZLOFF, M. L„ Toxicon, 3, 107-110, 1965.

20. GERHARDT, U., und KASTNER, A. in KÜKENTHAL, Traité de Zoologie,

De Gruyter, Berlin, 1937, pp. 394, 656.

21. MILLOT, J., in GRASSÉ, Traité de Zoologie, Vol. 6, Masson, Paris, 1949,

pp. 386-436.

22. SAVORY, TH., Arachnida, Acad. Press, 1964, p. 289.

23. McLEOD, J„ Arch. Biol., 1, 573-587, 1880.

24. BERLAND, L., Les Araignées, Stock, Paris, 1938.

25. BORDAS, L., Ass. fr. Av. Sei., Congr. Ajaccio, 1901, Recherehes sur les glandes,

venimeuses du Latrodectus tredecimguttatus.

26. BORDAS, L., Ann. Sei. Nat. Zool., 79, 147-164, 1905.

27. BERLAND, L., Rev. Sei., 65, 267-271, 1927.

28. ANCONA, L., An. Inst. Biol. (Méx.), 3, 77-84, 1931.

SciELO

10 11 12 13 14 15 16

L. ARVY

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Mem. Inst. Butantan

Simp. Internac.

33(3): 711-724, 1966

29. D’AMOUR, F„ BECKER, E., and RIPER, W. VAN, Quart. Rev. Biol., 11, 123-

160, 1936.

30. BÜCHERL, W. Mem. Inst. Butantan, 31, 77-84, 1964.

31. TROISE, E., C. R. Soc. Biol., 99, 1431, 1928.

32. SAMPAYO, R., J. Pharmacol. exp. Ther., 80, 309-322, 1944.

33. LEVY, R„ Ann. Sei. Nat. Zool., 1, 344-379, 1916.

34. BRAZIL, V., et VELLARD, J., Mem. Inst. Butantan, 2, 5-77, 1925.

35. CALVO, R., CHIONETTI, J., FASCIOLO, J., BANIA, A., PUEBLA, M., ZAN-

GHERI, E„ y FERNANDEZ, F„ Rev. Soc. argent. Biol., 33, 309-319, 1957.

36. CICARDO, V., Rev. Soc. argent. Biol., 30, 19-24, 1954.

37. DENIS, J., Buli. Soc. entomol. Nord-Fr., Suppl. 30, 1-15, 1947.

38. GAUBERT, P„ Ann. Sei. Nat. Zool., 13, 31-184, 1892.

39. GRASSO, A„ et TOSCHI-FRONTALI, N„ Boll. Soc. ital. Biol. sper., 39. 2079,

1963.

40. HOUSSAY, B. A., Buli. Soc. Patli. exot., 11, 217-239, 1918.

41. KAIRE, G. H„ Med. J. Aust., 50 (2), 307-311, 1963.

42. MARETIC, Z., und JELASIC, F., Acta Tropica, 10, 209-224, 1953.

43. MARETIC, Z„ Toxicon, 1, 127-130, 1963.

44. McCRONE, J. D., and PORTER, R. J„ Quart. J. Fia. Acad. Sei., 27, 307-310,

1965.

45. MILLOT, J„ C. R. Acad. Sei., 139, 119, 1929.

46. MILLOT, J., Buli, Soc. Zool, Fr., 60, 460-462, 1935.

47. OZANAM, CH., Étude sur le venin des arachnides, son emploi en thérapéutique

suivié d’une dissertation sur le tarentulisme et le tigretier, Bailiiere, Paris,

1856.

48. PHYSALIX, M., Buli, Mus. Hist. Nat., Paris, 132-134, 1912.

49. RAIKEN, A., Ann. Sei. Nat. Zool., 11, 5, 1839.

50. SCHIMKEWTTSCH, W., Ann. Sei. Nat. Zool, 17, 44. 1884.

51. SHAPIRO, H„ SAPEIKA, N„ and FINLAYSON, M., S. Afr. J. med. Sei., 4, 10-17,

1939.

52. SHULOV, A., and WEISSMAN, A., Ecology, 40, 515-518, 1959.

53. TROISE, E„ Rev. Soc. argent. Biol., 5, 605-615, 1929.

54. WILSON, W. H„ Rec. egypt. gov. School. Med. Cairo, 1, 141-150, 1901.

SciELO

10 11 12 13

Mem. Inst. Butantan

Simp. Internac.

33(3):725-734, 1966

ABDEM RAMÓN LANCINI VILLALAZ

725

9. CROTALUS VEGRANDIS KLAUBER, REDESCRIPCIÓN

Y DISTRIBUCIÓN

ABDEM RAMÓN LANCINI VILLALAZ

Museo de Ciências Naturales, Caracas, Venezuela

En 1941, Klauber (1) describió una nueva especie de serpiente de cascabel

de Venezuela, basado en dos ejemplares (Hololipo: Carnegie Museum, Pittsburgh,

n.° 17.384 y Paratipo: en poder dei Dr. L. M. Klauber, San Diego, Calif.).

Tanto ei holotipo como el paratipo, fueron colectados por Harry A. Beally en

1939. La procedência: “Maturin Savannah, near Cracoa, Sotillo District, State

of Monagas, Venezuela”.

En la diagnosis de dicba especie, Klauber senala que se diferencia de Crolalus

durissus terrificus y otras subespeeies dei grupo durissus, por tener un “pattern”

peculiar, en el cnal, las manchas dorsales roml>oidales están siempre obliteradas

por la presencia de un punteado de blanco en muchas escamas dorsales. Klauber

también dedujo que se trataba de una especie mucho más pequena que Crolalus

durissus terrificus debido a que, tanto el tipo como el paratipo, poseen los anilos

de la sonaja completos (10 segmentos en el holotipo y seis segmentos en el

paratipo) y, la talla de los mismos, es inferior a los C. d. terrificus con ese

mismo número de segmentos en la sonaja.

Más tarde, en 1956, el mismo Klauber en su magnífica obra: "Rattle-

snakes” (2), reconoce a Crolalus vegrandis como una subespecie válida dei grupo

durissus. Fué en 1958 cuando se vino a conocer un nuevo ejemplar de C.

vegrandis, donado por un comerciante al Parque Zoológico El Pinar, Caracas,

aunque sin procedência geográfica lamentablemente. El ejemplar en referencia

media poco más de 500 mm, fué fotografiado y luego, depositado en el Museo

de Biologia de la Universidad Central de Venezuela.

Anos más tarde, un comerciante colectó en El Tigre, Estado Anzoátegui,

Venezuela, un ejemplar de 640 mm. de longitud y, lo donó a los Laboralorios

Behrens, Caracas. El poblado de El Tigre dista unos 200 kms. en línea recta

al oeste de la terra typica: Cerca de Uracoa, Estado Monagas, Venezuela.

Todo esto desperto un gran interés entre nosotros y nos propusimos efectuar,

a fines de 1965 y princípios de 1966, una investigación sobre esta serpiente de

cascabel en su propia área de distribución. El presente trabajo es el resultado

de dicba investigación.

Material y métodos

En este trabajo se utilizan los métodos usuales empleados en sistemática de

serpientes. El material estudiado consta de una muestra de 55 ejemplares, re¬

partidos así: 28 machos (50,5%) y 27 hembras (49,5%), procedentes de 10

localidades diferentes, incluyendo la terra típica. En la muestra hay 51 ejem-

SciELO

10 11 12 13 14 15

726

CROTALUS VEGRANDIS KLAUBER, REDESCRIPCIÓN Y DISTRIBUCIÓN

|)lares adultos y apenas 4 ejemplares juveniles. Tamhién se incluyen eu este

trabajo los dalos dei holotipo y dei paratipo, dados por Klauber en la descripción

original. El autor tuvo el privilegio de examinar cl holotipo en el Carnegie

Museum, Pittsburgh, cuando visito los Estados Unidos durante el invierno de

1964, gracias a un “leader grant” concedido por el gobierno norteamericano.

Redescripción

Crotnlus vegrandis Klauber, 1941.

Crotalus vegrandis Klauber, Trans. San Diego Soc. Nat. Hist.,

9(30) :334, 1941.

Crotalus durissus vegrandis Klauber, Rattlesnakes, Tlieir Habits,

Life Histories and Influence on Mankind, 1:34, 1956.

Holotipo: Carnegie Museum

cerca de Iracoa, Estado Monagas

(véase Fig. 1).

Pittsburgh, n.° 17384, macho adulto, colectado

Venezuela, por Harry A. Beatly, en 1939

Fig. 1 — Aspecto dorsal dei Holotipo de Crotalus vegrandis Klauber, 1941. (Foto¬

grafia cortesia dei Dr. M. Graham Netting, Carnegie Museum. Pitts.).

Paratipo: L. M. Klauber collection, San Diego, Calif. n." 17385, hembra

juvenil, con los mismos datos de procedência y recolección que el holotipo.

6 SCÍELO 10 n 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33 (3): 725-734, 1966

ABDEM RAMÓN LANCINI VILLALAZ

727

Localidad típica: Sahanas cerca de Uracoa, Distrito Sotillo, Estado Mona-

gas, Venezuela, + 30 m. sobre el nivel dei mar.

Diagnosis: Una especie que se diferencia de las dei grupo durissus, por

tener una talla inuy reducida (684 mm. de largo total para el ejemplar más

grande conocido hasta ahora) y un “pattern” formado por dibujos romboidales

(27 a 33), obliterados por tener numerosas manchitas blancas en el ápice de

muchas escamas dorsales.

Lepidosis: La escamación dorsal varia de 25-25-19 a 29-29-21 (promedio

27-27-21). Las escamas dorsales están fuertemente carenadas, especialmente en

la parte anterior dei cuerpo, pero Ias primeras y segundas hileras dorsales

son lisas.

La cabeza es corta y triangularmente acorazonada. La rostral triangular,

tan alta como ancha; un par de internasales triangulares y un par de prefron-

tales alargadas y, ovaladas posteriormente. La región frontal está ocupada por

una escama grande básica o varias pequenas y, otra corta superpuesta anterior¬

mente; dos supraoculares normales y dos postsupraoculares. El resto de la parte

superior de la cabeza está formado por escamas pequenas y carenadas.

Las nasales divididas y los orificios nasales formados por las nasales poste¬

riores. Hay dos loreales en cada lado; dos preoculares, dos o Ires suboculares

y tres postoculares. Hay tres series de escamitas entre las supralabiales y la

órbita.

Las supralabiales en número de 12 a 16 (promedio 13). Hay 13 a 16

infralabiales (promedio 14). La escama mental es triangular, seguida de un par

corto y un par largo de geneiales.

La escamación ventral varia de 162 a 172 (promedio 165) en los machos

y de 163 a 178 (promedio 169) en las hembras. La escamación subcaudal

varia en los machos de 25 a 31 (promedio 27) y de 18 a 25 en las hembras

(promedio 21).

Las ventrales totales varían en los machos de 188 a 201 y en las hembras

de 183 a 202. Se observa un marcado dimorfismo sexual en la relación ven-

tral-subcaudal (vease Fig. 2), pero no en las ventrales totales.

Hemipenes: Similar al de Crotalus durissus cumanensis *, aunque propor¬

cionalmente más pequeno. Cada hemipene ocupa “in situ” un promedio de

nueve (9) escamas subcaudales. Órganos divididos eon “sulcus spermaticus” bi¬

furcados. Parte basal dei hemipene (40% dei órgano) formado por espinas

cortas, algo curvadas y de puntas dirigidas hacia la base; zona media y distai

formada por cálices (60% dei órgano) microornamentados por papilas. Final

distai dei ápice más o menos desnudo. El largo (comprimido) de los hemipenes

varia de 20 a 24 mm.

Sonaja: En cascabel (sonaja) es pequeno y formado por una serie de

segmentos que varían de 2 a 10, según la edad de los indivíduos.

En 1811, Humboldt describió dos especies de serpientes de cascabel (Crotalus cuma¬

nensis y Crotalus loeflingii, de Cumaná, Estado Sucre, Venezuela. Ambas especies

íueron colocadas más tarde en la sinonimia de Crotalus durissus terrificus. Reciente-

mente, Hoge, revalido el nombre Crotalus durissus cumanensis para la serpiente típica

cascabel de Venezuela (comuntcación personal).

SciELO

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CROTALUS VEGRAND1S KLAUBER, REDESCRIPCIÓN Y DISTRIBUCIÓN

VENTRALES

Fig. 2 — Dlmorfismo sexual en el número de ventrales y subcaudales en Crotalus

vegrandis. (Los puntos negros representan los machos y los puntos claros repre-

sentan las hembras.

Coloración: La coloración <le fondo varia de un gris verdoso claro a un

gris pardo ceniciento, que en aleohol o formalina, se torna más pardo. En la

calieza, la rostral siempre se halla liordeada de blanco; borde de las supralabiales

e infralabiales, de color blanco. Loreal y preocular superior e inferior de bordes

inferiores blaneos; rostro salpicado por manehitas blancas; canthales a veces

manehadas de blanco. Parte superior de la cabeza con varias estrias longitudi-

nales de manehitas blancas. A lo largo dei cuerpo hay 27 a 33 rombos alar¬

gados lateralmente y, más o menos visibles. Cada rombo está formado por el

blanco dei ápice de escamas dorsales. Las escamas de la l. a y 2. a hileras dor-

sales están totalmente bordeadas de blanco. Las escamas ventrales también se

encuentran nitidamente bordeadas de blanco. Las geneiales, guiares y primeras

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3): 725-734, 1966

ABDEM RAMÓN LANCINI VILLALAZ

729

ventrales sou completamente Mancas; cola gris obscuro en su totalidad. No se

observa dimorfismo sexual en la coloración. En los ejemplares juveniles las

sal picadoras Idancas son más acentuadas.

Dimensiones: La talla de Crotalus vegrandis es bastante reducida si se

toma en euenta que en la muestra el ejemplar más grande mide: 684 mm. (ma¬

cho). En el material examinado se encontraron apenas 4 ejemplares juveniles

(N. os 2892, T-9. T-6) y el paratipo (N.° 17885). No se conocen ejemplares in-

fantiles de esta especie.

Reproducción: Se encontraron 5 hembras prenadas en Diciembre 1965.

Son las siguientes: N.° 2582 (3 buevos), N.° 2346 (3 huevos), N.° 2305 (3

buevos), N.° 2196 (4 buevos) y N.° 2374 (4 buevos), lodos en formación.

Parece ser que el número de hijos es muy pequeno, en comparación con

otras especies dei género Crotalus.

Distribución geográfica y altitudinal: Crotalus vegrandis habita en los llanos

y mesas de los Estados Anzoátegui y Monagas (Venezuela), en una superfície

global de unos 20.000 kms. cuadrados, comprendida entre los paralelos 8° y 10°

de latitnd norte y entre los meridianos 62" y 65“ Oeste. La región está for¬

mada por llanuras y altiplanicies (mesas) dei Cuaternario. que van desde los 30

metros sobre el nivel dei mar (sabanas de LIracoal etc.) basta unos 300 a 500

metros de altitud en las altiplanicies aluvionales (Mesas de Lírica, Santa Bár¬

bara. etc.). Las llanuras arenosas están cubiertas por gramíneas y sometidas

por un clima ecuatorial de sabanas, con temperaturas medias anuales superiores

a 28"C. La precipitación es más o menos abundante, pero poco efectiva. Las

noches son frescas y búmedas.

Las barreras naturales en la dispersión de Crotalus vegrandis , las consti-

tuyen, por el norte, el Macizo Oriental; por el sur, el rio Orinoco y, por el

este, el Delta dei Orinoco. Los limites de distribución, por el oeste, son difíciles de

precisar, aunque sabemos que esta especie no ha sido colectada más alia dei

meridiano 65“. Es interesante senalar que, al parecer, existe una barrera ecoló¬

gica en la cuenca hidrográfica dei rio Lnare.

Hemos localizado la periferia septentrional de la distribución de Crotalus

vegrandis , a unos 36 kms. al sur de Maturín, Edo. Monagas, en un caserío deno¬

minado La Morroccya (véase mapa de distribución). En este caserío ya no se

eneuentra C. vegrandis sino C. d. cumanensis. Un poco más al sur de La

Morrocoya, las típicas sabanas de gramíneas, comienzan a sufrir Iransición hacia

la vegetación de chaparral, qne se extiende hacia el norte hasta cerca dc las

faldas dei macizo oriental.

C. vegrandis no ha sido eolectado en Uracoa ni en otros puntos de las

rnárgenes de los rios que cruzan el área <le distribución, porque las selvas de

galeria de estos rios no constituyen sn habitat.

Habitat: Crotalus vegrandis es una especie de hábitos vespertinos y se

eneuentra en las "matas” y sabanas que se extienden por los Estados Anzoátegui

y Monagas. AI parecer, se alimenta de pequenos lagartos ( Cnemidophorus

SciELO

10 11 12 13 14 15

730 CROTALUS VEGRANDIS KI.AUBER, REDESCRIPCIÓN Y DISTRIBUCIÓN

Fig. 3 — Aspecto dorsal de un ejemplar vivo de Crotalus vegrandis Klauber.

lemniscatus lemniscatus , Tropidurus torquatus hispidus, Hemidactylus rnabouia,

etc.) que abundan en la región. Los pequenos roedores (ratones, etc.) son nniy

escasos en estas llanuras. La coloración de Crotalus vegrandis es protectora

(colores claros dei animal sobre fondo arenoso), y le facilita la búsqueda dei

alimento.

Material examinado: En dos gráficos anexos (machos y hembras) se in-

cluyen los datos de cada ejemplar estudiado y para los Museos o personas que

los poseen se utilizan las siguientes abreviaturas: M.C.N.C. = Museo de Ciências

Naturales, Caracas; L.B.C. = Laboratorios Behrens, Caracas; I.M.T. = Insti¬

tuto de Medicina Tropical de la U.C.V., Caracas; l.B.H. = luslilulo Butantan

(Herpetología), São Paulo; C.M.P. ■ - Carnegie Museum, Pillsburgh; M.B.U.C.V.

= Museo (!<■ Biologia de la Universidad Central. Caracas; L.M.K. = Lawrence

M. Klauber; A. R. Iloge (cedido); A. R. Lancini (cedido); A. Pons (cedido).

Mapa. de distribución geográfica: En un tercer gráfico anexo, se destacan

triângulos negros, los puntos geográficos donde han sido colectados, hasta el

ejemplares de vegrandis. Las localidades de San Tomé (Edo. Anzoá-

Anzoátegui ) y .jíiiiiu udiiiaia ^iíuu. inuuaga

la cole

se pudo establecer la localidad exacta para

presente,

tegui), Santa Rosa (Edo. Anzoátegui) y Santa Bárbara (Edo. Monagas), corres-

ponden a especímenes de la colección dei Dr. Tidio Briceno Maaz. aunque no

establecer la localidad exacta para cada ejemplar.

SciELO

N.o

Museo

1961

M. C. N. C.

2520

M. C. N. C.

2069

M. C. N. C.

2081

M. C. N. C.

2118

M. C. N. C.

2148

M. C. N. C.

2075

M. C. N. C.

2150

M. C. N. C.

1936

M. C. N. C.

2036

M. C. N. C.

2127

M. C. N. C.

1939

M. C. N. C.

1493

M. C. N. C.

2577

M. C. N. C.

2070

A. Pons

1955

A. R. Hoge

8447

M. B. U. C. V.

A. R. Lancini

17384

C. M. P.

26103

I. B. H.

26101

I. B. H.

26102

I. B. H.

26104

I. B. H.

T-4

I. M. T.

T-6

I. M. T.

T-8

I. M. T.

MACHOS ( c ’)

material examinado

N.°

Museo

Localidad exacta

Colector y Fecha

cab

(mm)

cola

(mm)

corp

(mm)

total

(mm)

Dorsales

1961

M. C. N. C.

Uverito, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

615

682

27-27-21

164

191

13-13

14-14

2520

M. C. N. C.

El Calmán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

544

610

27-27-21

164

195

14-14

15-15

20159

M. C. N. C.

Uverito, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

611

684

27-27-21

165

193

13-14

16-16

20S1

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

28,5

564

630

27-27-19

169

197

13-14

14-14

2118

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

25,5

444

495

27-27-21

166

194

13-13

15-16

2148

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

27,5

526

584

27-27-21

162

1S8

13-14

15-15

2075

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

560

621

27-27-21

162

191

14-13

14-14

2150

M. C. N. C.

Das Pumarrosas, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

504

560

27-27-19

166

193

13-13

14-15

1936

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

27,5

56,5

523,5

580

27-27-19

164

192

12-12

14-14

2036

M. C. N. C.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Angel Delgado, 3-66.

447

500

27-27-19

166

193

12-13

13-15

2127

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

27,5

571

638

27-27-21

167

196

13-13

15-14

1939

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

27,5

569

638

27-27-21

165

192

13-15

15-16

1493

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

490

545

27-27-19

165

193

13-13

15-14

2577

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

44,5

442,5

487

29-29-21

170

197

13-13

14-15

2070

A. Pons

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

29,5

580

648

27-27-19

162

189

13-13

14-14

1955

A. R. Hoge

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

28,2

539

598

27-27-19

170

199

12-12

13-14

8447

M. B. U. C. V.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

535

600

29-29-21

165

195

13-13

A. R. Lancini

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

27,5

549

611

27-27-21

162

190

14-14

15-16

17384

C. M. P.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Harry A. Beatty, 1939

26,3

572

636

28-27-19

169

199

15-15

14-14

26103

I. B. H.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

515

585

27-27-21

166

194

13-13

14-14

26101

I. B. H.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

23,5

470

530

27-27-19

170

199

14-13

15-14

1 26102

I. B. H.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Angel Delgado, 3-66

530

600

27-27-21

167

195

14-14

15-15

1 26104

I. B. H.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

465

530

27-27-21

166

196

14-13

15-14

1 T-4

I. M. T.

Tulio Briceno Maaz,

555

630

26-26-20

163

189

13-13

15-16

1 T-6

I. M. T.

Tulio Briceno Maaz,

363

410

28-28-20

165

193

13-13

16-16

1 T-8

I. M. T.

Tulio Briceno Maaz,

567

626

27-27-19

164

189

12-14

14-14

1 S/N

I. M. T.

Tulio Briceno Maaz,

530

583

27-27-20

168

193

13-13

14-14

1 S/N

L. B. C.

EI Tigre, Estado Anzoátegui.

572

640

27-27-19

172

201

13-13

2 3

10 11 12

SciELO

L 6 17 18 19 20 21 22 23 24 25 26 27 21

HEMBRAS ( Ç ) 1

N.°

Musgo

Localidade exacta

Colector y Fecha

2392

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2532

A. R. Hoge

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65. !

2346

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2305

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2196

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2374

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2440

A. Pons

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2459

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2518

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2445

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2526

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2512

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2517

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

2543

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

8448

M. B. U. C. V.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

A. R. Lancini

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

T-l

I. M. T.

Tulio Bricefio Maaz,

T-3

I. M. T.

Tulio Briceno Maaz,

T-7

I. M. T.

Tulio Briceno Maaz,

T-9

I. M. T.

Tulio Briceno Maaz,

T-2

I. M. T.

Tulio Briceno Maaz,

Ex. 17385

L. M. K.

Maturin Savannah, n. Uracoa.

Harry A. Beatty, 1939

2173

M. C. N. C.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

494

M. H. N. L. S.

Tomblador, Estado Monagas

1296

M. C. N. C.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Angel Delgado, 3-66.

2032

M. C. N. C.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

26100

I. B. H

La Dominga, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

2 3

SciELO

3 11 12 13 14 15 16

HEMBRAS ( Ç )

MATERIAL EXAMINADO

N.o

Museo

Localidade exacta

Colector y Fecha

cab

(mm)

cola

(mm)

corp

(mm)

total

(mm)

Dorsales

2392

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

23,5

30,5

416,5

447

27-27-21

172

193

13-13

14-14

2532

A. R. Hoge

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

27,5

490

525

27-27-21

169

189

13-13

14-14

2346

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65,

24,5

27,5

467,5

495

27-27-21

171

191

13-13

14-14

2305

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

27,5

519

558

27-27-19

170

193

14-14

14-15

2196

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

526

561

25-25-19

169

190

13-13

14-14

2374

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

471

510

25-25-19

171

192

13-13

15-14

2440

A. Pons

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

521

559

27-27-19

167

190

13-13

16-16

2459

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

451

490

27-27-21

170

191

14-14

16-15

2518

M. C. N. C.

Paso Nuevo, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

26,5

471

510

27-27-21

167

189

13-13

14-14

2445

M. C. N. C.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

490

525

27-27-21

170

190

15-15

16-16

2526

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

560

606

27-27-21

171

196

14-14

15-15

2512

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

465

496

27-27-21

170

191

14-13

14-14

2517

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

38,5

561,5

600

27-27-21

170

191

13-13

15-15

2543

M. C. N. C.

El Caimán, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

513

546

27-27-21

174

196

14-14

8448

M. B. U. C. V.

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

556

600

27-27-21

172

196

13-13

14-14

A. R. Lancini

Sabanas de Uracoa, Distrito Sotillo, Estado Monagas.

Pedro J. Ricardi, 12-65.

501

540

27-27-21

168

190

12-12

13-13

T-l

I. M. T.

Tulio Briceno Maaz,

492

530

27-27-19

168

188

13-14

15-16

T-3

I. M. T.

Tulio Briceno Maaz,

540

575

28-28-19

170

190

14-13

14-15

T-7

I. M. T.

Tulio Briceno Maaz,

610

650

27-27-21

166

189

13-14

16-15

T-9

I. M. T.

Tulio Briceno Maaz,

415

440

28-28-20

163

183

14-14

15-15

T-2

I. M. T.

Tulio Briceno Maaz,

49S

532

27-27-20

166

1S8

13-13

16-14

E *. 17385

L. M. K.

Maturín Savannah, n. Uracoa.

Harry A. Beatty, 1939

23,2

422

454

27-27-19

172

194

14-14

15-16

2173

M. C. N. C.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

575

613

27-27-19

171

192

13-13

15-14

494

M. H. N. L. S.

Temblador, Estado Monagas

455

495

27-27-21

178

202

14-14

15-15

1296

M. C. N. C.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Angel Delgado, 3-66.

26,5

33,5

448,5

482

27-27-21

169

187

15-13

15-15

2032

M. C. N. C.

Las Pumarrosas, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

503

536

27-27-19

173

194

13-13

14-15

26100

I. B. H

La Dominga, Distrito Sotillo, Estado Monagas.

Pedro Castillo, 3-66.

28-28-21

164

185

14-14

15-14

2 3

10 11 12

SciELO

L 6 17 18 19 20 21 22 23 24 25 26 27 21

cola

(mm

30,5

27,5

38,5

33,5

XAMINADO

corp

(mm)

total

(mm)

Dorsales

416,5

447

27-27-21

172

193

13-13

14-14

490

525

27-27-21

169

189

13-13

14-14

467,5

495

27-27-21

171

191

13-13

14-14

519

558

27-27-19

170

193

14-14

14-15

526

561

25-25-19

169

190

13-13

14-14

471

510

25-25-19

171

192

13-13

15-14

521

559

27-27-19

167

190

13-13

16-16

451

490

27-27-21

170

191

14-14

16-15

471

510

27-27-21

167

189

13-13

14-14

490

525

27-27-21

170

190

15-15

16-16

560

606

27-27-21

171

196

14-14

15-15

465

496

27-27-21

170

191

14-13

14-14

561,5

600

27-27-21

170

191

13-13

15-15

513

546

27-27-21

174

196

14-14

556

600

27-27-21

172

196

13-13

14-14

501

540

27-27-21

168

190

12-12

13-13

492

530

27-27-19

168

188

13-14

15-16

540

575

28-28-19

170

190

14-13

14-15

610

650

27-27-21

166

189

13-14

16-15

415

440

28-28-20

163

183

14-14

15-15

498

532

27-27-20

166

188

13-13

16-14

422

454

27-27-19

172

194

14-14

15-16

575

613

27-27-19

171

192

13-13

15-14

455

495

27-27-21

178

202

14-14

15-15

448,5

482

27-27-21

169

187

15-13

15-15

503

536

27-27-19

173

194

13-13

14-15

28-28-21

164

185

14-14

15-14

SciELO

10 11 12 13 14 15 16

Mrm. Inst. Butantan

Simp. Internac.

3S<3):725-734, 1966

ABDEM RAMÓN LANCINI VILLALAZ

731

Fig. 4 — Vista dorsal de un topotipo de Crotalus vegrandis Klauber.

PrOPIEDADES DEL VENENO

El veneno de Crotalus vegrandis es de color amai i 11o y cuando se deseca

toma iin aspecto de color cromo con marcada tendencia a fiío y con tonalidades

verdosas de cádmio.

Cantidad media de extracción: Se utilizo una muestra de 30 ejemplares

de C. vegrandis, tomados al azar y, recién capturados (Una semana).

Se les extrajo el veneno por el procedimiento usual y suministraron en total

0.8 cc de veneno líquido, lo cual representa un promedio de 0.0266 ce de vene¬

no líquido de promedio para cada ejemplar.

Kl veneno, desecado en presencia de CaCK, dio un produeto final de

181 mgs, correspondiendo a cada ejemplar un promedio de 6,03 mgs de veneno

seco por extracción.

En la muestra, los 181 mgs de veneno sólido, representan el 22,625%

sobre el veneno líquido.

Determinación de la D.L.M.: Se determino la dosis

lomas adultas (250 a 320 gramos) y controladas durante

inyectadas. Se preparo una solución madre con

81.25 cc de solución fisiológica isotónica.

letal mínima en pa-

24 horas después de

16,25 mgs de veneno seco y

lo cual da una concentración de 1 mg

de veneno por cada 5 cc de solución. Con esta solución se hizo una dilución

1/10, lo cual dió una concentración de 1 mg de veneno en 50 cc de solución,

o sea, de 20 gammas por cc de solución.

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(3):725-734, 1966

ABDEM RAMÓN LANCINI VILLALAZ

733

Se inocularon las palomas por via i.v. (vena clel ala), con canticlades de-

crecientes de veneno (20, 15, 10, 5, 4, 3, 2, y 1 gamma, respectivamente) a

cada ejemplar, lo cual dió como resultado la muerte de las palomas que reci-

bieron entre 2 y 3 gammas a 20 gammas de veneno, sobreviviendo solamente los

ejemplares que recibieron 1 a 2 gammas de veneno de C. vegrandis, en las 24

horas siguientes a la inyección. Por tanto, podemos fijar la D.L.M. dei veneno

de Crotalus vegrandis en 2 a 3 gammas.

De terminación de crotamina: Se practicó la prueba de la detección de cro-

tamina, siguiendo los métodos empleados por Moura Gonçalves (3) y Schen-

berg (4). Se utilizo una solución de 0,5 mgs de veneno por cada cc de

solución fisiológica isotónica.

Se inyectó un centímetro cúbico de solución, por vía subcutânea, a 8 raton-

cilos blancos de laboratorio, de un peso promedio de 25 a 30 gms, no obser-

vándose parálisis dei tren posterior en un lapso de 20 minutos, después de

haberse practicado la inyección.Todos los ratones murieron en un plazo de 2

horas, excepto el n.° 1, que murió en 24 minutos.

Se concluyó que el veneno de Crotalus vegrandis es erotamino-negativo.

Discussion

.r - •

Crotalus vegrandis fue descrita por Klauber, en 1941, como una especie y

en 1956, el mismo autor la considero como una subespecie válida de Crotalus

durissus. No obstante, no liemos encontrado ejemplares intergrados en la peri¬

feria de su distribución. En el caserío La Morrocoya encontramos solamente

dos ejemplares típicos Crotalus durissus cumanensis y ningún ejemplar de C.

vegrandis.

Creemos que C. vegrandis se comporta como una especie alopátrica, que

ocupa un território exclusivo, porque su habitat no ofrece alimento suficiente a

C. d. cumanensis (barrera ecológica!. En cambio, C. vegrandis se alimenta de

pequenos lagartos, lo que satisface las exigências de su talla reducida.

Al contrario, es muy probable de que C. vegrandis penetre en el habitat

de C. d. cumanensis, especialmente por el oeste, y se comporte allí como una

especie simpátrica. Ello se explicaria debido a que en los llanos de chaparral

abundan tanto los pequenos roedores como los lagartos. Una investigación eco¬

lógica más exhaustiva contribuiría a esclarecer todo esto y permitiría establecer

hien la distribución de tan interesante serpiente de cascabel de Venezuela.

SüMMARY

There was studied a number of 50 specimens of Crotalus vegrandis Klauber.

1941, a rattlesnake proceeding from Uracoa, Sotillo District, State of Monagas,

Venezuela, which has been known only by its holotype and its paratype. From

this number 50,5% were males and 49,5% females, mostly adults. They were re-

described as a species and the following variabilities were given:

Dorsal scales:

Upper labiais:

Lower labiais:

Ventrals:

25-25-19 to 29-29-21 (médium 27-27-21)

12 to 16 (médium 13)

13 to 16 (médium 14)

162 to 172 (médium 165) in males

163 to 178 (médium 169) in females

í, | SciELO

734

CROTALUS VEGRANDIS KLAUBER, REDESCRIPCIÓN Y DISTRIBUCIÕN

The subcaudal scales vary from 25 to 31 (médium 27) in males and from 18

to 25 (médium 21) in femaies.

Sexual dimorphism was observed in lhe relation of the ventral-subcaudal

score. The relation of body-length to head-length was studied.

In this work the geographic distribution of Crotalus vegrandis was enlarged

and the problems of distribution limits were mentioned, which are between the

parallels 8 o and 10° north and the meridians 62" and 65", west longitude.

This species can be found in the savannas and alluvial plains of the States:

Anzoátegui and Monagas, Venezuela. The climate of this region is macrothermic

equatorial of savannas, with a médium annual temperature higher than 28°C.

Studying some properties of this snake’s venom, it has been verified that it

ih yellow, crotamin-negative and its M.L.D. is of 2 to 3 micrograms injected intra-

venously.

It is discussed if it is a species or subspecies of Crotalus durissus and it can

be conciuded that probably it is an alopatric species, since it excludes from its

territory other rattlesnakes ( Crotalus durissus cumanensis) by alimentary com-

petition.

Agrudecimientos — Debemos manifestar nueslros más sinceros agradecimientos

a las siguientes personas e instituciones: Dres. M. Graham Netting, Director y Neil

Richmond, Curator, dei Carnegie Museum, Pittsburgh, quienes generosamente sumi-

nistraron varias fotografias dei holotipo y permitieron su examen; Dra. Carmen

M. Antonetti, médico de la U.C.V., quien efectuó el estúdio dei veneno de Crotalus

vegrandis, cuyos resultados se incluyen en este trabajo; Dr. A. R. Hoge, Jefe de

la Sección de Herpetología dei Instituto Butantan, São Paulo, quien, durante su

permanência en Venezuela, nos acompahó a la localidad típica y nos oriento sobre-

manera; Dres. J. A. de Argumosa y Guenther Berthold, de los Laboratorios Behrens,

Caracas, por su amable colaboración en todo lo referente al veneno; Drs. Félix

Pifano y Alejandro Mondolfi, dei Instituto de Medicina Tropical de la U.C.V., por

habernos permitido examinar la Colección Briceho Maaz; Dr. César Alemán, dei

Museo de Historia Natural La Salle, quien nos permitió examinai- un ejemplar

depositado en dicho Museo; Dr. Tulio Briceno Maaz, quien nos suministró la pro¬

cedência de algunos ejemplares de su colección; Oscar Alemán y Nelson Gómez,

quienes colaboraron en el procesamiento de datos cuantitativos; Carlos Rivero,

quien colaboro en la preparación de mapas y gráficos, Dra. Adelaida G. de Diaz

Ungria, dei Museo de Ciências Naturales, Caracas, quien nos ayudó siempre con sus

valiosos consejos; Pedro J. Ricardi, colector de la mayoría de los ejemplares es-

tudiados.

Por último, debemos hacer extensivo nuestro reconocimiento al Instituto Na¬

cional de Cultura y Bellas Artes, por sua ayuda económica en esta investigación.

Bibliografía

1. KLAUBER, L. M., Trans. San Diego Soc. Nat. Hist., 9 (30), 333-335, 1941.

2. KLAUBER, L. M., Rattlesnakes, Their Habits, Life Histories and Influence on

Mankind, Vol. 2, 1476 pp„ 1956.

3. MOURA GONÇALVES, J., An. Acad. bras. Cienc., 22, 140, 1950.

4. SCHENBERG, S., Science, 129, 1361-1363, 1959.

5. ARAÚJO, P. e BELLUOMINI, H. E„ Mern. Inst. Butantan, 39, 133-142, 1960-62.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3):735-744, 1966

M. LATIFI, A. R. HOGE and M. ELIAZAN

735

10. THE POISONOUS SNAKES OF IRAN

M. LATIFI*, A. R. HOGE** and M. ELIAZAN*

Department of Herpetology and Antivenin of Razi Institut, Iran

Department of Herpetology of Instituto Butantan, Brazil

During the years 1960-1905 identification of more than 10 000 snakes was

made in Razi Institute. This work deals with the poisonous snakes (including

OPISTHOGLYPHA) captured in various loealities of Iran.

Included are 17 species and subspecies with a distribution rnap containing

loealities of the examined material; also a graph showing the number of snakes

collected during a period of five years. Zoological exploration of Iran is far

from complete and much laxonomic work is lo be done.

OPISTHOGLYPHA

Boiga trigonata melanocephala (Annandale): Sag-mar

Head very distinct from neck, one praeocular nearly extending to the upper

surface of the head; two postoculars (sometimes 2 + 1); scales smoolh, more

or less obliqúe with apical pits, in 21 rows, vertebral row enraged narrowing

lalerally, 8 upper labiais, third, fourth and fifth entering lhe eye, eleven lower

labiais; temporais 2 + 2 or 2 + 3. Ventrals 216-229 in males, and 232 to 236 in

females, subcaudals 74-88 in males and 78 to 96 in females. Yellowish olive

or pale brown, with a white black-edged zigzag band along lhe back, or with

a dorsal series of white, black-edged spots, head blackish, helly white, with or

without a series of small brown spots along each side.

Total length: 1120 mm., tail: 170.

Loealities: Kerman, Balouchestan and Sistan.

Malpolon monspessulanus insignitus (Gcoffroy Saint Hilaire) : Yelé-mar

Head long, angular, 8 upper labiais, fourth and fifth entering the eye, 10

or 11 lower labiais, two loreals, frontal very narrow, one large praeocular, reach-

ing the frontal, temporais 2 + 3. Dorsal scales in 17 rows, ventrals 169-183,

subcaudals 63-93 in females. Brown, olive, green, gray or bluish gray, with dark

lighll >rown edged spots.

Sometimes scales with blackish and whitish spots. Belly white or yello¬

wish, punctuated with black and marked with large yellowish spots.

Total length: 1360 mm., tail : 290.

Loealities: Azerbaijan, Central State, Kermanshahan and Khuzistan.

SciELO

10 11 12 13 14 15 16

736

THE POISONOUS SNAKES OF IRAN

Malpolon moilensis IRouss): Yelé-mar

Head long and thin, with angular conthus, 10 or 11 lower labiais, 8 or 9

upper labiais, the fourlh and fiflli entering the eye. Frontal as broad as the

supraocular, one loreal as long as deep or deeper than long. One praeocular not

reaching the frontal, three postoculars, temporais 2 + 2 or 2 + 3. Dorsal scales

in 17 rows, ventrals 173-178 in females. Snbcandals 53-58 in females. Brown,

yellow or sandy gray, spotted darker, tvvo obliqúe brown or blackish bars on

each side of the head behind the angle of the niotith. Belly white or yellowish.

Total le n g t h : 1020 mm., t a i l : 120.

Locality : Khuzistan.

Psammophis schokari (Forskal)

Bostral broader than deep, visible from above, neck defined, internasals

much shorter than the praefrontals, the fang is followed by an interspace, one

praeocular and two postoculars, nine upper labiais, fifth and sixtb (rarely fourlh

and fifth) entering the eye, 10 or 11 lower labiais, temporais 2 + 2 or 3 (rarely

1 + 2). Dorsal scales in 17 rows, anal divided, ventrals 177-189 in females, sub-

caudals 85-125 in females. Yellowish, grayish, pale olive or reddish above. a

dark streak on each side of the head, passing through the eye, a white spotted

line between two brownish lines from occipital until lhe end of the tail. the center

of the supraocular white, surrounded by brownish color, two dark lines on each

side of lhe body. Belly usually white, yellowish. with dark s])ots.

Total le n g th : 1090 mm., tail : 360.

L o c a l i t i e s : Khorasan, Khuzistan and Kerman.

Psammophis lineolatus (Brandtl : Tier-mar

Snout moderately prominent, grooved above and outsides. Bostral broader

than deep, jnst visible from above, internasal much shorter than the praefrontal.

The fangs not separated from other teeth by an interspace. One praeocular and

two postoculars. Nine upper labiais; fourlh. fifth and sixtb entering the eye,

10 or 11 lower labiais. Temporais 2 + 2 or 2 + 3. Dorsal scales in 17 rows,

ventrals 178-195, subcaudals 76-97 in females. Anal divided. Yellowish or pale

gray above, with four olive hlack-edged stripes, the median pair extending until

the top of the head, the outer pair lo the noslrils. passing through the eye; a

dark median streak from the interorbital region to the oecipit. Belly white,

dotted with gray or olive, and with one or two dark lines on each side.

Total l e n g t h : 830 mm., / a i I : 200.

Locality : Khorasan.

Telescopus fallax iberus (Kichwald): Afyi-sousan

Head distinct from neck, rostral broader than deep. nasal semidivided,

vertically pupil, loreal entering the eye below lhe praeocular, which is in contact

with the frontal, 8 or 9 upper labiais, third, fourth and fifth entering the eye.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3):735-744, 1966

M. LATIFI, A. R. HOGE and M. ELIAZAN

737

Ten or eleven lower labiais, (rarely 12), one praeocular, two postoculars (some-

times 2+1), scales smoolh, obliqúe in 19 or 21 rows, ventrals 204-228 in males,

187-227 in females, subcaudals 61-67 in males, 50-64 in females; subcaudals in

Iwo rows. Anal single or divided, usually enlire. Dark gray, with belly almosl

uniformly black, pinkish brown, belly blotches lilack and white. 32-40 black

hars on lhe body, a lateral series of smaller s]>ots allernating with dorsais,

lower pari speckled whitish, spotted with dark brown.

Total le ng t h : 650 mm., t ai l : 120.

Local ities : Azerbaijan, Central State, Kerman-Shahan and Khorasan.

Telescopus tessellatus tessellatus (Wall): Afyi-sousan

8 or 9 upper labiais; third, fourth and fifth entering the eye, 10 or 11

lower labiais, the scales are in 19 or 21 rows, ventrals 215 to 261 in males, 211

to 244 in females, subcaudals 64 to 81 in males, 53 to 71 in females, anal

entire or divided. Il is possible, that some of the specimens here referred to

as tesselatus are T. martini Schmidt, but more specimens are needed to solve

lhe question.

HYDIIOPHIINAE

Hydrophis cyanocinctus (Peters) : Mar-mahi

Head moderate, body elongated. llostral slightly broader than deep; fron¬

tal much longer than hroad. One praeocular and two postoculars; seven upper

labiais, ten lower labiais, scales 41 round the middle of the body and 31 at

the neck. Two specimens were examined from CHAHBOHAR (Persian Gulf),

ventrals in one male: 410, and in one female: 365. Greenish olive above, with

dark olive or black crossbars or annuli, broader on the back, black annuli com¬

plete and connected by a black hand along the belly.

Total length: 1120 mm., tail: 100.

Local ity : Persian Gulf.

ELAPIDAE

Naja naja oxiana (Eichwald): Kafché-mar

Eye moderate, internasal as long as or shorter than praefronlal, one prae¬

ocular, 3 or 2+1 (rarely two) postoculars, temporais 2 + 3 or 2 + 4 (2 + 5 or

2 + 6). Seven upper labiais; third and fourth entering the eye, eight lower

labiais. Dorsal scales smoolh in 21 rows. Ventrals 194 to 204 in males, 196

to 206 in females, subcaudals 57 to 69 in males, 51 lo 64 in females, subcaudals

all in two rows. Anal entire. Yellowish to dark brown or pale brown or gray

to blackish, no marking on the hood, one or more dark crossbands on the an¬

terior part of lhe belly; the young with dark rings.

Total length: 1710 mm., tail: 350.

Localities : Khorasan (Meshed ), Gorgan.

í, | SciELO

738

THE POISONOUS SNAKES OF IRAN

Walterinnesia aegyptia (Lataste) : Cobra

Heacl distinct from lhe neck, one praeocular resting on lhe third laliial, one

suhocular, two postoculars, lem|)orals 2+3, seven upper labiais, third and fourth

entering the eye; nine Iower labiais, scales smoolh anteriorly, feebly keeled on

lhe posterior part of the hody, strongly so on lhe lail, 23 rows across the body,

subcaudal single anteriorly, divided posteriorly. Ventrals 188 to 198 in females,

suhcaudals 42+1 to 47+1 in females. Characters differentiating Walter¬

innesia from Naja are as follows:

Anal plate divided in W alterinnesia, entire in Naja

2 — Posterior dorsal scales of body and lail distinctly keeled in W'alter¬

innesia , smooth in Naja.

3 — Subcaudals divided and entire in Walterinnesia, all divided

in Naja.

Upper surface dark brown, blackish, entire under surface dark brownish,

dack.

Total l e n g t h : 990 mm., t ai l : 115.

Local ities : Khuzistan and Fars.

VIPEKIDAK

Vipera ursinii subsp: Afyi

Snout ohtusely pointed, flat above, rostral as deep as broad. visible from

above, in contacl vvith two apical shields; supraocular well developed, separated

from frontal by two shields. Il is similar to Vipera ursini Renardi, hut the

hody is more lliick and the head is more distinct from the neck, nine or len

scales round the eye, five scales between the eyes, 9 upper lahials. Dorsal scales

in 21 rows, strongly keeled on lhe hack. Ventrals 130 to 136 in males, 128

to 134 in females, suhcaudals 22 to 33 in males, 26 to 36 in females. Anal

entire. Yellowish or pale brown, reddish above, a vertebral series of more or

less regular spots, transversely oval, some or all of which may be confluent and

lorm an undulous or zigzag band, two or three longitudinal series of dark

brown or blaek spots along the sides, lhe Iower ones on the outer row of scales,

an obliqúe dark stripe from lhe eye to the angle of the mouth. Ventrals and

subcaudals with transverse series of small white spots; gray, checkered with

blaek and white.

Total length: 420 mm., tail: 50.

Loca li ty : Alhorz mountains (Tehran).

A subspecies of V. ursinii ebneri was described from the Alhorz Mountains.

The validity of ebneri will be discussed in another paper.

Vipera lebetina obtusa (Dwigubsky) : korze-mar or Afyi

Snout rounded, rostral as deep as broad, reaching the upper surface of the

snout and contact with two or three apical shields. Upper surface of head co-

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 735 - 744 , 1966

M. LATIFI, A. R. IlOGE and M. ELIAZAN

739

vered vvith small subimbricate scales, whicli are all more or less distinctly keeled.

7 lo 12 longitudinal series of seales between the eyes, supraocular narrow, usually

broken up into two or more small shields, 12 to 19 scales around the eyes, tvvo

or three series of scales between the eye and the labiais, upper labiais 10 to 11,

lower labiais 12 to 14. Dorsal scales in 23 or 25 or 27 rows (usually 25 rows),

strongly keeled. Ventrals 126 lo 278 in males and 151 lo 181 in females,

subcaudals 35 lo 53 in males and 39 to 51 in females. Anal entire. It seems

that V.l. schweizeri, turanica, mauritanica, deserti and euphratica are nearly

the same as V .1. obtusa. The very variable coloration and size depends upon lhe

localities, climate and geographical conditions.

Gray, olive, or brown, with darker dorsal hlotches and lateral spotling;

usually grayish buff or pale brown above with dorsal series of darker spots

which may stand in pairs, alternate or unite to form cross-bars and a lateral

series of large dark spots or bars. A more or less distinct dark hand on each

side of the head, passing through the eye and than extending lo the neck. Some-

times dark dots or spots on the head present; belly white, speckled (powdered)

brown, with or without dark brown spots.

Total le n g t h : 1600 mm.. / ail : 200.

Localities : lt is a common poisonous snake in Iran. nearly distributed

in every part: Azerbaijan, Kordestan, Tehran, Lorestan. Kermanshahan, Khuzis-

tan, Khorasan, Kerman, Balouehestan and Sislan.

Vipera xanthina subsp: Afy

i atnorzi

Head covered with feebly keeled scales; supraocular well developed, erectile,

the free edge angular, separated from the eye by small scales. 13 or 14 (rarely

12) around the eye, two series of scales between the eye and labiais, nine or

ten (rarely eight) upper labiais, 11 lo 13 (usually 12) lower labiais, 7 to 10

scales between the eyes. Dorsal scales in 23 rows strongly keeled; ventrals 162

to 171 in females, subcaudals 27 to 34 in females. Anal entire. Pale brown or

grayisb above, with dorsal series of somewhat lighter reddish roundish spots, which

are dark-edged on the sides; lhese spots may be in pairs or alternative; two dark

lines on lhe back of the head and a dark streak behind lhe eye, in some speci-

mens pale brown or grayish above, with a dark brown, reddish vertebral line

from the end of the head along the dorsal until the end of the tail; belly

yellowish beneath, powdered with black, each shield with a transverse series of

black and white spots.

Total le n gt h : 700 mm., / a i I : 35.

Locality : Alborz mountains (Tehran).

Vipera pérsica pérsica (Duméril, Bibron and Duméril) : Mar-shakhadar

Snout very short and hroadly rounded, supraocular "horns” above the eye

present. 15 lo 19 (usually 17 to 19) scales around lhe eye, three series of

scales between the eye and labiais, one series of scales between nasal and rostral,

SciELO

10 11 12 13 14 15 16

740

THE POISONOUS SNAKES OF IRAN

12 to 13 (usually 12) scales between the eye. Dorsal scales 23 to 25 (usually

23) rows at mid-body, keels of lateral scales not serrated; ventrals 150 to 155,

subcaudals 39 to 49. Anal entire.

Grayisb or brownish or pale ycllowish brown above, with four series of

large dark spots, the tvvo median sometimes confluent and forming transverse

darker bars across the back; sides of the head darker brown, due lo two very

obscure obliqúe dark bars below the eye; ventral surface uniform pale straw

color, sometimes whitish beneath, dotted, with dark and a lateral series of

dark spots.

Total length: 850 mm., tail: 110.

Localities: Khorasan, Balouchestan and Sistan.

Echis carinatus pyramidum (Geofftoy Saint-Hilaire) : Mar dzafari or kockmar

Snout very short, rounded, head covered with keeled scales, and very dis-

tinct from the neck, a narrow supraocular present, 10 to 12 upper labiais; 11

to 13 lower labiais; 15 lo 19 scales around the eyes; usually two series (rarely

three or one) scales between the eye and upper labiais, 11 or 12 scales across

from eye to eye. Dorsal scales 31 to 37 (usually 37) rows round the middle of

lhe body, and 27 at the neck; ventrals 170 to 183, subcaudals 26 to 37 in a

single row. Anal entire. Yellowish,

jrayish, br

rown or

mish

gray

/ith

longitudinal series of whitish, dark-edged spots; enclosing a round dark brown

lateral one, a cruciform shaped whitish mark present on the head. llelly uni¬

form whitish, speckled with brown dots, or with small round black spots.

Total length : 660 mm., t a i l : 65.

Localities : Khorasan. Kerman. Fars, Khuzistan, Balouehistan and

Sistan.

CROTALINAE

Agkistrodon halys caucasicus (Nikolsky) : Afyi kafkazi

Snout ohtusely pointed, slightly turned up at the end. roslral as deep as

broad or broader than deep; a ])air of internasals, and a pair of praefrontals;

frontal as long as or little shorter than the parietal, upper praeocular separated

from posterior nasal hy a loreal; one praeocular, one subocular, two postoculars;

loreal pil separated from labiais; three large teni|)orals, 7 or 8 (usually 8)

upper labiais, scales keeled in 23 rows; ventrals 149 to 166; subcaudals 31 to

56 pai rs.

Yellowish. grayish. reddish or pale brown above, with darker spots forming

transverse cross-bars; two lateral streaks on the back of the head; lips speckled

with brown ;lower parts whitish, more or less speckled with gray or brown.

Total length: 560 mm., tail : 70.

Locality : Alborz Mountain (Tehran).

10 11 12 13 14 15 16

I Hem. Inst. Butantan M. LATIFI, A. R. HOGE and M. ELIAZAN

II Simp. Internar.

I 33(3):735-744, 1966

741 ,

1 Acknowledgements — The authors are grateful to Prof. Dr. M. Kaveh, General

E Director, Dr. H. Mirchamsy, Assistant Direclor of Razi Institute, for their encou-

| ragements and for providing us the facilities and materiais needed for this work. i

■ We wish to extend our thanks to Prof. Dr. A. Rafyi, Dean of Veterinary College, 1

| Prof. Dr. N. Ghaffary, Biology Dept. of Medicine College, University of Tehran,

1 for their encouragements and valuable help and interest. We are grateful to |

■ Mr. J. C. Battersby (Brit. Mus. Nat. Hist. London), for his useful data and helpful !

I comments. Also we are thankful and indepted to the following friends and colle- 1

I agues: Dr. R. Farzanpay, Mr. A. Nafici and Mr. H. Darakhshani for their help and

I assistance.

References

ANDERSON, ST. C., Proc. Acad. Sei., Ser. 4, 31 (16), 417-498, 1963.

BOULENGER, G. A., Catalogue of the snakes in the British Museum

ral History), Vol. 3, London, 1896.

(Natu-

CORKILL, N. L., J. Bom. Nat. Hist. Soc., 35 (3), 1932.

CORKILL, N. L., Indian J. med. Res., 20 (3), 1933.

1 5.

FITZSIMONS, F. W., and VÍVIAN, Snakes of Southern África, McDonald,

London, 1962.

FORCART, L. ; Verh. Naturforsch. Ges. Basel, 61, 1950.

GUENTHER, A. C., The Reptiles of British índia, London, 1864.

GUIBÊ, J., Buli Mus., Ser. 2, 29 (2), 1959.

HHALAF, K. T., Reptiles of Iraq, with some note on the Amphibian,

1959. I

10.

HYMEN, M„ Field. Mus. Nat. Hist. Zool., Ser. 18, 39, 1958.

11.

HYMEN, M., and GEORGE, B. R., Field. Mus. Nat. Hist. Zool., Ser.

1965.

21, 44,

12 .

MAX, H„ Field. Mus. Nat. Hist. Zool., Ser. 16, 34, 1953.

I 3 .

MERTENS, R., Rev. Fact. Sei. Univ. Istambul, (B), 17 (1), 1952.

14.

MERTENS, R„ Rev. Fact. Sei. Univ. Istambul, (B), 18 (3-4), 1953.

15.

MERTENS, R., Jb. Ver. Vaterl. Naturk, Würtenberg, III Jahrgang,

Heft, Stuttgart, 1956.

Erstes j

16.

POPE, C. H„ Amer. Mus. Nat. Hist., N.Y., 1935.

17.

SCHMIDT, K. P., Field. Mus. Nat. Hist. Zool,, Ser. 7, 24, 1939.

18.

STEJNEGER, L., Buli, U.S. Nat. Mus., 58, 1907.

19.

TERENTEV, P. V., and CHERNOV, C. A., Reptiles and Amphibians from the 1

USSR, 1940. 1

20.

WERNER, F., Verh. zool. bot. Ges. Wien, 67, 1917.

21.

WETTSTEIN, O., S. õst. Akad. Wiss. math-nat. Kl., Abt. I, 160, 1951.

SciELO

10 11 12 13 14 15 16

THE POISONOUS SNAKES OF IRAN

3500

3000

2000

1000

Graph showing the number of snakes collected (1960-1965)

Harmless snakes

Poisonous snakes

Each millim. 20 snakes

SciELO

10 11 12 13 14

DISTRIBUTION OF POISONOUS SNAKES IN DIFFERENT STATES OF IRAN

a — Malpolon

b — Telescopus fallax iberas

c — Telescopus tessellatus tessellatus

tf — Psammophis schokari

e — Psammophis lineolatus

f — Boiga trigonata melanocephala

g — Hydrophis cyanocinctus

h — Vipera ursinii subsp.

i — Vipera lebetina obtusa

j — Echis cari natas pyramidum

k — Vipera pérsica pérsica

1 — Naja naja oxiana

m — Vipera xanthina subsp.

n — Agkistrodon halys caacasicas

o — Walterinnesia aegyptia

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internar.

33(3):745-760, 1966

M. STANIC

745

II. DIE SANDOTTER, VIPERA AMMODYTES AMMODYTES LINNAEUS

M. STANIC

Schweizerisches Serum & Impfinstitut, Bem, Schweis

Geograehische verbreitung

Von den Giftschlangen Ieben in Euroj)a nur die

Eamilie Viperiden

gehürenden ViiPERiNAE-Arten. Eine der grõssten und gifligsten Arten ist di

Vipera amnwdytes ammodytes Linnaeus, die Sandotter (V.a.).

Die Heimat der Sandotter ist die Balkanhalbinsel, besonders Jugoslawien,

und zwar die Rrovinzen Herzegowina. Dalmatien, Montenegro und das alpinische

Slowenien (Ahb. 1). Man findet sie auch in den westlich und nordlich an-

grenzenden Berglãndern wie Oesterreich, Italien, Rumãnien und Bulgarien. Es

soll noch erwàhnt werden, dass man ihr auch in den bayerischen Alpen begegnet

ist. In Albanien und Griechenland wird sie von der ihr sehr àhnlichen Vipera

ammodytes meridionalis Boulanger abgelcist. Auch in Nordafrika, in Algerien,

ist die Vipera ammodytes gefunden worden (1, 2, 3, 4, 5).

Abb. 1

Geographische Verbreitung der Vipera ammodytes in Jugoslawien

und angrezenden Lándern.

SciELO

746

DIE SANDOTTER, VIPBRA AMMODYTES A MMODYTES LINNAEUS

Die Namen Vipc.ru urnmodyles sowie Sandotter sind für diese Schlange

unpassend. da sie auf sandigem Gelande nic vorkommt. Besser wiirde der Name

Karstviper, Vipera saxosa, saxea, zutreffen, wird doch das Karstgebiet vou ihr

besonders bevorzugt. In der serbokroatischen Sprache gibt es für diese Viper

iiljer 200 Bezeichnungen. Die haufigste ist “Poskok” — der Springer — eine

Bezeichnung, die ebenfalls nieht besonders zulrifft (6, 7, 8, 9). Diese Bezeich-

niing rührt von den zahlreichen Legenden iiber diese Schlange her, die besa-

gen, dass dch die Schlange aus der Angriffsposition mit einem Sprung, Absprung,

auf den Menschcn wirft. Tomasini (6) fiihrle in die deutsche Terminologie

den Narnen Modrass ein, den er der slowenischen Sprache entliehen hat (modras).

Interessant ist, dass jede dieser 200 Bezeichnungen nac-h irgendwelehen Eigen-

schaften dieser Schlange gewahlt worden ist. So gibt es Namen, die die Mor-

phologie dieser Schlange hervorheben: Einrnal ist es die Zeichnung, dann die

Farbe oder die Aehnlichkeit mit der Umgebung. Weiter war für die Pragung

des Namens die Bewegungsart, das Verhalten, die Giftigkeit und zuletzt auch das

Zischen ausschlaggebend.

Morphologie

Die Vipera ammodytes ammodyl.es wird bis zu 90 em gross, sogar Exern-

plare von über einen Meter Kõrperlange sollen sehon vorgekommen sein(10).

Der Kopf dieser Schlange ist sehr charakteristisch; neben der ausgepriigten

herzfõrmigen Kontur des Kopfes hat die Schlange and der Nase eine mil einigen

Schnppen bedeekte Schriauzwarze, die oft ais Horn bezeiehnet wird. Daher auch

der irreführende Name “Hornviper” für diese Schlange, der eigentlich der nordafri-

kanischen Viper Cerastes comutas zukommt. Die Vipera ammodytes meridionalis

hat ein ahnliches “Horn”. Begegnet man also einer Schlange mit der beschrie-

benen Schnauzwarze, sei es in Europa oder Afrika, so steht man zweifellos einer

Giftschlange gegenüber!

Die Sehnauze wird vom Rüsselschild, Rostrale, gebildet, dann folgen links

nnd rechts je ein Nasorostrale, die bis an den Canthus rostralis reichen. lm

Gegensatz zur Vipera ammodytes meridionalis ist der Rostrum bei der Sandotter

relativ breit, kurz und ziemlich unregelmassig in seiner Forni (11).

Unler dem Nasorostrale beginnt eine Reihe von Supralabialia. Manchmal

findet man zwischen dem Nasorostrale und dem ersten Oberlippensehild eine kleine

Einlage, Squama praenasalis. Das Nasenschild, Nasale, ist ungeteilt. Das Na-

senhorn ist, wie sehon gesagt, ein aus weiehem Bindegewebe bestehender Nasen-

fortsatz, der mit 3-4 quergelegten Sehuppenreihen gepflastert ist. Das “Horn”

kann fast senkrecht (aufrecht) stehen, manchmal auch etwas nach vorn oder

sogar nach hinten gerichtet sein. Dieser Nasenfortsatz hat die Phantasie der

Serben und Kroatcn angeregt zur Schaffung einer ansehidichen Zahl von volks-

lümlichen Namen für die Vipera ammodytes ammodytes, wobei für den Forlsatz

alie moglichen Varianlen und Synonima wie Nase, Sehnabel, Horn und ãhnli-

ehes zur Anwendung kamen.

Ueher den Augen befindet sich der Scutum supraculare, der ziemlich gross

ist. Um die Augen herum befindet sich ein skleraler Ring, der aus zwei Reihen

konzentrisch angeordneter kleiner Schuppen gebildet wird. Die Pupille ist, wie

das für die Vipern charakterislich ist, eine vertikale Spaltpupille. Auf dem oberen

Teil des Kopfes findet man manchmal einen Scutum frontale und zwei Seuta

parietalia. Die untere Lippe hat in der Mitte einen Mentale und ein Kinnschild,

von weiehem aus sich zu beiden Seiten 4-5 Sublabialia ansehliessen. Unter und

SciELCVo

748

DIE SANDOTTER, VIPERA AMUODYTES AMMODYTES LINNAEUS

Gescheechtuche unterschiede

Die Unterscheidung des Geschlechtes ist ziemlich schwierig. Im allgemei-

nen gilt die Regei, dass das Mánnchen, lat. serpens mas, etwas grõsser ist. Die

Farbe der Schuppen am Rücken ist sehr oft grau oder braun, der Bauch

lichtgrau bis schwarz. Beim Weibchen ist die Zeichnung etwas verschwommen,

besonders in den kaudalen Partien. Das Mánnchen hat sehr oft an der Unter-

lippe, an den Sublabialia, Pigmentflecken. Aehnliche Pigmentflecken setzen sich

fort an den Kõrperflanken. Auch diese sind beim Weibchen weniger ausgeprãgt.

Die Schwanzspitze ist riillich, korallenrot, bis gelblich.

Das eigenartige Begattungsorgan des Mannchens, der paarig eingelegte,

vorstülpbare Penis, in der Schwanzwurzeltasche untergebracht, kann iiusserst selten

heobachtet werden.

Die Paarung

Die Paarung erfolgt irn spiiten Frühling. Auf sonnigen Felsen findet man

oft Schlangenknáuel, in. semet convolvi serpentis more, serpentes circumvolutae

sibi ipsae, das Mánnchen und das Weibchen in der Paarung begriffen, liegen

stundenlang intim aneinandergeschmiegt.

Nach vier Monaten wirft das Weibchen 6-16 vollentwickelte Junge, von

10-15 cm Grõsse. Die Jungen gleichen den Allen vollkommen und sind schon

votn ersten Tag ihres Lebens auf sich selhst überlassen. Sie ernahren sich durch

verschiedene Kiifer. Spater lernen sie auch allmiihlich junge Miiuse und Vogei

zu toten und zu verschlingen.

Der Biotop

Die Sandotter bevorzugt gebirgiges Gelande, besonders das trockene und

h« usse Klima in den kahlen Bergen des Karstgebirges. Dort sieht man, wie sie

sich zu Knaueln vermengt, pila serpentina, auf dem heissen Gestein sonnen.

Die Sandotter bewohnt die Berge Herzegowinas, Dalmatiens, des Montenegro.

Man findet sie auch in den Bergen Serbiens, Kroatiens (das Lika-Gebiet, Istrien)

und in den slowenishen Alpen. Die Abb. 3 zeigt uns das typische Gelande,

wo die V. ammodytes haust. Meistens ist das in den Hõhen bis zu 700 Metera,

doch wurde sie auch schon in 1700 Metera Hôhe heobachtet und gefangen ge-

nommen. In diesen Hôhen ist sie wohl auch die einzige Giftschlange. Die

Kreuzotter hingegen bevorzugt Taler und feuchteres Gelande wie Wiesen, Walder,

Gebüsch und die Nahe von Wasser. Damit soll aher niclit gesagt sein, dass auch

die Sandotter schon in den eben geschilderten und fiir die Kreuzotter charakte-

ristischen Gegenden gefunden worden ist. Sie klettert sogar auf Bãume, was

fiir den Menschen besonders gefahrlich ist. Dort hebt sie die Nester der Vogei

aus und verschlingt die Jungen. Von diesem Versleck aus lauert sie auf das

Vorbeifahren eines Bauernwagens, der mit Heu, Stroh oder Holz heladen ist.

So wird sie dann bis in den Bauernhof verschleppt. Dort, wenn sie kein Unheil

verursacht, begnügt sie sich mit Mãusejagd; ist jedoch eine standige Gefahr fiir

Mensch und Vieh. Mir wurde schon ein Fali mitgeteilt, wo sich eine Sandotter

von einem Asl aus auf einen vorbeiziehenden Reiter warf und ihn in den Nacken

biss; eine Bisstelle, die stets zu einem katastrophalen Ende fiihrt. In Gegenden,

wo diese Schlange haust, ist es also sehr gefahrlich, in einem Busch nach einem

í, | SciELO

Mem. Inst. Butantan

Simp. Internac.

3»(3) :745-760, 1966

M. STANIC

749

Abb. 3 — Typisches Wohngelande der Sandotter.

Vogelnest oder nach sonst etwas zu greifen. Oft tritt ein barfüssiger Hirlen-

junge, sein Hund oder das Vieh auf eine im Gras oder im Gerdll liegende

Sandotter.

Die Sandotter pflegt sich nicht weit von ihrer Unterkunft zu entfernen.

Die Behausung liegt unter einem Stein oder Felsen. Dort verbringt sie die

Halfte des Jahres, vom Herbst bis zum Frühling. Oft findet man ais Zeichen

ihrer Gegenvvart die abgestreifte Schlangenhaut.

Im allgemeinen ist die Sandotter ein angstliches Tier und verkriecht sich

sehr schneil, sobald sie einen Menschen wahrnimmt. Erst in der Todesangst beisst

sie zu. Bei gewissen Gelegenheiten zischell die Sandotter, so z.B. bei der Ver-

folgung eines Opfers, in Wut oder Angst ( sibilus , stridor serpentis).

Die Sth ei fu ng

Die Sandotter streift ihre Haut, exuvia serpentis seriecta vernatio, die

eigentlieh die oberste, keratinisierte Epithelschicht ist und mil der Zeit in tuto

desquamiert, mehrmals im Jahre und besonders wáhrend des Wachstums ab.

Sie beginnt mit der Streifung an der Schnauze, indem sie die Haut durch

rbsichtliche Reibung an festen Gegenstanden der Umgebung zerreisst. Diesem

Vorgang hilft sie durch Kriechen zwischen engen Stellen im Gestein so lange nach,

bis sie schliesslich die Haut umstülpen und abziehen kann. Gelegentlich beob-

achtet man, dass die Sandotter sogar das Wasser aufsucht, damit die Haut weicher

wird und so die Streifung schneller zustandekommt.

750 DIE SANDOTTER, VIPERA AMMODYTES AMMODYTES LINNAEUS

Bei der Betrachtung der Haut sehen wir deullieh die abgestossene Hornhaut,

wodurch uns das Phanomen des starren und “hypnotischen" Blickes der Schlange

verstandlich wird. Gesunde Schlangen streifen die Haut im ganzen ah. vviihrend

sich bei kranken die Hiiutung in Fetzen vollzieht.

Die Ernaehrl.ng

Die Nahrung der Sandotter besleht aus Mausen, Vogeln, Eideehsen, Maul-

würfen und manchmal sogar aus eigenen Verwandten. In der Abb. 4 bringen

wir ein Beispiel des Schlangenkannibalisrmis: eine Sandotter verscblingt eine

Abb. -1 - Foto eines "Kannibalen”.

andere, ein durchaus nicht kleineres Exemplar.

Falles (Abb. 5) gibi deutlich den geschliingelten

Interessanl ist indessen, dass der gierige Kannibal

Die Hontgenaufnahme dieses

Schatten des Opfers wieder.

nach diesem Mahl hald selbst

emaeearmen

ist. Eine ahnliche Erfahrung bat auch Dittmars gemaeht (12).

Der Giktarparat

Der Giftapparat der Sandotte

init ihrem Ausführungsgang,

iniindet, und dem Giftzahn,

viperae. Kalliariner hat in

besteht aus der Giftdrüse, Glandula venenosa,

der in die Duplikatur der Mimdsehleimbaut ein-

dens venenatus viperae, telum venejieum, telum

seinen ausführlichen Studien über die Entwickluna

SciELO

Mem. Inst. Butantan

Simp. Intcrnac.

33(3):745-760, 1966

751

Abb. 5 — Rõntgenbilrl des “Kannibalen”.

und den Ersatz der Giftzãhne. sowie auch über die Mechanik des Bisses, be-

richtet (13, 14).

Der Giftapparat dient der Schlange zuerst zur Sicherstellung ihrer Nahrung

und zur Tõtung der Lebewesen, die ihr ais natürliche Nahrung gelten. Erst in

zweiter Linie steht ihr der Giftapparat fiir die Abwehr und zum Angriff zur

Verfügung. Die Abh. 6 zeigt uns den Schadel einer Sandotter.

Die Giftdrüse ist eine Speicheldrüse, die anatomisch der Glândula labialis

superior entspricht (15). Auf der Mikrofolographie (Abb. 7) erkennt man deut-

lich die weiten Tubuli, deren Epithel relativ niedrig und zylindrisch ist. Der

Ausführungsgang dagegen ist mit eiii('m hohen zylindrinschen Epithel ausgestattet.

Der Giftzahn ist in eine Duplikatur der Mundschleimhaut eingehüllt. in die

sich der Ausführungsgang der Giftdrüse offnet. Von liier gelangt das Gift in

die obige Oeffnung des Zahnes und fliesst durch dessen zentral gelegenen Kanal,

der in eine nahe der Zahnspitze gelegene Oeffnung mündet, beim Biss tief in

die Wunde hinein. Diese Gifteinspritzung gleicht fast einer künstlichen Injektion.

Der Zahn wird bis zu 5 mm gross und in Ruhestellung durch ein Gelenk nach

hinten gerichlet; sonst konnte die Viper ihr Maul gar nicht sehliessen. Die

Auspressung des Giftes erfolgt durch kriiftige Kontraktion der Masseter-Muskeln.

Dem Mechanismus des Bisses liegt, wie man aus obiger, kurzer, schema-

tisierter Beschreibung entnehmen kann, ein sehr kom])liziertes durch eine Anzahl

von Reflexen bedingtes Muskelspiel zugrunde.

SciELO

Abb. 7 — Mikrofotogram <lcr Giftdrüse der v. ummodytes.

SciELO

752 DIE SANDOTTER, V1PERA AMMODYTES AMMODYTES LINNÀEUS

Abb. 6 — Schadel einer Sandotter.

Mem. Inst. Butantan

Simp. Internac.

33(3):745-760, 1966

M. STANIC

753

Die Sandotter heisst in Ruhestellung meist nur zur Abwehr. Falis sie ihr

Opfer verfolgt, kriecht sie ihm langsam nach, um aus einer giingstigen Stellung

heraus zuzupacken. Eine salte Sandotter kümmert sich üherhaupt nicht um eine

weisse Maus im Kiifig, die sich sogar vor der Nase herumtummeln kann.

> Beim Angriff wirfl die Viper den Kopf mit grosser Wucht vorwãrts, õffnet

das Maul, richtet die Giftzáhne auf, heisst zu, zuckt mit dem Kopf rückwiirts

lin cl liisst dann das Opfer wieder los. Sie bezweekt damit die Erweiterung

und die passive Auspressung des Giftes aus der Duplikatur iu

All das gesehiehl blitzartig, mit einer Fertigkeit, welche man

Natur der Schlange nicht zutrauen wiirde. Das gebissene Opfer,

z.B., lliiift nach dem Biss winselnd davon und sucht ihr Heil in einem

Versteek. Doch das Gift vvirkt rasch und tõdlich. Die Sandotter weiss das

und kriecht dem Opfer langsam nach. Dann beginnl die dramatische Mahlzeit.

Opfer wird vom Kopf her angefasst und langsam und schubweise verschlun-

Es ist fast unvorstellbar, wie weit die Schlange ihr Maul aufreissen kann.

was für ein grosses Opfer sie zu verschlingen vermag. In spãtestens 20-30

Stichwunde

Zahnkanal.

kriechenden

Maus

Das

gen.

und

Minuten

in Form einer

noch aus dem

für die Umwe

siel

man nur

langlichen

noch einen stark erweiterten Te iI des Schlangenleihes

Kontur und ais letzle Spur den Schwanz der Maus, der

Maul ragt. Nach solch einem Schmaus wird die Schlange triige,

lt ziemlich uninteressiert und kaurn noch angriffslustig. Ich hahe

auch schon heobachtet, dass eine sehr hungrige Sandotter sogar zwei Mãuse

hintereinander verschlungen hat.

Dlili ScH LANGENFANG

Für den Schlangenfang gibt es eine Anzahl verschiedener Gerate (siehe

Abb. 8), welche sich der Mensch in seinem milenischen Ringen mit den Gift-

schlangen konstruiert hat (16). Meistens sind es verschiedene Modifikationen von

Spaltzangen und Holzgabeln. Die Schlangen wurden jedoch am haufigsten mit

einer Keule oder einem Slock getotet. Mit dem Schuhabsatz eine Schlange zu

toten, gehõrte schon zu den Heldentaten.

Die Sandotter wird von der stets fortschreitenden Zivilisation mehr und

inehr verdrangt; sie flüchtel in die Berge, in die Hohe. Heute ereignen sich

Schlangenbisse beim Menschen sehr selten, etwas haufiger beim Vieh.

Die Sandotter war in den besagten Gegenden jahrzentelang eine wahre

Plage für die Bevolkerung. Wahrend der osterreichischen Okkupationsherrschaft

in den Gebieten Bosniens und Herzegowina wurden von deren Administration

sogar eine Belohnung für jeden überbraehten Schlangenkopf ausbezahlt. Laut

damaligen Statistiken wurden in den Jahren 1907-1911 780 Menschen gebissen.

wovon 40 starben und 53.378 Stück Vieh gebissen, wovon 10.712 Stiick eingingen.

In der erwahnten Zeitspanne wurden den Behõrden 790.612 Schlangenkopfe über-

gcben; eine imposante Zahl (1). Um so viele Schlangen zu fangen, bedarf es

Geschicklichkeit und guter Fanggerate, vor aliem aher ein tapferes Herz. Tapfer-

keit schon deshalb, weil sich die Menschen im allgemeinen vor Schlangen fürchten,

sogar vor toten Schlangen. Wie überall in der Welt ist die Schlange auch in

diesen Gebieten von viel Mystik umgeben.

Seitdem die Schlangen zur Giftentnahme massenhaft gefangen werden, sind

auch die Fanggerate wieder in die Mode gekommen und in ihrer Ausführung

vcrfeinert worden. Die Schlange muss unversehrt gefangen werden, da nur un-

beschadigte Exemplare angekauft werden.

SciELO

10 11 12 13 14 15 16

754

DIE SANDOTTER, V1PERA AMMODYTES AMMODYTES L1NNAEUS

Ohwohl (lie Schlangenjagd und der Schlangenfang ein gefahrlicher Beruf

sirul, liefert er den Bauern und llirten jener Gegenden ein belrachlliches Nehenein-

kornmen. So wurde im Laufe der Zeit eine Plage in einen gewissen Segen

umgewandell.

Abb. S — Die primitiven Fanggeráte.

Die Sandotter in der Gefangexsch\FT

In den Terrarien werden die Sandottern in Holz- oder Metallkafigen. mil

Glas kombiniert, gehalten. Die Temperatur rnuss stets auf 22-26°C bleilien, wenn

die Scblangen immer frisch, gesund und rege sein sollen. Das ist die Vorausset-

zung fiir eine õkonomische Giftausbeute. Oft werden am Plafond der Kiifige

starke Glühlampen angebracht, die den Sehlangen Sonnenschein vortauschen

sollen. Tatsachlich schlângeln sich die Vipern unter den Lampen und bilden

dort Schlangenknãuel und -teller. Damil sie sich noch wohler fühlen, legl man

trockene Baurniiste oder frische Fichtenáste sowic auch eine Schiissel mit Wasser

in die Kiifige.

Die Sandotter nimmt in der Gefangenschaft zuerst nur selten Nahrung.

Einige verharren in stetem Hungerstreik. Solche Exemplare leben monatelang,

ja bis zu einem Jahr nur von Wasser. Selbstverstiindlich magern sie sehr ab;

eine solche Sandotter erkennt man an der stark gerunzelten Haut am Rücken.

Die andern, welche sich mit der nenen Lebensweise abfinden. bckommen durch-

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan M. STANIC 7 EE

Simp. Internac.

.13(3) :745-760, 1966

schnittlich alie 14 Tage eine Maus. Das Wasser wird taglich gewechselt. In

(ler Gefangenschaft lebt die, Saudotter im Durchschnitl 2-4 Jahre.

Ich habe wiederholt versuchl, die Jungen, die in unserem Institut in Gefan-

genschaft geboren wurden, aufzuziehen. jedoch ohne Erfolg. Die Jungen nehmen

in der Gefangenschaft keine Nahrung zu sieh. auch wenn mau ihnen ihre natiir-

liche Nahrung anbietet. Der Versuch, sie künstlich mit Kafern. Mehlwürmern

u.a. zu füttern, scheiterte ebenfalls: entweder würgten sie die mit etwas Gewalt

verabreichte Nahrung zurück. oder sie starben am nãchsten Tag ohne anato-

mischen Befund! Auch das Einführen der Milch oder emer nahrhaften Brühe

mittels einer Sonde konnte diese Wesen nicht langer ais drei Monate am Lehen

erhalten.

Die Giftentnahme

Die Giftentnahme geschieht in zweiwochentlichen Abstãnden; die Abh. 9

zeigt uns diesen Vorgang. Die Schlange wird mil dem Daumen und Zeigefinger

der linken Hand dicht hinter dem Kopf angepackt. Im Zorn õffnet sie das

Maul, vor welehes man eine Petrischale hall. Mit dem Hand der Schale werden

mm die Ziihne berührt. Durch diesen Heiz wird reflektorisch das Giftausstossen

bedingt, ein his zwei Tropfen, in Form einer gelldieh zahen Flüssigkeit. lasst

man aus jedem Zahn in die vorgehaltene Petrischale fliessen. Die das Gift ent-

haltenden Schalen werden nachher in einem Exsiccator mit Chlorcalcium unter

Vakuum getrocknet. Im trockenen Zustand hal das Gift eine glitzernde zitro-

nengelhe Schuppen- und Pseudokristall-Beschaffenheit. Ein weisses Gift, wie es

Kornalik und Master kürzlich beschrieben haben, konnten wir nie heohachten (17).

(liftcntnahme.

SciELO

756

DIE SANDOTTER, VIPERA AMMODYTES AMMODYTES LINNAEUS

Die Sandotter gibi hei einer Giftentnahme ca. 20-40 mg Gift ah (Trok.cn-

substanz). Diese Schwankungen sind dem Gesundheitszustand, der Gròsse der

Schlange und der Saison unterworfen. An heissen Sommerlagen sind die Erlriige

arn ausgiebigsten.

Die Haeueigsten Frkrankungen deu Sandotter

In der Gefangenschaft erkranken die Schlangen wahrscheinlich am hiiiifigsten

durch die nicht immer sanften Manipulationen der Schlangenwarter bei der

Giftentnahme, wobei ali und zu ein zahn abgehroehen vvi rd. Dadurch kommt

es leicht zu ínfektionen der Zahnwurzel und auch der ganzen Mundschleimhaut.

Oft finden wir in dem entnommenen Gift ahgestossene oder ahgehrochene

erkrankte Schlange zeigt uns die Abh. 10, die nicht nur

Giftzahne. Eine

Abb. 10 — Kupt einer erkrankten V. ammodytes : zahnlos und geschwollen.

eine durch die Entzündung geschwollene und livide Mundschleimhaut. sondem

auch einen durch die Schwellung deformierten Kopf aufweist. Das Mikrofolo-

gramm der Abh. 11 zeigt einen Schlangen blutausstrich, in dem man deutlich

Streptokokken erkennt. Leider haben wir die Streptokokken nicht niiher identi-

fizieren kõnnen. Das Blutbild stammt von der Schlange, welche die ohen ge-

schilderte Krankheit hatte und wahrscheinlich an den Folgen einer Sepsis einging.

Burtscher (18) hat einen ahnlichen Fali beschrieben, in dem er einen zur

Gruppe des Bacillus liquefaciens gehorenden Keim für den inôglichen Errcger

hall (Abb. 11).

Die Schlangen tragen an ihrem Korper sehr oft Milben. Bei den Sektionen

findei man im Darm manchmal Bandwürmer.

SciELO

Mem. Inst. Butantan

Slmp. Intcrnac*.

33(3):745-760, 1966

M. STANIC

757

Abb. 11 — Mikrofoto des Blutes einer an Streptokokkensepsis

eingegangenen Sandotter.

Der biss der Sandotter

Der Schlangenbiss bei Mensch und Tier ereignet sich fast immer unerwartet

und blitzschnell. Gefahrdet sind die Bauern, Hirten, Touristen und spielende

Kinder, die Vogelnester ausheben oder ahnliche Streiehe ausüben. Das Vieb

ist beim Weiden gefahrdet, ebenso der Hund (besonders der Schiifer- oder

Jagdhund). Die bevorzugten Bisstellen beim Menschen sind die Beine, besonders

der Fuss, ferner auch die Hánde. Nach dem Biss sieht man zunáchst nur zwei

winzige, 5-6 mm voneinander entfernte Einstiche, die etwas bluten. Der Allge-

meinzustand des Befallenen verschlimmert sicb aber zusebends. Das gebissene

Glied schmerzt, schwillt an und wird rot. Hiiufig sieht man die roten Auslãufer

der enlziindeten Lymphl)ahnen. Der Patient weist eine Blasse der ganzen Haut

auf, zittert, bekommt Angstzustánde und Schweissausbrücbe, hat beschleuniglen

weichen Puis und bald darauf folgt der Kollaps. Der Atem wird kurz, schnell

und mübsam. Vielfach weint der Patient; auch andere Symptome einer gewissen

Reizung der Psyehe sind feststellbar. Es ist wahrscheinlicb nicht gerechtfertigt,

diese Angstzustánde ais simple Folge des iiberstandenen Schreckens aufzufassen.

Nacb einigen Stunden wird das Oedem enorm, der Umfang des betroffenen

Gliedes kann sich verdoppeln. Bings um die Bisstelle sieht man rõtlichblaue,

dunkle Flecken, verursacht durch den Blutaustritt ins Gewebe. Das Glied ist

kalt, und wenn dem Patienten nicht fachgemãss gebolfen wird. nekrotisiert die

Umgebnng der Bisstelle oder sogar das ganze Glied, vor aliem aber die distalen

Teile einer Extremitãt. Deswegen sind ofl Amputationen notwendig. Das

Allgemeinbefinden besserl sich nur langsam. Erfolgt der Biss ins Gesicht, liais

SciELO

758

DIE SANDOTTER, VIPERA AMMODYTES AMMODYTES L1NNAEUS

oder Nacken,

rasch ein.

schlimmern sich

setzl der Tod infolge eines sich akul entwickelnden Hirnodem

das Gift ungliicklicherweise direkt

die oben beschriehenen allgemeinen

tritt miter Alemstillstand ais Folge der Lahmung des

sind Todesfãlle glücklicherweise sehr selten. Oftmals

Giftquantum in die

durchdringen miissen

uns eine Frau, eine

eines Schlaneenbisses

Wimde, weil die Giftziihne die

und somil einen Teil des Giftes

Biiuerin aus dem dalmatischen

und verspâteter Behandlung ihren

sehr

in die Blutbahn, so ver-

Symptome und der Tod

Alemzentrnms ein. Doch

gelangt nicht das ganze

Kleidung des Betroffenen

abfangen. Abb. 12 zeigl

Carslgehiel, welcbe infolge

rechten Arm bis auf einen

restlichen Oberarmstumpf amputieren lassen musste. Es war schon eine weitge-

bende Nekrose des Armes eingetreten, verbunden mit einer Infektion und dro-

bender Sepsis. Eine Infektion der Wunde ist namlich haufig zu beobachten.

Das rasche Fortschreiten der Infektion ist durchaus verstãndlich, wenn man

bedenkt, dass das Gift der Sandotter reich an Hyaluronidase ist. Dem letzteren

ist zu verdanken, dass sich das Oedem so schnell entwickelt und so enorme

Dimensionen annimmt.

Abb. 12

- Bãuerln, der infolge des Bisses einer V. animo -

dytex der rechte Arm verloren ging.

SciELO

Mem. Inst. Butantan

Slmp. Internac.

33(3): 745-760, 1966

M. STANIC

759

Die Therapie des Schlangenbisses

Die Therapie richtet sich heute nach der einzigen und bewâhrten Methode,

die moglichst rasche Einspritzung von antitoxischem Serum, am besten homo¬

logem Antiloxin. Tatsãchlich ist es in den bedrohten Gebieten heute moglich,

dem Verunglückten sofort Serum zu verahreicnen. Es hat sich ais praktisch

und nützlich erwiesen, dass nicht nur jede Apotheke, jeder Arzt und Veterinar,

f-ondern auch der Lehrer. Pfarrer und Agronom in diesen Gehieten wenigstens

eine Ampulle Serum hat oder Bescheid weiss, vvo es sicher und rasch zu beziehen

isl. Audi die Touristeu haben sich die Gewohnheit angeeignet, in ihrem Ruck-

sack eine Ampulle antiviperines Serum und eine sterile Injeklionsspritze mit-

zutragen.

Die anderen therapeutischen Miltel verdienen meistens nur historisches Inte¬

resse. Was man hei einem Verunglückten nebst oder auch ohne Serum tun

kann und soll, ist die Bekampfung des Kollapses, allerdings nicht mit reichlichen

Gaben von Alkohol, wie es früher der Fali war. In grõsseren Mengen verah-

reicht. íõrdert er hloss den Kollaps. Soíortige Erweiterung und Oeffnung der

Bisstelle oder das Aussaugen des Giítes mit dem Mund hat nur einen relaliven

Wert. Das heste und sicherste Mittel hleiht, den Erkrankten mit oder ohne

sofort verahreichtem Serum schleunigst in ein Spital einzuliefern.

Literatur

1. CURCIC, V., Nase zmije otrovnice, Sarajevo, 1931.

2. SCHWARZ, E., Behringwerkmitt, 7, 159-355, 1953.

3. KRAUS, R., WERNER, F., Gifschlangen und die Serumbehandlung der Sclilan-

genbisse, G. Fischer Verlag, Jena, 1931.

4. BOQUET, P., Venins de serpents et antivenins, Ed. Médicales Ftammarion,

Paris, 1948.

SERGENT, E., Arch. Inst. Pasteur, Aigér., 25, 71-76, 1947.

TAMASINI, O., Wissenschaft. Mitt. Bosnien Herzegowina, 2, 630, 1894.

HIRC, M., Zbornik za nar. zivot i obicago juz., (Siavena), 1, 21, 1896.

HOVORKA, O., Glasnik zem. muz. Bosne i Herceg., 12, 134-135, 1900.

RADIC, DJ., Nase otrovne zmije i biljke, Beograd, 1909.

BOLKAY, S. u. CURCIC, V., Glanisk zem. muz. Bosne i Herceg., 32, 155-204,

1920.

11. BOLKAY, S., Glasnik zem. muz. Bosne i Herceg., 32, 1-9, 1920.

12. DITMARS, L. D., Snakes of the World, The MacMillan Co. New York, 1957.

13. KATHARINER, L„ Zool. Jahrbuch, Abt. f. Anat., 10, 55-92, 1897.

14. KATHARINER, L„ Biol. Zbl., 10, 45, 1900.

15. RADOWANOWITSCH, M„ Jen. Z. Naturwiss., 63, 559-616, 1928.

16. DOJMI, L., Priroda, 24, 1-5, 1934.

17. KORNALIK, F„ und MASTER, R. W. P., Toxicon, 2, 109-111, 1964.

18. BURTSCHER, J„ Zbl. Bakt., Abt, 2, 77, 544-551, 1929.

SciELO

10 11 12 13 14 15 16

Mcm. Inst. Butantan

Simp. Internac.

38(3):V61-7H6, 1906

II. E. BELLUOMINI, R. FRANCO DE MELLO, A. M. 7C1

PENHA e G. SCHREIBER ml

12. ESTUDO CITOLÓGICO E PONDERAL DO TESTÍCULO DE CROTALUS

DL/RISSUS TERRIFICUS DURANTE O CICLO REPRODUTIVO ANUAL

H. E. BELLUOMINI*, R. FRANCO DE MELLO*, A. M. PENHA**

e G. SCHREIBER***

* Instituto Butantan, São Paulo, ** Instituto Biológico, São Paulo e *** Instituto

de Biologia Geral da Universidade Federal de Minas Gerais, Belo Horizonte, Brasil

Os trabalhos sôbre o ciclo de maturação sexual das serpentes das regiões

temperadas são, relativamente, escassos. Em lai assunto foram consultadas as

publicações fundamentais de Volsõe(l), Fox (2) e H. Saint Cirons e colabora¬

dores (3, 4). Naquilo que diz respeito ao interessante campo de pesquisa dos

ofídios brasileiros, não se conhece absolutamente nada sôbre o assunto. Foi

com o fim de preencher a falta de dados, nesse campo de estudos, que colhemos

os testículos de uma série de indivíduos da cascavel. Crotalus durissus terrificus

(Laurentius) durante os anos de 1964 e 1965, no Biotério Experimental de Ser¬

pentes Peçonhentas do Instituto Butantan. Em cada mês do ano, foram colhidos

cêrca de 10 indivíduos. Os testículos eram pesados e fixados em Bouin. Cada

serpente foi pesada, individualmente e o comprimento total do seu corpo era

medido. Ante os dados obtidos foi analisada a variação anual do testículo da

seguinte maneira:

a) Estudo histológico da atividade espermatogenética;

b) Em cada grupo de indivíduos colhidos, no mesmo mês, foi determinada esta¬

tisticamente. a regresão entre o pêso dos testículos e o comprimento total

do corpo, considerando-se êste último parâmetro, como aquêle correspon¬

dente, aproximadamente, à idade do indivíduo;

c) Foi determinado o coeficiente de correlação entre o pêso testicular e o com¬

primento corporal dos indivíduos de cada grupo mensal.

Os resultados obtidos dêsse modo, estão apresentados na Tabela I e no

gráfico da Figura 1. Infelizmcnte, 110 momento da colheita do material não foi

conseguido o lugar de procedência da serpente, informação essa que poderia elu¬

cidar alguns dados anômalos desta pesquisa. Apesar disso, pela análise dos

dados obtidos, chegamos às seguintes conclusões:

1 . O pêso do testículo (média de mais ou menos 10 indivíduos em idades dife¬

rentes) varia durante o ciclo anual de um mínimo de 377 mg em Setembro

de 1964, alcançando o máximo de 1106 mg em Abril de 1965 e reduzin¬

do-se, novamente ao valor de 397 mg em Setembro de 1965;

Trabalho realizado com o auxilio do Fundo de Pesquisas do Instituto Butantan.

SciELO

10 11 12 13 14 15 16

762

ESTUDO CITOLÓGICO E PONDERAL DO TESTÍCULO DE CROTALUS

DURISSUS TRRR1F1CUS DURANTE O CICLO REPRODUTIVO ANUAL

TABELA I

Valores da média do pêso do testículo (Y), do comprimento do corpo (X), do coefi¬

ciente de regressão <b’), do coeficiente de correlação (r) e do número de indivíduos (n.).

O coeficiente de regressão (b') foi calculado na base de que ambas variáveis estão

sujeitas a êrro.

Setembro

1964

86,10

377,5

11,19

0,111

Outubro

”

82,83

595

84,44

0,577

Novembro

”

84,1

769

10,51

0,177

Dezembro

”

7S9.67

6,80

0,473

Janeiro

1965

86,2

923

46,67

0,457

Fevereiro

”

85,81

929

27,09

0,367

Março

”

87,8

987

58,47

0,664

Abril

86,8

1106,5

33,94

0,497

Maio

”

87,72

1014,54

6,06

0,244

Junho

”

88,08

790

39,19

0,642

Julho

”

88,60

844

2,46

0,027

Agosto

”

88,10

487,5

11,51

0,766

Setembro

”

88,90

397,5

4,84

0,093

Pêso testículo.

Pêso testiculo/comprimento.

Fig. 1 -

Variação do valor médio do pêso testicular e do coeficiente de regressão

durante os meses do ano.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3) :761-766, 1966

H. E. BELLUOMINI, R. FRANCO DE MELLO, A. M. 7 C 0

PENHA e G. SCHREIBER 1

2. Constala-se com algumas exceções (Outubro de 1964 e Junho de 1965) que

o coeficiente de regressão (1/) é mais elevado nos meses de espermatogê-

nese ativa que nos meses de repouso. Isto pode ser expresso da forma se¬

guinte: o aumento ponderai do testículo é maior nos meses de atividade

sexual que nos meses de repouso sexual, para um aumento unitário de com¬

primento do corpo (idade).

Devemos, aqui, considerar a significação “biológica” do coeficiente de regres¬

são: em geral êste coeficiente mostra a variabilidade do pêso do testículo em

relação ao crescimento corporal (idade). Nos meses de repouso sexual a va¬

riação é, em geral, relativamente pequena, mas no período da maturidade sexual,

intervém um fator novo, que altera complctamentc esta regressão, aumentando

enormemente a variação de pêso dos testículos, em função do comprimento do

corpo. Embora a análise histológica do material não esteja ainda completa,

podemos pensar que êste novo fator de variabilidade do pêso testicular seja o

crescimento do epilélio germinativo com a onda de espermatogênese que ocorre,

no período de atividade sexual. Não excluímos, igualmente outros fatores con¬

comitantes, como o acúmulo de sêmen e o fenômeno de embebição que podem

acompanhar a maturidade espermatogenética.

Não comentamos por enquanto, a variação do coeficiente de correlação (r)

que é extremamente variável durante os meses do ano por serem as amostras

mensais constituídas de indivíduos de diferentes procedências e. como já disse¬

mos a propósito da situação da espermatogênese, altamente variável entre os indi¬

víduos de um mesmo mês.

Um exame superficial da situação da espermatogênese, nos diferentes indi¬

víduos, durante vários meses, indica que embora haja uma notável variabilidade

de indivíduo para indivíduo, as serpentes colhidas, nos meses de Janeiro a Maio

mostram plena atividade espermatogenética, que está em declínio nos outros meses

do ano. Porém, nos meses de inatividade, alguns dos testículos colhidos apre¬

sentavam atividade espermatogenética. Esta variabilidade de situação histológica

do testículo de indivíduos colhidos, no mesmo mês, poderia ser explicada, even-

tualmente, pela diferente procedência das serpentes, pois a área de colheita do

Instituto Butantan cobre climas muito diversos.

Aqui, apresentamos duas microfotografias (Figs. 2 e 5) de testículos, res¬

pectivamente em repouso e em atividade espermatogenética. 0 estudo sistemá¬

tico do processo espermatogenético na cascavel, nos moldes da descrição das

etapas feitas nos mamíferos pela Escola de Leblon e Clermonl e por Roosen-

Runge, está em andamento. A aplicação dêstes princípios aos répteis com pe¬

ríodos anuais de variação de atividade espermatogenética exige um estudo mais

detalhado.

Podemos salientar, como conclusão geral, que êste estudo difere dos traba¬

lhos executados pelos autores precedentes, que levaram em conta somente as

variações estacionais do testículo. As pesquisas presentes combinam o estudo

da variação do testículo com a idade do indivíduo e com a maturidade sexual.

Êste modo de apreciar o problema nos permitiu averiguar a existência de dois

fatores de correlação: um primeiro fator que aparece, na regressão dos meses

inativos e que corresponde ao crescimento anual do testículo em relação à idade

e um segundo fator, que se sobrepõe nos meses ativos e que altera totalmente

esta regressão básica.

SciELO

10 11 12 13 14 15 16

SciELO

cm 1

764 ESTUDO CITOLÓGICO E PONDERAL DO TESTÍCULO DE CROTALUS

nURlSSUS TERRIFICUS DURANTE O CICLO REPRODUTIVO ANUAL

Fig. 3 —- Testículo de cascavel em plena atividade espermatogenítica.

Fig. 2 — Testículo de cascavel em repouso espermatogenétlco.

Mem. Inst. Butantan

Simp. Internac.

33(3):761-766, 1966

H. E. BELLUOMINI, R. FRANCO DE MELLO, A M 7CK

PENHA e G. SCHREIBER ‘ DO

SUMMARY

The cytological and ponderai variations of the testicle of the rattlesnake,

Crotalus durissus terrificus (Laurentius) taken from the Butantan Institute during

the year, have been studied from Septemher, 1964 lo September, 1965. Earh

month, about 10 individuais were studied. The regression line hetween the total

length (age) and the testicle weight was calculated.

The weight of the testicle increases from Septemher to April and decreases

from then onward. In the same way, the regression coefficient increases from

September to April and decreases during the inactive months.

The maximum of spermatogenetic activity is found during the period from

January to May.

Some considerations are made concerning the hiological significance of these

variations of lhe regression coefficient.

Bibliografia

1. VOLSÕE, H., spol. Zool. Mus. Haun. (Copenhagen), 5, 7-172, 1944.

2. FOX, W., J. Morphol., 90, 481-553, 1952.

3. SAINT-GIRONS, H., et KRAMER, E., Rev. suisse Zool., 70, 191-221, 1963.

4. SAINT-GIRONS, H., Ann. Sei. Nat. (Zool. et Biol. Animale), 5, 462-476, 1963.

5. DAVIS, O. L., Statistical Methods in Research and Production, 3rd. Edition,

Oliver and Boyd, Edinburgh and London, 1961, p. 205.

SciELO

10 11 12 13 14 15 16

Mcm. Inst. Butantan

Simp. Internac.

33(3): 767-774, 1966

P. J. DEORAS

767

13. PROBABLE SICNIFICANCE OF VENOM YIELD RECORD STUDIES

P. J. DEORAS

Haffkine Institute, Bombay, índia

The venom yield records are necessary for sera producing laboratories lo

knovv the output and numbers required for immunization. They are useful lo

tbe clinician to know the maximum amount of venom given by a snake so that

in lhe case of a snake bite he is prepared for neutralizing that much amount

of venom. Il is also necessary for a biochemist lo know the periodicity during

the year and the loxicily thereby, to plan experiments for enzymes.

In the production of venom by snakes some of the factors that are supposed

to affect the yield are, environment, sex, seasonality and frequency of milking;

provided there is nniformity of feeding and age.

This Institute lias been working on these aspects for some years and

Deoras (1) gave the description of a snake farm wherein Naja naja, Vipcra

russclli, and Bungarus coeruleus were kept, after giving them a habitat which

vvas seen for these snakes in nature. The snakes were milked for venom from

the snake farm and this yield after lyophilization was compared to similar yield

by the same snakes kept in cages in the room. The results of the comparison

of these venom yield records have been given in the same paper and in the

proceedings of the symposium held al lhe Pan Pacific Science Congress (2).

In lhese comparative studies specific attention was given to taking different

number of samples and sexes separately. The results of these individual studies

showed that the output of venom in the farm by a cobra and Krait snakes bolh

for male and female sexes was more than in rooms. This was not the case with

the viper as is shown by table below which indicates significance or otherwise

TABLE I — STATISTICAL ANALYSIS OF AVERAGE VENOM PRODUCTION DATA TO

SHOW WHETHER THE ENVIRONMENTAL DIFFERENCE IS SIGNIFICANT OR NOT

Year of

Production

M/F

Cobra

(Naja naja)

M/F

R. Viper

(Vivera

russclli)

M/F

Krait

(Bungarus

coeruleus)

1953

Vf-Vr

Significant

Not significant

Vf-Vr

Significant

1954

Vf-Vr

Significant

Not significant

1955

Vf-Vr

Significant

Vr-Vf

Significante

Not significant

1956

Vf-Vr

Significant

Not significant

Vf-Vr

Significant

SciELO

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768

PROBABLE SIGNIFICANCE OF VENOM YIELD RECORD STUDIES

TABLE II — STATISTICAL ANALYSIS OF AVERAGE VENOM PRODUCTION DATA TO

SHOW, WI1ETHER THE SEX DIFFERENCE IS SIGNIICANT OR NOT

Year of

Production

M/F

Cobra

( Naja naja )

R. Viper

M/F {Viper a

russelli )

M/F

Krait

(Bungarus

coeruleus )

1955

Vm-Vf

Significam

Not significant

Vm-Vf

Significant

1957

Vm-Vf

Significant

Not significant

Vm-Vf

Significant

1958

Vm-Vf

Signifieant

Not significant

Vm-Vf

Significant

Vf = Venom

of farm

snake

M/F = Vm verses Vf

Vr = Venom oí room snake

Vm = Venom of male snake

VI = Venom of female snake

The actual figures appear at Table No. V and VI of reference No. 2 quoted in

this paper.

only; lhe figures are not given. A periodicity iu milking and tIre production

of venom output during different months as well as differenlial output of venom

from different fangs especially hy lhe Russell’s viper vvas eslahlished (2). Since

then improvements have heen done in lhe keeping of snakes in lhe rooms and

milking lias heen done in groups of both male and female snakes of different

lenglhs together as will normally he practical for any lahoralory. In the routine

working of a lahoralory, routine work cannot always he done with statistical data.

It was lherefore necessary to see if the residis ohtained in previous studies with

controlled conditions are available in routine working. Vipers yield better in

rooms. The caging conditions were improved as such it was necessary lo see if

the yield was slill maintained. The present studies give an account of these

subsequent ohservations and their probable significance.

Materials and Metiiods

The snakes in the rooms were kept in uniform size of tin cages. These had

on two sides, a 16 mesh wire gauze and a 2 inch lall partition inside the cage,

dividfng it into two chambers. One that is helow the cover, has a drinking

water in a brass pot (clamped) and a number of rough stones. The other side

has a small platform lo allow lhe snake to resl on a dry surface. The stones are

for rubbing the body during casting. The details aboul lhe rearing have heen

mentioned in the proceedings of the symposium on Animal husbandry in Inter¬

national Congress of Lahoralory Animais, Dublin 1965. The room 46' X 26'

contained 300 snake cages on raeks. It had two exhaust fans running 12 hours

during the nighl when doors and Windows were closed. The maximum and rni-

nimum temperatures in room and farm were 3T’C, 25.8°C, 31°C, and 24.9°C,

respectively. The maximum and minimum relative humidity during the year

in the room and the farm was 81%, 64%, 82%, and 67% respectively.

Total number of times that snakes were rnilked for these studies are 7960.

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Mem. Inst. Butantan

Simp. Internac.

83(3):767-774, 1966

DEORAS

769

Fig. 1 — Naja naja biting a

plastic coverstretched over a

stainless Steel funnel (whose

stem leads into an ampoule

for eollection of venom).

Observations

Having seen significanee in lhe output of venom hy taking a group of snakes

and even individual snakes of differenl sexes, \ve have novv seen tliis in a large

mixture group as vvill normally be available in any laboratory. Table III below,

gives lhe observations from 1961 lo 1965 wherein both male and female snakes

have been taken together from farm and room. The residis again shovvn as in

lhe past work statistically analysed, lhat lhe output of venom given out hy cobra

and krait snakes is significantly more in lhe farm than the room. This is not

lhe case with RusselTs viper.

lhe periodicily of milking in the case of lhesc snakes was one monlh.

lahle 111 gives the maximum and mininum output of venom for lhe differenl

snakes. Table IV gives the entire yearly data showing the periodicily of output

of venom in the room and farm in these snakes. The maximum outputs being

concentrated during April to September months.

Individual snakes of differenl lengtli and sexes were taken and milked in

farm and room separately. Table V gives the data in detail for Krait and

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Mem. Inst. Butantan

Simp. Internar.

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P. J. DEORAS

771

TABLE IV — TABLE SHOWING THE AVERAGE OUTPUT AND PERIODICITY OF

VENOM YIELD IN DIFFERENT SNAKES. (VENOM OF ONLY 13

SNAKES IN ONE AMPOULE).

1954

Cobra

(Naja naja)

Viper

(Vipera russelli )

Krait

(Bungarus coeruleus )

Farm

Room

Farm

Room

Farm

Room

January

0.1480

0.0933

0.1110

0.0650

0.0167

0.0414

February

0.1710

0.1240

0.1310

0.0770

0.0121

0.0200

March

0.1810

0.1120

0.1290

0.1040

0.0250

0.0180

April

0.1810

0.1250

0.1310

0.1130

0.0290

May

0.1810

0.1180

0.1670

0.1000

0.0280

0.0220

June

0.1700

0.1400

0.1670

0.1050

0.0275

0.0192

July

0.1390

0.1280

0.1250

0.0890

0.0240

0.0180

August

0.1327

0.CS26

0.1760

0.0605

0.0354

0.0180

September

0.15S9

0.1220

0.1564

0.1346

0.0360

0.0240

October

0.1235

0.0904

0.1237

0.1187

0.0260

0.0207

November

0.1714

0.0966

0.1280 ■

0.1067

0.0210

0.0160

December

0.1193

0.1497

0.1062

0.1343

0.0314

0.0126

Average

0.1564

0.1160

0.1376

0.1015

0,0261

0.0216

average for HusselFs viper an<] colira indicating tliat the sexes and length in.~pitt*

°f being different, the results show that the output of venom in farm is hetter

tlian in room for Krail and cohra and not significant fo the RusselFs viper. It

may here he noticed that beyond eertain length, there is not much difference in

lhe output of venom and that similar sizes may give more venom lhan bigger ones.

Venom collected from snakes vvas centrifuged and the supernatant Iiquid as

well as the dehris lyophilised. There was no or extremely small dehris left in

lhe ease of eobra and krail snakes, but Kussells vipers gave a lot of dehris.

Iable VI below shows lhe amount of venom in volume hefore centrifuging and

lhe toxicity of the supernatant as well as the dehris part. The Iable indicates

that there is dehris in vipers and the amount of dehris left over may he onlv a

dead 1 mlk which is not loxic. Venom used in horses for immunization recently

has shovvn that the present purified venom where dehris has heen discarded does

not give the sloughing and other side reaetions in horses as was available when

this had not heen removed.

Discussion

Stadelman (3), Fairley and Splatt (4), Amaral (5), and Schoettlerló) have

reeorded the differenees in the output of venom in vipers of different locations.

Koehwa (7) has tried to study the yield of venom in Vi per a palestinae, but he

has not made any attempts lo keep snakes under natural surroundings. These

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P. J. DEORAS

773

TABLE Vr — SIIOWING THE WEIGHT IN GMS OF LYOPHILIZED SUPERNATANT

LIQUID AND DEBRIS IN VENOM UNDER PURIFICATION

Snake

No.

snakes

milked

Volume

Venom

Weight

Venom

Weights

Wt. oi

super¬

natant

after centrifugation and

Wt. of M. L. D.

debris of super¬

natant

Lyophilizing

M. L. D.

debris

Naja naja

(Male)

3.0 cc.

2.8307

1.0230

Nil

1:120000

No debris

Naja naja

(Female)

2.0 cc.

1.9291

0.7059

Nil

1:135000

Do.

Vivera russelli

(Male)

5.5 CC.

5.1790

0.5371

0.0104

1:140000

Not toxic

even at

1:10

Vipera russelli

(Female)

4.7 cc.

5.0679

0.8553

0.0149

1:130000

Not toxic

even at

1:10

surroundings were therefore made after studying lhe natural habitat and the

results of experiments published in 1961 and 1963 (1, 2). These observations

needed confirmation under routine conditions and data established for taking

on over-all view about lhe keeping of snakes in the laboratory. There are a

number of Institutions which are keeping snakes for immunological studies or

for venom in connection with biochemical studies. Many have a structure called

snake farms, in which the snakes are kept and exhibited. Some expenses are

involved in keeping and exhibiting lhem. Our present studies have sliown that

the production of venom from cobra and Krail snakes is more from snakes

kept in the farm where natural habitat is given than lhose kept in the rooin.

Both these snakes have neurotoxic poison. The vipers which have haemotoxic

venoms give more venom in the rooms. It has also been observed by us that the

mortality of vipers kept in the farm is more than those kept in rooms as com-

pared to cobra and krait snakes. Jl is therefore argued that if a snake farm

is to he maintained in an institution for the production of venom, then it should

have the natural surroundings of the habitat of that snake. Secondly the snakes

like cobra and krait may lie reared in such a kind of farm because there is

more production of venom. For RusselFs viper. the production is more in the

room and the conditions there he improved to prevent the infection of viper

throals by giving them cages where living and feeding compartments are sepa-

rate. Laboratories working on immunological aspects should have venom pro¬

duction data for the year to plan

required number of snakes. It wil

period when maximum amount of venom is produced is also the time to get

most toxic venom. If so then the maximum collection he done during this

lime and venom used for antigenicity. The same could he available for bioche¬

mical studies. The figures for the maximum and average venom output for

snakes give an indication lo clinicians as to how much venom to expect in

the worst cases when that particular snake biles. Lastly venom used for immu-

nization he better centrifuged and supernatant liquid ordy used for the purpose.

immunological programmes for keeping lhe

be worthwhile experimenling whether the

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10 11 12 13 14 15 16

774

PROBABLE SIGNIFICANCE OF VENOM YIELD RECORD STUDIES

Acknowledgements — We are extremely thankful to the Indian Council of

Medicai Research for initially sponsoring these studies and the Director, Haffkine

Institute as well as the Govt. of Maharashtra for giving facilities to continue

these studies. Mr. N. E. Vad and Mr. T. G. Tare my colleagues were most

helpful to me and without their active cooperation this project would never

have been completed.

References

DEORAS, P. J., J. Univ. Bombay, 30 (3), 50, 1961.

2. DEORAS, P. J., Snake farm venom yield records and their significances.

Venomous and Poisonous Animais and Noxious Plants of the Pacific Area,

Pergamon Press, 1963.

3. STADELMAN, R. E., Buli. Antivenin Inst. Amer., 3 (1), 29, 1929.

4. FAIRLEY, N. H., and SPLATT, B„ Med. J. Aust., 1 (11), 338, 1929.

5. AMARAL. A. do, Mem. Inst. Butantan, 4, 1929.

6. SCHOETTLER, W. H. S., Amer. J. trop. Med. Hyg., 31 (4), 389, 1951.

7. KOCHWA, E., Amer. J. trop. Med. Hyg., 9 (4), 381, 1960.

8. KLAUBER, L. M., The Rattlesnake, Univ. Calif., Los Angeles, 1956.

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WILLY BECAK

775

14. CONSTITUIÇÃO CROMOSSÔMICA E MECANISMO DE DETERMINA¬

ÇÃO DO SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS

SEXUAIS E EVOLUÇÃO DO CARIÓTIPO.

WILLY BEÇAK

Secção de Genética, Instituto Butantan, São Paulo, Brasil

O estudo cariotípico das serpentes cujos resultados já foram apresentados (1),

serviu de Rase para a elaRoração do presente trabalho.

Especial ênfase foi dada ao problema dos cromossomos sexuais visando es¬

clarecer êsse aspecto ainda pouco conhecido nos répteis. Os resultados relativos

à constância da massa cromossômica e à proporção do cromossomo Z em relação

ao lote cromossômico são de interesse, principalmente quando comparados à

observações semelhantes feitas em outros grupos animais. Os microcromosso-

mos, cuja natureza cromossômica nas aves foi negada por alguns autores, são

discutidos em um tópico separado demonstrando-se que êsses elementos são cro¬

mossomos característicos, que se diferenciam dos macrocromossomos só pelo ta¬

manho reduzido. Finalmente, quanto à evolução, são correlacionadas as obser¬

vações cariotípicas, sendo sugerido um esquema de evolução para as serpentes

estudadas.

Cromossomos sexuais

O padrão de herança típico dos genes ligados ao sexo está relacionado, fre-

qüentemente, com um heteromorfismo do par de cromossomos portadores desses

fatores. Essa diferenciação morfológica dos heterocromossomos permite correla¬

cionar aspectos filológicos e distribuição gênica, e tem sido uma das bases da

teoria cromossômica da hereditariedade. No entanto, em numerosos casos de

vertebrados, a demonstração da existência de uma heterogametia quanto aos cro¬

mossomos não foi até agora possível.

Nos peixes, o estudo de herança ligada ao sexo revela a existência de dois

tipos de digametia. Gordon (2, 3), demonstrou que os peixes Platypoecilus rnu-

culatus ‘"selvagens” do México têm um mecanismo genético de determinação do

sexo (XX = fêmea; XY = macho) oposto ao encontrado nas criações de aquá¬

rio da mesma espécie (ZW - fêmea; ZZ ■= macho). Gordon (4) verificou que

o Platypoecilus das Honduras Britânicas pertence ao último tipo (ZW,

ZZ). Caslle, em 1936(5), sugeriu que ZW e ZZ poderiam ser expressos como

YX o YY respectivamenle. Segundo Gordon (3) apesar do W dos peixes ‘‘do¬

mesticados” não ser homólogo ao X dos “selvagens”, o Z e o Y eram aparen¬

temente homólogos. Portanto, existiriam populações de Platypoecilus

(com fêmeas heterogaméticas) nas quais, como condição normal, os machos se¬

riam ZZ (YY). Yamamolo (6, 7) por sua vez verificou que no peixe Oryzias

latipes, ocorre heterogametia masculina (XY), aparecendo excepcionalmente tam-

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776 CONSTITUIÇÃO CROMOSSÓMICA E MECANISMO DE DETERMINAÇÃO DO

1 lu SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS SEXUAIS E

EVOLUÇÃO DO CARIÔTIPO

l>ém machos YY viáveis. A ocorrência de fêmeas WW em casos de heteroga-

melia feminina tamhém foi relatada. Belammy e Queal (8) criando o peixe

“domesticado” l‘laty[>oecilus maculalus, encontraram vários machos Z\V, o que

confirma as observações anteriores de Breider (9). Verificaram que êsses ma¬

chos quando cruzados com fêmeas normais ZW, davam origem a fêmeas WW,

além tias fêmeas ZW e machos ZZ normais. Progénies dessas fêmeas V W cru¬

zadas com machos normais ZZ consistiram somente de fêmeas.

Nos anfíbios, sob o ponto de vista genético, ocorrem dois tipos de digamelia,

podendo esta variar dentro do mesmo gênero(lO). Humphrey (11), demons¬

trou na salamandra Amblystoma mcxicanum que a fêmea é geneticamente diga-

mética. Quando fêmeas experimentalmente transformadas em machos eram cru¬

zadas com fêmeas, a proporção na descendência era de três fêmeas para um

macho. Os descendentes WW dêsse cruzamento (um têrço das fêmeas) eram

fêmeas viáveis, pois, ao serem cruzadas com machos normais ZZ, produziram

somente progénie feminina. Humphrey (12), utilizando a mesma espécie, conse¬

guiu a reversão do sexo de fêmeas WW, através de implantação experimental de

preprimórdio de gônadas de embriões. Essas observações em peixes e anfíbios

sugerem que nas espécies de vertebrados inferiores os cromossomos sexuais encon¬

tram-se ainda em estágio tal de diferenciação que o X ou Z pode ser substi¬

tuído pelo Y ou W, respectivamente.

Nas aves, o estudo experimental indicou a existência de uma digametia femi¬

nina. Yao e Olsen (13) observaram, por partenogênese, no peru, embriões exclu¬

sivamente masculinos o que seria explicado, segundo Poole e Olsen (14), admi¬

tindo uma digametia feminina; os ovócitos que retivessem o cromossomo Z na

maturação dariam nascimento, após desdobramento do complemento haplóide, a

indivíduos ZZ, portanto a machos, enquanto que os embriões 00 (ou WW), re¬

sultantes de ovos onde o cromossomo Z passou ao glóbulo polar, morreriam.

Nenhum dêsses métodos foi utilizado para elucidar o tipo de digametia entre

répteis; somente Dantchakoff (15) tentou relacionar os resultados de suas expe¬

riências endocrinológicas em mamíferos, aves e répteis com o tipo de digametia

que ela presumia feminina nesses últimos.

Quanto aos estudos citológicos, NogusadG) descreveu no peixe Mogrunda

obscura um par fie cromossomos XY no macho, cujo heteromorfismo poderia

ser observado na metáfase I. Êsse heteromorfismo não é, no entanto, convincente,

segundo vau Brink(17).

Nos anfíbios, Witschi (18) e Yosida (19) descreveram respectivamente em

Rana temporária e líyla arbórea dimorfismo dos cromossomos sexuais no macho,

mas também, as diferenças encontradas foram extremamente pequenas. Já no

Xenopus laevis, que é um dos anuros mais primitivos, no qual a heterogametia

feminina havia sido provada experimentalmenle por transformação do sexo, Chang

e Witschi (20), Gallien (21) e Weiler e Olmo (22) identificaram citològica-

mente um grande dimorfismo dos cromossomos sexuais, na fêmea. () cromos¬

somo Z é o menor elemento tio cariótipo, enquanto que o W excede em tamanho

o maior autossomo. Segundo êsses autores, em algumas espécies de vertebrados

inferiores, e êste seria o caso de Xenopus laevis , a diferenciação morfológica

tios elementos sexuais pode ter precedido sua diferenciação genética real.

Nas aves, em Gallus domesticus, a primeira confirmação filológica da hete¬

rogametia feminina deve-se a Suzuki (23), que identificou o quinto elemento,

em ordem de tamanho como sendo o cromossomo Z. Subsequentemente vários

investigadores confirmaram êsses achados. Miller (24), estudando uma galinha

com transformação de sexo, demonstrou que o cromossomo Z, ímpar no sexo

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83(3): 775-798, 19H(Í

WILLY BEÇAK

777

heterogamético, passa a somente urn pólo do fuso, durante a primeira divisão da

meiosc. Posteriormente verificou que o Z não é pareado com qualquer dos cro¬

mossomos menores na placa equatorial, favorecendo assim a hipótese da constitui¬

ção ZO e não Z\V no sexo licterogamético. Oguma (25) fixou em 78. o número

diplóide para o macho e 77 para a fêmea de Gallus domesticus, admitindo, tam¬

bém, a constituição cromossômica ZO para o sexo helerogamético. Yamashi-

na (26) numa série de trabalhos descreveu condições análogas, num grande

número de espécies estudadas. Ohno (27) estudando células meióticas e mitóti-

eas de Gallus domesticus , encontrou também só um Z na fêmea beterogamética.

Segundo êle, o número de cromossomos é pouco acima de 70, mas admite, como

o fizeram Matthey e van Brink (28), que a determinação exata do número de

cromossomos nessa espécie é ainda impossível. Segundo van Brink (17), das 29

espécies de aves estudadas por vários autores, 18, pertencentes a 8 famílias dife¬

rentes, apresentam digametia feminina reconhecível ao nível eromossômico. Nas

11 espécies restantes é impossível, à base dos dados existentes, um pronuncia-

Oguma, em 1934(29), relatou o primeiro caso de digametia feminina nos

répteis, constatando que o macho de Lacerta vivipara possui 36 e a fêmea 35

cromossomos, admitindo assim a existência de uma digametia ZO também entre

os répteis. Em 1937 (30) encontrou o mesmo tipo de digametia na tartaruga

Amyda japonica (macho = 64; fêmea = 63). Makino e Asana (31) descreve¬

ram o mesmo fenômeno em Calotes versicolor (macho '= 34; fêmea = 33) e

em Sitana ponticeriana (macho 46; fêmea = 45). Nakamura (32) confirmou

a existência do mecanismo ZO na fêmea da tartaruga Carctta carelta olivaceii

(2n = 57) e na Chelonia japonica, Makino (33) encontrou 56 elementos no

macho e 55 na fêmea. Nesse mesmo trabalho, relata ter reexaminado as prepa¬

rações de Oguma confirmando as conclusões dêsse autor. Também Suzuki (34)

relata cromossomos sexuais do tipo ZO-ZZ em Amyda maacki.

Por outro lado Matthey (35) não observou nenhuma diferença cromossô¬

mica nos dois sexos de Chamaeleon vulgaris, que possui 2n = 24 cromossomos.

Margot (36) também não encontrou diferenças cromossômicas nos dois sexos de

Anguis jragilis (2n = 44) e Lacerta vivipara (2n = 36). Num outro sáurio

Chamaeleon hitaeniatus, cuja análise cromossômica é muito fácil pelo pequeno

número de cromossomos (2n = 24) e por ter apenas 4 microcromossomos,

Matthey e van Brink (37) também não observaram digametia. Em 1957. Mat-

they (38) relatou a ausência de digametia feminina em outros CHAMAELEON-

TIDAE: Brookesia stumpfii (2n = 24), Chamaeleon campani (2n = 26), Cha¬

maeleon brevicornis (2n = 32) e Chamaeleon nasutus (2n = 34). Êsses estudos

levaram-no à convicção da inexistência generalizada de cromossomos sexuais mor¬

fologicamente diferentes nos répteis. Em 1962, Kobel(39) relata ter encontrado

um par dimórfico de cromossomos sexuais numa fêmea estudada de Vipera berus.

A raridade de observações de digametia feminina é provavelmente devida,

em parte, às dificuldades concernentes aos métodos de estudo até há pouco utili¬

zados. Para evidenciar os heterocromossomos os autores têm utilizado, geral¬

mente, técnicas que envolvem o estudo da meiose. A digametia seria identificada

pela presença de um bívalente assimétrico nos estados meióticos que precedem a

anáfase I e pelo comportamento eventualmente aberrante, alocíclico, dêste hiva-

lente durante a prófase da meiose, assim como durante a anáfase I. Ora, as

células em meiose são geralmente abundantes nos testículos do macho, onde um

heteromorfismo morfológico é facilmente observável. Na fêmea, porém, as divi-

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77 X CONSTITUIÇÃO CROMOSSÔMICA E MECANISMO DE DETERMINAÇÃO DO

1 ‘ SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS SEXUAIS E

EVOLUÇÃO DO CARIÓTIPO

sões meiólicas, além de serem menos numerosas que no macho, ocorrem num

período onde o ôvo se encontra repleto de substâncias de reserva, o que dificulta

uma fixação perfeita do material. Portanto, o melhor meio para se investigar a

presença de um par heteromorfo na fêmea é a análise numérica e morfológica

dos cromossomos na mitose; no caso de digamelia do tipo ZZ-ZW encontraremos

números pares em ambos os sexos, mas na fêmea um dos pares será constituído

de elementos desiguais.

Nossas observações nos ofídios indicam que as espécies não venenosas estu¬

dadas, da família BOIDAE, não apresentam cromossomos sexuais morfologica¬

mente diferentes que permitam sua distinção em relação ao restante do carió-

tipo. Essa ausência de diferenciação morfológica não indica a inexistência de

cromossomos sexuais, pois, através do processo evolutivo, podem, nessas espé¬

cies, ter-se acumulado genes masculinizantes o feminilizanles em um ou mais

pares de cromossomos, que condicionariam a determinação do sexo.

As espécies incluídas nas famílias COLUBRIDAE e CROTALIDAE, por outro

lado, caracterizam-se por apresentarem no cariótipo um par de heterocromosso-

mos. que foi por nós identificado como sendo o quarto par de macrocromossomos

em ordem de tamanho. Os machos constituem o sexo homogamético apresen¬

tando, no quarto par, dois cromossomos metacêntricos idênticos, que correspon¬

deriam aos cromossomos ZZ, ou XX dos outros animais. As fêmeas por sua vez

constituem o sexo heterogamético apresentando, no quarto par, dois cromossomos

morfologicamente diferentes, sendo um semelhante ao cromossomo metacênt rico

Z ou X encontrado no macho, enquanto que, o outro cromossomo é menor, acro-

cêntríco e corresponderia ao cromossomo \V ou Y dos outros animais.

Nos COLUBRIDAE e CROTALIDAE, o mecanismo cromossômico de deter¬

minação do sexo é mais diferenciado do que nos BOIDAE. Além do acúmulo

diferencial de genes, já leria ocorrido uma diferenciação morfológica entre os

cromossomos sexuais. Essa hipótese, que supõe um mecanismo cromossômico

de determinação do sexo mais diferenciado nos COLUBRIDAE e CROTALIDAE,

estaria em perfeito acordo com a posição sistemática ocupada pelas espécies estu¬

dadas, pois, não resta dúvida que, por exemplo, as espécies peçonhentas repre¬

sentam um grupo mais evoluído, em relação as espécies que não são venenosas.

A simples presença de um aparelho inoculador de veneno altamente especiali¬

zado nas serpentes peçonhentas, caracteriza o seu grau de evolução em relação

as outras espécies, dentre as quais algumas ainda apresentam vestígios rema¬

nescentes da sua evolução a partir de grupos sistematicamente mais primitivos.

Por exemplo, nos BOIDAE é ainda evidente a presença de membros locomoto-

res atrofiados.

Na família COLUBRIDAE estão incluídas as serpentes que apresentam as

maiores variações de número e morfologia dos cromossomos, inclusive do par

de cromossomos sexuais. Porisso mesmo constituem excelente material para es¬

tudo dos mecanismos de alterações cariotípicas nas serpentes. No que diz res¬

peito aos cromossomos sexuais, que pretendemos discutir neste capítulo, verifica¬

mos ([ue tôdas as espécies de colubrídeos que estudamos apresentam um par

heteromórfico na fêmea. A sua morfologia e tamanho em relação aos outros

cromossomos é, no entanto, variável.

Em Spilotes pullutus anomatèpis, Spilotes pu-llatus maculatus, Phylodrias

olfcrsii olfcrsii, Dryadophis bifossalus bifossalus, Drymarchon corais corais e

Chirordus bicarinahis, ambos os cromossomos sexuais têm aproximadamente o

mesmo tamanho, diferindo somente pela posição do centrômero, que é terminal

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Mem. Inst. Butantnn

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no Z c submediano no W. Aparentemente, a quantidade de material genético é

a mesma nos dois cromossomos. Baseando-nos no aspecto morfológico do par

fie heterocromossomos, poderíamos sugerir uma explicação para a diferenciação

dêsscs cromossomos sexuais. É possível que tenha ocorrido uma inversão peri-

cênlrica num dos homólogos do quarto par de cromossomos. Em consequência

dessa inversão, a permutação entre os homólogos ficaria reduzida considerável-

mente. Êsse mecanismo de isolamento, pode ter resultado num maior acúmulo

diferencial de genes relacionados à determinação do sexo, nos cromossomos em

questão.

O tipo de cromossomos sexuais apresentado, por exemplo, pela S pi lotes,

pode representar um grau intermediário na evolução dêsse par cromossômico em

direção ao apresentado pelos crotalídeos. Nestes últimos, o tamanho relativamen¬

te menor do cromossomo W parece ser conseqüência de uma redução por elimi¬

nação de material cromossômico. É possível que a redução de tamanho do W

tenha conduzido nos animais mais evoluídos como os mamíferos, ao diminuto

cromossomo Y, que teria como função a de induzir o desenvolvimento das gôna-

das masculinas.

Em Clelia occipitolutea, o par de cromossomos sexuais é representado por

um Vi. que é o maior cromossomo do cariótipo e por um Z, que ocupa o quarto

lugar em ordem de tamanho em relação aos autossomos. Essa situação peculiar,

na qual o cromossomo que ocorre só no sexo heterogamético é muito maior que

o outro cromossomo sexual, foi descrita recentemente também num outro orga¬

nismo, Xenopus laevis ( 22). 0 tamanho maior do cromossomo W pressupõe

um acúmulo também maior de genes, que aparentemente não ocorreriam no sexo

homogamético, que possui somente cromossomos Z.

A observação de Weiler e Ohno (22) sôbre a existência dêsse dimorfismo

cromossômico acentuado num anuro tão primitivo como o Xenopus laevis poderia

ser explicada, segundo êsses autores, como conseqüência de uma diferenciação

morfológica dos cromossomos sexuais antecedendo a uma maior diferenciação

genética real fios mesmos. Em outras espécies, apesar da impossibilidade de

distinguir morfologicamente os cromossomos sexuais, pode na realidade existir

um grande acúmulo diferencial de fatores masculinizantes e feminilizantes. Pode¬

ríamos, também, com base nessas observações e nas nossas sugerir a hipótese de

que a evolução quanto à diferenciação dos cromossomos sexuais não é sempre

uniforme, no sentido de acompanhar a escala zoológica. Dependendo das con¬

dições em que se desenvolve determinado grupo animal, o seu mecanismo cro¬

mossômico de determinação do sexo pode ser mais diferenciado do que o de

um grupo zoologicamente superior como é o caso, por exemplo, do anfíbio

Xenopus laevis em relação às serpentes BOIDAE.

Espkumatocknese

Para o estudo da espermalogênese nos ofídios, foi escolhida a espécie Bothrops

jararaca, pela facilidade local de obtenção de material e porque nessa espécie o

cariótipo tem 36 cromossomos, que é o mais comumente encontrado nas serpen¬

tes, além de que o heteromorfismo dos seus cromossomos sexuais é evidente (40).

O método utilizado foi o de esmagamento do testículo e coloração pelo

Giemsa ou orceína acética (1). As preparações foram feitas no segundo semes¬

tre dos anos de 1962 e 1963, na época de reprodução dessas serpentes, que

apresentaram então abundância de células em meiose I Fig. 1).

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E s p e r m a t o g ô n i a s

Na gônada masculina, as espcrmalogônias apresentavam-se principalmcnte

nos estágios de prófase e metáfase. Nas células em prófase os cromossomos en¬

contram-se distendidos soli a forma de filamentos longos provàvelmentc devido

ao afrouxamento da espiral. Pôde-se, no entanto, distinguir facilmente a presen¬

ça de Hf) elementos, que consistem do 16 macrocromossomos e 20 microcromos-

somos.

Na prófase da gônia os cromossomos encontram-se distendidos e em certos

cromossomos pode-se observar a associação com o nucléolo, através das assim

chamadas regiões organizadoras do nucléolo. Nas espcrmalogônias de Bothrops

jararaca tanto na prófase precoce como na prófase tardia verificamos que o

nucléolo encontrava-se associado a I ou 5 mierocromossomos (Fig. 2). Em al¬

gumas células encontrou-se um macrocromossomo próximo ao nucléolo. mas de¬

vido à inconsistência dêsse achado cm outras células, êsse aspecto foi considerado

como fortuito.

Devido ao tamanho reduzido dos mierocromossomos de Bothrops jararaca.

não foi possível com as técnicas utilizadas, identificá-los, no sentido de verificar

se os que se encontram associados ao nucléolo pertencem a pares homólogos ou

se são 4-5 mierocromossomos pertencentes a pares diferentes.

Na cobaia, Cavia cobaya, foram observados um ou dois nueléolos nas pró-

fases diplóides de ambos os sexos. Apesar de ambos os membros do par de

aulossomos de maior tamanho possuírem aparentemente capacidade inerente de

organização do nucléolo, verificou-se que somente um dos homólogos apresenta-se

associado a um nucléolo. É. portanto, possível que certas regiões de cromosso¬

mos homólogos da mesma célula não funcionem sincrônicamente (41).

Na galinha, Gallus domesticas, foi também observada a existência de um

só nucléolo e êste apresenta-se associado aos mierocromossomos, mas nesse orga¬

nismo o número de mierocromossomos associado foi avaliado em aproximada¬

mente 12 (42).

Mais de 60 prófases somáticas e de espcrmalogônias examinadas, em várias

preparações de testículo de Bothrops jararaca , não demonstraram a existência de

qualquer macrocromossomo inteiramente heteropicnótico. Essa análise permite con¬

cluir que, tanto nas células somáticas como nas espcrmalogônias do macho dêsse

ofídio, nenhum dos cromossomos sexuais apresenta heteropienose positiva.

No testículo, as metáfases somáticas e das espermatogônias apresentam 36

elementos, sendo 16 macrocromossomos e 20 mierocromossomos, como jú descre¬

vemos nas metáfases somáticas de culturas de leucócitos (40). Também nesse

estágio nenhum dos cromossomos exibe heteropienose positiva, lodos os macro¬

cromossomos apresentam estrutura de duplas eromátides, que se apresentam co¬

nectadas somente pela região do centrômero. Os mierocromossomos têm estru¬

tura idêntica, apesar de que em alguns, devido ao tamanho reduzido, é difícil

localizar-se a posição do centrômero.

Na anáfase os centrômeros, através dos quais as eromátides mantinham-se

juntas, dividem-se, deslocando-se para pólos opostos da célula. Em consequência,

as eromátides separam-se. afastando-se. Observa-se divisão equitativa do material

cromossômico, isto é, tanto as eromátides que constituem os macrocromossomos

como as que formam os mierocromossomos separam-se igualmente migrando para

os pólos (Fig. 3). Os mierocromossomos por terem tamanho menor apresentam

às vêzes as suas eromátides completamente separadas, antes dos macrocromossomos.

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789 CONSTITUIÇÃO CIIOMOSSÔMICA E MECANISMO DE DETERMINAÇÃO DO

SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS SEXUAIS E

EVOLUÇÃO DO CARIOTIPO

Na lelófase os dois complementos cromossômicos, morfologicamente idênti¬

cos, separam-se completamenle dando origem a dois novos núcleos diplóides.

Mciose

Nos esperniatócilos de primeira ordem foi possível encontrar Iodos os estágios

que caracterizam a prófase meiótica. A primeira evidência do início da raeiose,

numa fase que chamaremos de pré-leplóteno, é, em comparação com a célula em

interfase, uma ligeira rarefação do material nuclear, com um princípio de deli¬

neamento dos filamentos cromossômicos. Na fase de leptóteno êsses filamentos

tornam-se mais evidentes, mas ainda finos, apresentando pequena afinidade pelo

corante.

Na fase de zigóteno, os filamentos homólogos atraem-se, ocorrendo o parea-

mento, simultâneamente, em vários pontos ao longo tios cromossomos e nas extre¬

midades. É freqüente observar-se nessa fase células em que os filamentos en¬

contram-se quase completamenle pareados, exceto em alguns pontos intersti¬

ciais (Fig. 4).

Células em paquíteno são muito freqiientes. Nessa fase os cromossomos ho¬

mólogos apresentam-se mais condensados e conjugados, formando hivalentes. Ao

longo dêles observam-se, às vêzes, regiões mais condensadas, que correspondem

aos cromômeros. Os microcromossomos apresentam-se, como o restante dos cro¬

mossomos, isolados, formando hivalentes, podendo-se, às vêzes, distinguir cromô¬

meros nos mesmos. Ao contrário do que foi descrito por Newcomer e Brant (43)

na galinha, os microcromossomos de ofídios não apresentam heteropicnose posi¬

tiva, que os distinga dos macrocromossomos nesta ou em qualquer outra fase

da meiose. Certas regiões dos macrocromossomos apresentam uma condensação

mais pronunciada. Essas partes mais condensadas parecem resultar do maior

acúmulo de heterocromatina e se localizam geralmente em regiões adjacentes aos

centrômeros e nas extremidades dos cromossomos (Fig. 5).

Na fase de diplóteno, os cromossomos homólogos, que no estágio precedente

apresentavam-se completamente conjugados, separam-se exceto nas regiões onde

provàvelmente ocorrem quiasmas (Fig. 6). Êsse fenômeno pode ser claramente

observado em todos os macrocromossomos. Apesar da dificuldade em determi¬

nar-se o número exato de quiasmas de cada bivalcnte, verificamos que o número

de quiasmas no primeiro bivalente é o mais alto, sendo por nós avaliado em seis

a oito aproximadamente por célula. No segundo e terceiro bivalente foram con¬

tados de quatro a seis quiasmas. No quarto bivalente, que corresponde em

ordem de tamanho aos cromossomos sexuais, ocorrem de três a cinco quiasmas.

Nos outros hivalentes de macrocromossomos observamos de dois a três quiasmas.

Os microcromossomos também apresentam quiasmas, mas em número reduzido,

sendo a sua avaliação mais difícil.

A fase de diacinese pouco se distingue do diplóteno, mas, pode ser caracte¬

rizada pela maior condensação dos hivalentes, que se coram mais intensamente

que nas fases anteriores. Ocorre uma reduzida lerminalização dos quiasmas, não

se observando a formação de figuras em anel. Nessa fase, a avaliação do número

de quiasmas é ainda possível, porém é mais difícil.

A existência de diplóteno e diacinese característicos e a ocorrência de fre¬

quência relativamente alta de quiasmas em Bothrops jararaca mostram que, quan¬

to à evolução genética, os ofídios são mais semelhantes às aves que aos anfíbios.

Realmente, Weiler e Olmo (22), estudando preparações de testículo adulto do

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WILLY BEÇAK

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anuro Xenopus laevis, não encontraram figuras de paquíteno e diplóteno precoce.

Nesse animal, na meiose do espermatócito de primeira ordem, os liivalentes apa¬

rentemente passam do estágio de zigóteno ao de diplóteno tardio ou diacinese,

apresentando nessa fase, todos os bivalentes forma de anel. Mesmo nos lii va len¬

tes grandes não foi observado quiasma intersticial. Essa observação está de

acordo com o trabalho de Wickhom (44) sôbre a baixa freqiiência de quiasmas

nos anuros, que segundo Witschi (45), estaria correlacionada ao reduzido ritmo

de evolução nesse grupo. Nas aves, por sua vez, não só ocorre paquíteno e

diplóteno, como nas cobras, mas também o número de quiasmas é alto. Segun¬

do Olmo (27), o primeiro e segundo pares de bivalentes apresentam de oito a

dez quiasmas e o terceiro e quarto têm seis quiasmas. 0 quinto par, que cor¬

responde aos cromossomos sexuais, apresenta forma de anel ou de 8 na diaci¬

nese, enquanto os bivalentes maiores apresentam vários quiasmas. Êsse fenôme¬

no. no entanto, é devido à rápida terminalização do bivalente sexual, pois no

diplóteno é possível observar que êsse bivalente tem de quatro a cinco quiasmas.

Por outro lado, a presença nos répteis e nas aves de microcromossomos, que

não ocorrem aparentemente nos anfíbios, é um outro falo que indica a maior

semelhança entre os dois primeiros grupos citados.

Na metáfase, os cromossomos apresentam-se uniformemente mais condensa¬

dos. Os bivalentes dispõem-se na placa equatorial, com os centrômeros no equa¬

dor. Cada cromossomo encontra-se dividido nesse estágio em duas cromátides,

portanto, cada bivalente é constituído de quatro filamentos, podendo o conjunto

ser denominado tétrade. Alguns dos bivalentes, principalmente os maiores, não

se apresentam ainda completamente lerminalizados (Fig. 7).

Na anáfase, os centrômeros homólogos dos bivalentes separam-se, deslocan¬

do-se para pólos opostos do fuso celular. Os microcromossomos separam-se como o

restante dos bivalentes, equitativamente, recebendo cada célula filha, dez micro-

cromossomos.

Na intercinese, os dois lotes de cromossomos encontram-se já completamenle

separados nos espermatócitos de segunda ordem. Os cromossomos, ou díades,

aparecem mais distendidos, sob a forma de finas estruturas filamentosas.

Na prófase II da meiose, os cromossomos encontram-se mais separados, nas

preparações. O número de elementos observados nessa fase é de 18, sendo 8

maiores que correspondem aos macrocromossomos e 10 menores que corres¬

pondem aos microcromossomos. Certas regiões dos cromossomos, principalmente

na proximidade dos centrômeros, parecem mais marcadas por heterocromatina.

Na metáfase II. os cromossomos, ou díades, num total de 8 macrocromos¬

somos e 10 microcromossomos aparecem fortemente condensados. Os braços são

relativamente mais curtos e largos que na fase anterior. Em tôdas as figuras

principalmente nos cromossomos maiores, observa-se que os braços estão bastante

separados, formando pràticamente ângulos retos entre si (Fig. 8).

Na anáfase II. os centrômeros dividem-se e as duas cromátides de cada cro¬

mossomo separam-se, deslocando-se a polos opostos do fuso (1'ig. 9).

Na telófase II. os cromossomos já se encontram completamente separados,

dando origem a duas células haplóides com 18 cromossomos cada. Os filamentos

começam novamente a alongar-se tornando-se aparentemente menos visíveis. É

comum observar-se nas preparações a divisão sincrônica das células, que se ori¬

ginaram do mesmo espermatócito, resultando em quatro células haplóides, pró¬

ximas e morfologicamente semelhantes.

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784 constituição cromossômtca e mecanismo de determinação do

' ' SEXO em ofídios sul-americanos. II. cromossomos sexuais e

EVOLUÇÃO DO CARIÓTIPO

Em nenhuma das fases acima descritas, quer na divisão das espermatogô-

nias quer na primeira e segunda divisões meiólicas, foi observado comportamento

alocíclico de um ou de ambos os cromossomos sexuais. Aparentemente êstes com¬

portam-se como o restante dos autossomos, durante a espermatogênese.

ClíOMATI\A SEXUAL

A eromatina sexual, também conhecida por corpúsculo cromatínico ou de

Barr, foi originalmente descrita por Barr e Bertram (46) em células nervosas

de gatas, lendo sido subsequentemente demonstrada também em células somáticas

humanas e de outros mamíferos.

A eromatina sexual é característica das células femininas, onde se apresenta

como uma massa basófila, Feulgen-posiliva, na face interna da membrana nuclear.

A presença dessa estrutura nos núcleos femininos, era interpretada, até recen¬

temente, como regiões heterocromáticas dos dois cromossomos X que na intérfase

somática apresentar-se-iam condensadas e forlemente coráveis, isto é, heleropic-

nóticas. No entanto, na célula feminina em intérfase apenas um cromossomo X

é heteropicnótico, sendo o outro indistinto dos autossomos.

Segundo Lyon(47), nas células somáticas femininas, somente um X seria

geneticamente ativo durante a intérfase, apresentando-se o outro condensado.

Numa fase precoce da embriogênese, provavelmente ao tempo da implantação, cm

cada célula seria “decidido” qual dos dois cromossomos X seria ativo. Nas cé¬

lulas masculinas normais os cromossomos X seriam presumivelmente ativos. A

linhagem germinativa da fêmea não participa, aparentemente, dêsse processo de

diferenciação do cromossomo X. A hipótese de Lyon de diferenciação do X

baseia-se citològicamente no fato de ter-se observado heteropienose de um dos

cromossomos X nas células femininas em prófase(48).

For outro lado, empregando-se técnicas de autoradiografia em culturas de

tecidos de hamster (49) e humanos (50), 151), (52) tratadas pela timidina tri-

tiada verificou-se que um dos dois X da célula feminina, replica o seu ADN

relativamente mais tarde que o outro X e os autossomos. Êsse cromossomo

“tardio” não foi encontrado nas células masculinas.

Para investigar a existência da eromatina sexual nos ofídios foram feitas

preparações de cérebro, baço, fígado, rim e gônadas. As lâminas foram prepa¬

radas pelas técnicas de esmagamento ou corte histológico, seguida de hidrólise e

coloração pelo Ciemsa ou Hematoxilina-Eosina. Nas preparações examinadas de

Boa constriclor amarali e Bolhrops jararaca não observamos em nenhum dos sexos

a existência de uma massa condensada, que correspondesse à eromatina sexual.

Em algumas preparações apareceram corpúsculos condensados nos núcleos de al¬

gumas células, mas êstes não se encontravam, geralmente, na periferia do núcleo

e eram esparsos. Além disso, essa ocorrência foi observada indistintamente nos

dois sexos.

Examinamos também preparações de baço e gônadas, obtidas por esma¬

gamento, em outras ordens de répteis. Foram estudados Anolis carolinensis

(LACEItTIEIA), Caiman sclerops (CKOCODILIA) e Arnyda jerox (CHELONIA).

Nesses animais também em nenhum dos sexos foi observado um corpúsculo que

pudesse ser considerado como eromatina sexual.

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Em Gallus domesticus, Kosin e Ishizaki (53) relataram uma diferença nu¬

clear entre o macho e a fêmea. Êsses autores identificaram um corpúsculo cor¬

respondente à cromatina sexual nos núcleos somáticos do sexo heterogamético.

Encontraram também um corpúsculo nos núcleos de machos, mas em frequência

muito menor. Ohno, Kaplan e Kinosita (54) confirmaram a existência da cro¬

matina sexual na fêmea de Gallus domesticus, demonstrando que a mesma re¬

presenta o cromossomo Z, ímpar, em heteropicnose positiva. Por outro lado.

Schmid (55) empregando timidina triliada em cultura temporária de medula

óssea, verificou, por autoradiografia, que os dois cromossomos Z do macho homo-

gamélico e o Z ímpar da fêmea heterogamélica não apresentam diferenças entre

si ou em relação aos autossomos, quanto à incorporação de timidina.

A condensação de um cromossomo X nas células somáticas femininas, nos

mamíferos, parece ser um mecanismo muito especializado. Aparentemente a di¬

ferenciação dos cromossomos X e Y seria tal que a maior parte dos genes conti¬

dos no cromossomo X se encontraria em dose simples na célula diplóide mas¬

culina, sem correspondência no cromossomo Y. Paralelamente a êsse fenômeno,

a condensação de um cromossomo X nas células femininas compensaria a situa¬

ção de hemizigose do cromossomo X, nas células masculinas. Nos répteis, apa¬

rentemente, não se desenvolveu êsse tipo de mecanismo compensador.

Massa cromossômica e proporção Z : Autossomos

0 estudo contínuo de material cromossômico em diferentes organismos des¬

pertou o nosso interêsse em encontrar algumas regras gerais que governassem s

variedade de cariótipos exibidos pelos animais. Estudamos os cromossomos de

sete espécies e um híbrido interespecífico de mamíferos placentários pertencen¬

tes às ordens PRIMATA, ARTIODACTYLA, PERISSODACTYLA, CARNÍVORA e

RODENTIA, cujo número cromossômico diplóide varia de 17/18 no Microtus

oregoni a 78 no Canis familiaris (56). Através de um método que nos permi¬

tiu um cálculo bidimensional da área dos cromossomos, obtivemos resultados que

nos levaram a conclusões interessantes.

Apesar da grande variação no número cromossômico, as oito espécies estu¬

dadas parecem conter quantidades semelhantes de material genético, que é de

aproximadamente 155 p, 2 . O cromossomo X da maior parte dessas espécies

(Homo sapiens, Canis familiaris, Bos taurus, Equus caballus, Equus asinus, delis

domestica , Mus musculus ), parece ter área quase idêntica e equivalente a apro¬

ximadamente 4,5 p 2 . que é um pouco mais de 5% do lote haplóide da espécie

considerada. No Mesocricetus auratus, o X apresenta aproximadamente o dôbro

de tamanho do X dessas espécies, sendo por nós considerado como duplo e no

Microtus oregoni o X tem mais ou menos três vêzes o tamanho do X dessas

espécies, sendo considerado como triplo. Estudando o comportamento hetero-

picnólico dos três tipos de cromossomos X nas células somáticas, verificamos que

êstes apresentam padrões diferentes. Se admitirmos que a heteropicnose de um

cromossomo ou parte dêle significa inércia genética então, apesar das diferenças

de tamanho dos três tipos tôdas as espécies estudadas apresentam aparentemente

a mesma quantidade de material cromossômico X funcional, em ambos os sexos.

Nos ofídios o cariótipo mais comumente descrito, incluindo o presente tra¬

balho, é o que possui 36 cromossomos compreendendo 16 macroeromossomos e

20 microcromossomos. No entanto, cariótipos com número cromossômico dife¬

rente têm sido encontrado cm várias espécies. Os valores extremos encontrados

são os das espécies Aenodoa merremii e Clelia orei pi tola teu. presentemente des-

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78fí CONSTITUIÇÃO CROMOSSóMrCA E MECANISMO DE DETERMINAÇÃO DO

SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS SEXUAIS E

EVOLUÇÃO DO CARIÓTIPO

eritos. Na primeira espécie ocorrem 30 e na última 50 cromossomos como nú¬

mero diplóitle.

Com o intuito cie verificar a significação dessa variação numérica quanto à

massa total de material cromossômico c a relação do cromossomo sexual Z com

o complemento cromossômico, resolvemos estudar êsses parâmetros, nos ofídios.

Para possibilitar o estudo comparativo com as espécies de mamíferos pesquisa¬

das usamos o mesmo método e ampliação que haviamos utilizado para o estudo

anterior.

Selecionamos preparações filológicas de sele espécies de serpentes perten¬

centes a três famílias diferentes. Cinco dessas espécies apresentam cariótipo com

36 cromossomos, que é o número mais característico dos ofídios, e duas outras

espécies apresentam a variação mínima e máxima do número de cromossomos

nesse grupo.

Os CROTALIDAE são uma família muito homogênea, sem grandes varia¬

ções, aparentemente. Tôdas as espécies que estudamos têm 36 cromossomos.

Escolhemos as espécies Bothrops jararaca e Bothrops atrox. Os BOIDAE também

são uma família homogênea, apresentando as espécies estudadas como número

diplóide, 36 cromossomos. A única exceção encontrada nos boídeos, foi a espé¬

cie Corallus caninus, que tem 44 cromossomos. Escolhemos para êsse estudo as

espécies Boa conslrietor amarali e Epicrates cenchria crassas. Quanto aos colu-

brideos a heterogeneidade é maior. Com o intuito de estudar a significação da

variação numérica dos cromossomos, escolhemos para essa investigação três es¬

pécies, Spilotcs pnllatus anornalepis , que tem como número diplóide 36 cromos¬

somos, com cariótipo basicamente semelhante ao das outras espécies de serpen¬

tes com 36 cromossomos; A enodoa merremii, cujo cariótipo nas células diplóides

apresenta só 30 cromossomos e Clelia occipitolutea, na qual o número cariotípico

é 50 cromossomos, que é o maior número descrito até o presente, nas serpentes.

Com exceção de Boa constrictor amarali e Epicrates cenchria crassus, os

cromossomos sexuais podem ser fàcilmente identificados nas outras espécies, de¬

vido ao seu heteromorfismo nas células das fêmeas, que constituem o sexo heté-

rogamético.

As metáfases somáticas, utilizadas para o estudo comparativo da massa cro-

mossômica e do cromossomo X em diferentes espécies, foram selecionadas de

preparações citológicas obtidas em condições semelhantes. Tôdas as células pro¬

vém de culturas temporárias de leucócitos, sendo as preparações citológicas feitas

de acordo com as técnicas já descritas anteriormente e que compreendem trata¬

mento hipolônico das células durante dez minutos. A coloração foi a de orceína

acética ou Giemsa. A seleção das metáfases utilizadas obedeceu aos seguintes

critérios: cada cromossomo deveria estar uniformemente corado, bem delineado

e com as cromátides irmãs claramente separadas. A célula deveria estar bem

achatada e os cromossomos no mesmo plano focal. Por êsses critérios pudemos

eliminar as variações decorrentes principalmente dos diferentes graus de hidra¬

tação sofridas pelas células durante a preparação citológica.

As medidas foram feitas da seguinte maneira: microfotografias de cinco

metáfases foram selecionadas para cada espécie. Os negativos foram feitos com

os aumentos 400 X e. 2.200 X. Os filmes utilizados foram o Microfile e o

Contrast Process Filrn, ambos da Kodak. O negativo era colocado no ampliador

e a imagem projetada numa fôlha de papel branco (“Victoria”, Ind. Brasileira

ou “Plover Bond”, U.S.A.). O aumento final era de 6.300 X. Os limites dos

cromossomos eram desenhados e as imagens recortadas e pesadas numa balança

analítica.

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Os recortes cios cromossomos Z e W eram pesados separadamente. A per¬

centagem do Z no lote haplóide (Z :AZ) foi calculada individualmente para

cada uma das cinco células e a média aritmética foi determinada. 0 pêso do

lote haplóide dos autossomos foi calculado da seguinte forma:

_ (pêso total) — (pêso do Z, + Z 2 (on W)

Pesando-se recortes de papel de 63 X 63 mm, que correspondem a área de

100 p 2 (10 p X 10 p) obtivemos um valor médio de 0,3458 g (0,3332-0,3640)

para o papel “Vietoria ' e 0.3332 g (0,3220-0,3440) para o “Plover Bond . Pelo

uso dêsses fatores de conversão, as áreas cromossômicas do Z e do lote diplóide

puderam ser calculadas pelos pêsos dos recortes.

As médias dos valores encontrados para a área cromossômica total, assim

como, para os cromossomos sexuais Z e W e as suas proporções em relação ao

lote haplóide, estão sumarizadas na Tabela que segue.

Na coluna correspondente ao pêso total dos cromossomos, os números entre

parêntesis representam os valores mínimo e máximo encontrados em cada espe-

eie. A média dos pêsos totais das sele espécies é de 0,2744 g. A segunda

coluna foi obtida por conversão da média do pêso total em área, em cada espé¬

cie. Comparando-se as áreas totais das sete espécies investigadas, verificamos

que em média êsse valor é de 81.04 p 2 , sendo que o limite de variação entre

os valores extremos é de aproximadamente 7 p 2 (78,81-85,86 p 2 ).

Na coluna correspondente ao cromossomo sexual Z, o pêso médio dêsse

cromossomo nas sete espécies é de 0,0128 g que corresponde em área a 3,78 p".

Nas duas espécies de boideos incluídas consideramos, para efeito de pesagem,

o quarto par de cromossomos, por analogia às espécies de colubrídeos e crota-

lídeos com 36 cromossomos, onde o cromossomo Z corresponde ao quarto par

em ordem de tamanho. A relação entre o cromossomo Z e o lote haplóide

(Z : AZ) é de 9,38% em média, nas várias espécies estudadas.

Na coluna correspondente ao cromossomo sexual W, a média dos pesos en¬

contrados foi de 0,0127 g. que corresponde em área a 3,78 p 2 , à semelhança

do valor encontrado para o cromossomo Z. A relação entre o cromossomo W

e o lote haplóide é de 9,41%, em média, variando porém de 6,46% na \ cnodon

merremii a 18,20% na Clelia occipitolutea.

Nas espécies de CROTALIDAE estudadas, fíothrops jararaca e Bothrops atro.x.

as áreas do cromossomo W foram de 2,85 p 2 e 2,5 p 2 , respectivamente, en¬

quanto que as áreas correspondentes do cromossomo Z foram de 9.57 p e

8,30 p 2 . Êsses resultados estão de acordo com as observações de Schreiber,

Menin, Cavenaghi e Fallieri (57). Êsses autores estudaram, por método eito-

fotométrico, as diferenças sexuais no conteúdo em ADN, dos^ núcleos de eiitró-

cilos de ofídios. Num grupo homogêneo de seis espécies do gênero Bothrops.,

verificaram que o teor de ADN é sempre maior nos machos do que nas fê¬

meas. Algumas espécies apresentaram esta diferença estatisticamente signifieante

(Bothrops jararaca , Bothrops cotiara e Bothrops jararacussu) ; nas demais ( Bo-

throps atrox, Bothrops alternatus e Bothrops neuwiedi) a diferença não foi

significativa, mas sua tendência foi no sentido esperado, isto é. de que o macho,

sendo o sexo homogamético (ZZ), apresenta maior quantidade de ADN que a

fêmea, que é o sexo helerogamético (ZWj.

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Determinamos também, para (‘feito de comparação, a área total eromossô-

mica em duas outras espécies pertencentes a subordens diferentes de répteis:

Anolis carolinensis (LACERTILIA), cujo número diplóide é de 36 cromossomos

e Amyda jcrox (CHELONIA), cujo número diplóide é de 66 cromossomos. En¬

contramos para a primeira espécie uma área total de 82,41 p. 2 e para a segunda

de 79,92 p 2 . Esses valores, apesar da variação do número diplóide, são seme¬

lhantes entre si e por sua vez também semelhantes aos determinados para os

ofídios.

A área total cromossômica em tôdas as espécies estudadas é praticamenle

constante, independente da variação do número de cromossomos do cariótipo.

Isso sugere que os rearranjos estruturais, envolvendo pouca ou nenhuma perda

na quantidade total de material genético, parecem ter desempenhado um papel

importante na espeeiação desses animais. Espécies próximas com diferentes nu¬

meros de cromossomos exemplificam hem êsse fenômeno (58). Mesmo rearranjos

mais complicados raramente parecem representar uma perda ou ganho substan¬

cial de material genético. Assim por exemplo, em três espécies de macacos do

Velho Mundo, o número cromossômico diplóide varia de 42 a 60, mas os valo¬

res médios de ADN são essencialmente os mesmos (59).

Na célula diplóide de serpentes, a área total de cromossomos (81 p 2 )

corresponde aproximadamente á metade do valor determinado para os mamíferos

placentários (155 p 2 ). No entanto, o valor absoluto da área do cromossomo Z

dos ofídios é somente 15% menor que o determinado jiara o cromossomo X dos

mamíferos.

As observações nos mamíferos e nos ofídios sugerem que o cromossomo X

ou Z, que aparentemente acumula fatores governando o desenvolvimento do sexo

homogamético, tende a reter, dentro de cada grupo, o tamanho original, através

da evolução.

Microcromossomos

Neweomer e llrant (43) estudando a espermatogênese de Gallus domesticus,

em material fixado ]>or uma mistura a base de ácido propiônico, concluiram que

o número de cromossomos nessa espécie não é constante e é muito menos elevado

do que o descrito, em 17 raças de galinha, por Yamashina (26), que encontrou

77 cromossomos na fêmea e 78 no macho. O êrro nas avaliações numéricas seria

devido ao comportamento aberrante dos microcromossomos, que se formariam

durante a prófase a partir de segmentos helerocromáticos dos macrocromossomos.

Os microcromossomos seriam supernumerários, teriam um eentrômero difuso e

se multiplicariam por simples fragmentação. Ao fim duma divisão êles seriam

novamente reabsorvidos pelos grandes elementos. Segundo os autores, na pré-

prófase do espermatóeito primário, massas heterocromáticas seriam visíveis no

núcleo e progressivamenle condensar-se-iam através do zigóteno e paquíteno, agru¬

pando-se ou loealizando-se entre os cromossomos, obscurecendo freqüentemente as

relações cromossômicas. Esses corpúsculos heteropicnóticos positivos poderiam

ainda ser observados em associações com os cromossomos através do paquíteno

e diplóteno, mas gradualmentc condensar-se-iam eoaleseendo freqüentemente no

início da diacinese num número variável de estruturas cromossomóides, as vezes,

com formações aparentes de quiasmas. Segundo Neweomer (60). também na pró¬

fase mitótica os microcromossomos não existiriam como entidades individuais,

agregando-se para formar massas heteropienóticas positivas.

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constituição cromossômica e mecanismo de determinação do

SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS SEXUAIS E

EVOLUÇÃO DO CARIÓTIPO

investigações an¬

de microcromos-

lambém nessas eélu-

Km contraste com essa descrição, Olmo (27), confirmando

teriores de van Brink (17) distinguiu perfeitamente os microcromossomos corno

tais em tôdas as prófases mitóticas de gônadas embrionárias de Galliis domes¬

ticas e segundo êsse investigador êles nunca se apresentam com hcteropicnose

positiva. Na fase de paquíteno, os elementos menores do grupo

somos podem ser reconhecidos como bivalentes individuais e

las não apresentam hcteropicnose positiva. Os microcromossomos retêm sua indi¬

vidualidade e não são heteropicnòticamcnte positivos em qualquer estágio da

mitose, ou da meiose.

Nossas observações em diferentes espécies de cobras levam-nos a concluir que,

nos ofídios, à semelhança do que van Brink (17) e Olmo (27) demonstraram na

galinha, os microcromossomos são realmente estruturas cromossômicas com com¬

portamento idêntico ao dos macrocromossomos é só diferindo dos mesmos pelo

tamanho. Mantém sua individualidade durante tôdas as fases da divisão celular

e não apresentam heleropicnose positiva quer na

células de ofídios, em que os cromossomos não se

distinguimos perfeitamente a existência de uma

cenlrômero nos microcromossomos. Portanto, não

trômero difuso imaginada por Newcomer e Brant,

sença de um cenlrômero individualizado nos

Na mitose final de certas metáfases, parte

mitose quer na meiose. Nas

apresentam muito condensados,

constricção correspondente ao

é aceitável a hipótese do cen-

que não identificaram a pre-

microcromossomos da galinha.

ou todos os microcromossomos

apresentam as duas cromátides já separadas. Êsse fenômeno é, porém, perfeita¬

mente explicável, levando-se em consideração que devido ao seu tamanho redu¬

zido as cromátides dos microcromossomos separam-se antes que as dos macro¬

cromossomos. Além do mais, é frequente encontrar-se nas metáfases também

macrocromossomos com as duas cromátides completamente separadas. Êsse as¬

pecto não serve, portanto, para diferenciar os microcromossomos dos cromossomos

verdadeiros como pretendem Newcomer e Brant.

Verificamos que a utilização de técnicas filológicas não apropriadas para o

estudo dos cromossomos de ofídios poderiam conduzir a resultados confusos. Em¬

pregando técnicas de cortes histológicos em parafina ,os microcromossomos, devido

ao seu tamanho reduzido, não são às vêzes incluídos no corte, o que pode difi¬

cultar a determinação precisa do seu número. O mesmo pode acontecer com a

técnica de esmagamento que, por rompimento mecânico das células, pode resultar

na perda de um ou mais cromossomos com distorsão na determinação do número

correio de cromossomos existentes no cariólipo. Por outro lado, verificamos que

a técnica de coloração empregada também influi nesse aspecto. Certos corantes

que, às vêzes, coram com pouca intensidade, como por exemplo o de Feulgen,

ou que descoram após algum tempo, como o azul de toluidina, podem dificultar

a observação dos microcromossomos, que pelo seu porte reduzido apresentam

freqüentemente resolução crítica ao microscópio óptico comum. Corantes como

a fucsina básica, quando não apropriadamente utilizados, resultam numa fusão

aparente de cromossomos muito próximos e no caso dos microcromossomos podem

aparentar coalescência. Como a constituição cromossômica das aves é comparável

à dos ofídios, no que diz respeito à existência de microcromossomos, é possível

que as técnicas usadas por Newcomer e Brant tenham levado êsses autores a con¬

clusões incorretas quanto à natureza dos microcromossomos.

Uma hipótese que explicaria a origem dos microcromossomos é de que êsles

derivariam de fragmentos provenientes da fusão cênlrica, de cromossomos aero-

cêntricos. Através de um mecanismo "Hobertsoniano a fusão de dois acro-

eêntrieos daria origem a um metacênlrieo. Poderíamos imaginar que em decor¬

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WILLY BECAK

791

rência dessa Iransloeação surgisse um pequeno fragmento portador de centrô-

mero (|iie viesse a constituir um microcromossomo. Essa hipótese, no entanto,

não é satisfatória para explicar os nossos achados em espécies nas quais ocorrem

somente macrocromossomos acrocêntricos mas que possuem ainda assim conside¬

rável número de microeromossomos. CoruHus caninus (BOIDAE), cujo cariótipo

tem 44 cromossomos na célula diplóide, apresenta 24 macrocromossomos lodos

acrocêntricos e 20 microeromossomos. Em Clelia occipitolutea (COLUBRIDAE),

cujo cariótipo tem 50 cromossomos, êsse aspecto é ainda mais acentuado, pois,

nessa serpente a célula diplóide apresenta 11 macrocromossomos todos acrocên¬

tricos, com exceção do cromossomo sexual Z, e 36 microeromossomos. Portanto,

nessas espécies, apesar de não ocorrerem macrocromossomos metacêntricos, o nú¬

mero de microeromossomos não é menor que o encontrado nas espécies em

que ocorrem vários metacêntricos, como por exemplo, Boa constrictor amaral i

(BOIDAE), fíothrops jararaca ( CROTALIDAE ) e Spiloles pullatus anomalepis

(COLUBRIDAE).

Evolução do Cariótipo

Antes de considerar o mecanismo e evolução cariotípica nos ofídios, é inte¬

ressante examinar soh um ponlo de vista geral a relação do número de cro¬

mossomos com outros fatores de significação taxonômica ou evolutiva. Segundo

Swanson (61), os dados existentes não permitem no reino vegetal ou animal

uma correlação direta entre o número de cromossomos e a posição filogenética.

Essas correlações, quando existentes, são geralmente válidas somente nos limites

da família ou do gênero.

Quando se trata de mudança do número cromossômieo e o fator poliploidia

pode ser excluído, o prohlema principal é o de determinar o mecanismo que

leva a essa mudança. Uma maneira teórica de explicar êsse fenômeno foi suge¬

rida por Darlington (62). O esquema proposto é de que o ganho e perda de

cromossomos de um lote básico envolve ganho ou perda de centrômeros e o êxito

dessa mudança dependeria da existência na região adjacente ao centrômero de

heterocromatina ou eucromalina.

Mudanças do número básico cm Drosophila foram extensivamente in¬

vestigadas e indicam que reduções no número são frequentes enquanto aumentos

são raros, sòmenle sendo conhecida em Drosophila trispina (n =: 7). O nu¬

mero básico primitivo seria 6. compreendendo 5 bastonetes com centrômero sub-

terminal e um cromossomo cm forma de grânulo. Essa configuração é encon¬

trada em espécies como Drosophila virilis, D. junehris, D. repleta e D. tripunc-

tata. A grande maioria de espécies, porém, mostra um complemento modificado

e as variações aparentemente teriam decorrido de translocações (incluindo as

fusões cêntricas), de inversões pericêntricas e de alterações da quantidade de

heterocromatina (63).

A importância do processo de fusão cênlrica foi evidenciada já em 1916(64)

por Hoberlson e é porisso também denominada por alguns autores como lei

ou regra de Robertson”. Êsse autor mostrou que nos ortópteros os cromosso¬

mos metacêntricos poderiam ser considerados como resultado de fusão apical de

dois cromossomos acrocêntricos não homólogos. Êsse conceito, que foi verificado

num grande número de casos, previa, no entanto, a existência de cromossomos

com centrômero terminal (telocêntricos). As fusões cêntricas podem, atualmente,

ser consideradas como um tipo especial de Iransloeação. envolvendo os braços

inteiros de cromossomos acrocêntricos e parecem ser peculiares do reino animal.

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*' J * SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS SEXUAIS E

EVOLUÇÃO DO CARIÓTIPO

Em Drosophila , as fusões cênlricas são possíveis e mais prováveis que

outros lipos de translocações talvez por causa dos grandes blocos de heterocro-

matina na região do centrômero. A perda de um centrômero e da sua hetero-

cromatina adjacente não deve aparentemente produzir efeito marcante na viabi¬

lidade quando comparado com a perda de eucromatina. Nos casos de animais

híbridos interespecíficos, em que é impossível uma análise meiótica ou de cro¬

mossomos do tipo glândula salivar, não se pode verificar se os cromossomos meta-

cêntricos provieram de fusão ou de inversão pericêntrica, a menos que os com¬

primentos dos braços nesses elementos possam ser usados para distinguir entre

os dois tipos de alienação.

Estudos recentes de Wahrman nos mantídeos do gênero A meles constituem

um bom exemplo de uma relação “Robertsoniana”, que se manifesta tanto intra

como interespecíficamente. As populações de três espécies de mantídeos do gê¬

nero A m e l e s são heterogêneas em relação aos complementos cromossômicos dos

seus membros. Em cada espécie êsse polimorfismo depende de um ou vários

cromossomos, que podem estar representados como um metacêntrico ou dois

acrocêntricos. Os números cromossômicos diplóides variam de 18 a 29, mas

o número total de 30 braços permanece constante assim como a quantidade

média de ADN por núcleo ( 65, 66). Também nos mamíferos, casos bem do¬

cumentados de filogenia cromossômica confirmando a hipótese de Robertson têm

sido relatados, por exemplo, no roedor do gênero Gerbillu s(58). A trans-

locação do tipo fusão cêntrica parece prevalecer como fator de mudança cromos¬

sômica nos animais, enquanto que nas plantas a maioria das translocações ana¬

lisadas envolvem somente uma parte e não todo o braço.

Mudanças podem também ocorrer se um cromossomo em forma de V se

quebra para formar dois cromossomos em bastonete. Não é sabido, porém,

nesse caso, se o centrômero extra deriva de um acessório ou se o centrômero

original fragmentou-se para dar dois centrômeros menores, mas ainda funcionais.

Centrômeros terminais são instáveis, pelo menos em milho, mas é possível que

a estabilidade possa ser adquirida, em certos organismos (67).

Outro tipo de mudança, que introduz variações cariotípicas, é o ganho ou

perda de heterocromatina. Onde consideráveis quantidades de heteroeromalina

são encontradas no cariótipo, alterações na forma, tamanho e número de cromos¬

somos podem ocorrer aparentemente sem sacrifício das porções eucromáticas.

Como já foi dito, as fusões cêntricas em animais, envolvendo, provavelmente, só

regiões heterocromátieas assim como perda ou ganho de centrômeros com hete¬

rocromatina adjacente, não têm efeito drástico no indivíduo. Nêsse sentido, uma

certa quantidade de heterocromatina é evolutivamente desejável como fator de

segurança, permitindo um grau de variabilidade cariotípica maior que o sistema

composto somente de eucromatina (61).

Parece, porém, que os rearranjos estruturais dos cromossomos com pequena

ou nenhuma mudança na quantidade total de material têm função muito impor¬

tante na evolução dos animais. Nos organismos capazes de reprodução sexual,

a formação de mediocêntricos a partir de acrocêntricos resulta em diferença no

número de grupos de permutação, nos índices de recombinação e nas posições

relativas de certos genes. Rearranjos estruturais dêsse tipo podem determinar

diferenças nos valores adaptativos de seus portadores.

Segundo White (68) as mudanças estruturais, que são positivamente selecio¬

nadas, seguem o que pode ser denominado de "princípio da Iranslocação homó¬

loga”, isto é, cromossomos passam pelo mesmo tipo de modificação na mesma

sequência. Isso torna-se compreensível quando consideramos os cromossomos não

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WILLY BEÇAK

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como simples conjuntos lineares de genes, mas, como entidades organizadas com

uma sequência que determina as suas relações com o fuso, as suas permutações

e seus efeitos de posição. É, portanto, natural que o rearraujo estrutural, que se

estabelece na evolução, deva ser de um ou dois tipos principais em todos os

cromossomos de um determinado grupo, resultando assim numa situação muito

comum, onde todos os cromossomos ou membros cromossômicos são aproxima¬

damente do mesmo tamanho e têm uma distribuição similar de segmentos hetero-

cromáticos.

Os resultados dos nossos estudos, em vinte espécies das famílias BOIDAE,

CROTALIDAE e COLUBRIDAE, quando justapostos aos estudos sistemáticos mais

recentes (69), permitem-nos sugerir um esquema de evolução para as espécies

por nós investigadas.

Dentre as serpentes que estudamos, a família mais primitiva seria a dos

BOIDAE. Nessa família, Corallus caninus é a única espécie em que encontra¬

mos 44 cromossomos, apresentando as outras 36 cromossomos. Os macrocromos-

somos de Corallus caninus são todos acrocêntricos e poderiamos, à base de um

mecanismo “Robertsoniano”, imaginar que êsse tipo de constituição cromossô-

mica teria dado origem a cariótipos encontrados em outros BOIDAE. Fusões

cêutricas de alguns cromossomos, acompanhadas de outras alterações como inver¬

sões e translocações, explicariam os cariótipos com 36 cromossomos encontrados

nos gêneros Roa, Eunectes e E pie rates. Neuhuma cobra dessa famí¬

lia apresentou heteromorfismo cromossômico em qualquer dos sexos, o que suge¬

riria um primitivismo do mecanismo cromossômico de determinação do sexo

dessas serpentes em relação às que possuem mecanismo mais diferenciado.

Nos COLUBRIDAE, as espécies estudadas dos gêneros S p i l o t e s , Phi-

l o d r y a s , Dryadophis, Drymarchon e C hi r o niu s , apresentam

36 cromossomos, semelhantes aos dos BOIDAE. No entanto, nesses gêneros já

encontramos heteromorfismo dos cromossomos sexuais na fêmea. 0 aspecto dêsses

cromossomos sexuais parece-nos particularmente interessante. O Z e W, apesar

de morfologicamente diferentes, têm tamanho semelhante. Admitindo que uma

inversão pericêntrica teria ocorrido num dos homólogos do quarto par, a troca

de genes seria reduzida entre êsses cromossomos. Em conseqüência dêsse meca¬

nismo de isolamento os cromossomos poderiam tornar-se cada vez mais diferen¬

ciados quanto aos fatores sexuais. Uma hipótese é de que êste tipo de consti¬

tuição cromossômica seria dentre os COLUBRIDAE um elo com os BOIDAE

Se admitíssemos que as opistoglifas são mais evoluídas que as áglifas, as ser¬

ão mecanismo cromossômico de determinação do sexo.

Os gêneros S pilotes, Dryadophis, Drymarchon e. Chi-

ronius são constituídos de espécies áglifas, enquanto que o gênero Phi lo-

dryas, cujo cariótipo e cromossomos sexuais são semelhantes, é opistóglifa.

Se admitíssemos que as opistoglifas são mais evoluídas que as áglifas, as ser¬

pentes do gênero Philodryas seriam mais evoluídas que as outras. No

entanto, atualmente, muitos sistematas consideram que êsse critério nem sempre

é verdadeiro (70).

O colubrídeo Xcnodon merremii apresenta como número diplóide somente

30 cromossomos. Recorrendo ainda ao mecanismo “Robertsoniano” poderíamos

imaginar que o seu cariótipo se tenha originado, principalmente, por fusões

cêntricas do tipo da constituição cromossômica apresentada pelas espécies de

COLUBRIDAE, com 36 cromossomos. Também o grau de heteromorfismo dos

seus cromossomos sexuais é maior, pois já teria ocorrido no cromossomo sexual

W uma redução de tamanho por eliminação de material cromatínico.

I, | SciELO

7Q4 CONSTITUIÇÃO CKOMOSSÕMICA E MECANISMO DE DETERMINAÇÃO DO

SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS SEXUAIS E

EVOLUÇÃO DO CARIÓTIPO

For sua vez, o cohibrídeo Clelia occipitolutea, com 50 cromossomos, é a

espécie com o maior número diplóide que encontramos. Todos os cromossomos

são acrocêntricos, com exceção do cromossomo sexual Z que é metacêntrico.

Nessa espécie, excepcionalmente, o cromossomo sexual W é maior que o Z, cons¬

tituindo o maior cromossomo do eariótipo. Essas características de número di¬

plóide alto e cromossomo W grande, torna difícil a sua correlação com as outras

espécies. Talvez o tipo de constituição eromossômica apresentada por Clelia

occipitolutea decorra de uma linha paralela de evolução cujos cariótipos ances¬

trais desconhecemos.

Na família CROTALIDAE, que é aparentemente a mais evoluída, as espé¬

cies do gênero Bothrops e C r o t alu s apresentam 36 cromossomos e, pro¬

vavelmente, derivaram dos COLUBRIDAE com número diplóide igual. Essas

espécies apresentam acentuado heteromorfismo dos cromossomos sexuais, na fê¬

mea. O cromossomo W resultaria de um W do tipo encontrado, por exemplo,

na S pilotes, por eliminação de material eromossômico (vide proporções na

tabela).

Resumo e Conclusões

() cromossomo Z, nas serpentes apresenta morfologia muito semelhante; a

quantidade de material dêsse cromossomo foi avaliada através de um método in¬

direto. O seu valor absoluto é semelhante cm sete espécies investigadas e tam¬

bém a proporção do Z em relação ao lote haplóide (Z : AZ) é constante. Pelo

mesmo método foi avaliada a quantidade de material de lodo o lote cromossô-

mico, encontrando-se valores similares para as diferentes espécies. Na célula

diplóide de serpentes, a área total dos cromossomos corresponde, aproximada¬

mente, à metade do valor determinado para os mamíferos placentários. A cons¬

tância da área total eromossômica sugere que os rearranjos estruturais acompa¬

nhados de pouca ou nenhuma perda na quantidade total de material genético

parecem ler desempenhado papel importante na especiação dêsses animais.

Nas espécies de COLUBRIDAE dos gêneros Spilot.es, /' h ilodryas,

Dryadophis, Drymarcon e Chironiu s , os cromossomos sexuais

têm o mesmo tamanho, diferindo pelo centrômero, que é submediano no Z e

suhterminal no W. Êsse aspecto morfológico sugere que tenha ocorrido uma

inversão pericêntrica num dos homólogos do quarto par de cromossomos, redu¬

zindo assim consideràvelmente a permutação. Esse mecanismo de isolamento

teria possibilitado um maior acúmulo diferencial de genes relacionados a deter¬

minação do sexo nesses heterocromossomos. Nos CROTALIDAE, o tamanho re¬

lativamente menor do cromossomo W pode ler decorrido de uma redução por

eliminação de material cromatínico.

Em duas espécies investigadas das famílias BOIDAE

foi observado em nenhum dos sexos a existência de um

dente à cromatina sexual, na periferia interna do núcleo,

somáticas e de espermatogônias não demonstraram a existência de qualquer ma-

erocromossomo heteropicnótico. Nenhum dos cromossomos sexuais apresenta he-

teropienose positiva, nessas fases. Nos répteis, aparentemente, não existe um

mecanismo de compensação de dose para os cromossomos sexuais, como nos

mamíferos.

Eoi estudada a espermalogênese de Bothrops jararaca, observando-se nessa

c em outras espécies de ofídios a ocorrência de um número relativamente alto

dc quiasmas, o que sugere a sua maior semelhança com as aves do que com os

e CROTALIDAE nao

corpúsculo correspon-

amhem as

nrófa;

ses

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33(3):775-798, 1966

WILLY BEÇAK

795

anfíbios que lêrn número relativamente baixo de quiasmas. A presença nos

répleis e nas aves de microcromossomos que não ocorrem, aparentemente, nos

anfíbios é também um fator que sugere a proximidade entre os dois primeiros

grupos citados.

Nas serpentes os microcromossomos são realmente estruturas cromossômicas

com comportamento idêntico ao dos macrocromossomos, dos quais diferem só

pelo tamanho. Mantêm sua individualidade durante tôdas as fases da divisão

celular e não mostram heteropicnose positiva quer na mitose quer na meiose.

Foi identificada nos microcromossomos a presença de uma constricção, correspon¬

dente ao centrômero dos macrocromossomos. Em Bothrops jararaca verificou-se

que o nucléolo encontra-se associado a quatro ou cinco microcromossomos.

Dentre as serpentes estudadas, a família mais primitiva seria a dos BOIDAE.

Nessa família, Corallus caninas com 2n ■= 44, sendo todos macrocromossomos

aerocêntricos, seria a espécie mais primitiva. Êsse tipo de constituição cronios-

sômica teria dado origem aos cariótipos encontrados nos gêneros Boa. Ba¬

ne c t e s e Epicrates.

Nos COLUBRIDAE várias espécies apresentam 36 cromossomos, semelhan¬

tes aos dos BOIDAE, mas já possuem cromossomos sexuais heteromórficos. ()

tipo de constituição cromossômica de S pilotes, Philodryas, Drya-

dophis, Drymarchon e Chironius representaria um elo com os

BOIDAE. Êsse tipo de constituição teria dado origem ao de Xenodon mer-

remii, que tem 2n = 30 e maior heteromorfismo dos cromossomos sexuais. A

espécie delia occipitolutea tem 2n = 50 e um cromossomo Y\ com aproxima¬

damente o dôbro do tamanho em relação ao X; a sua correlação com as outras

espécies de COLUBRIDAE é difícil, decorrendo talvez de uma linha paralela de

evolução.

Na família CROTALIDAE, que é a mais evoluída, os gêneros Bothrops

e C r o I a I u s têm 2n = 36 com heteromorfismo acentuado dos cromossomos

sexuais, na fêmea. Êsses gêneros derivaram provavelmente dos COLUBRIDAE,

com igual número de cromossomos.

SUMMARY AM) CONCLUSIO.NS

amount of material

seven species

The X-chromosomes in snakes are very similar. The

of Ihis chromosome calculated by an indirect method is lhe sana

investigaled; the ratio of lhe Z to the haploid set of chromosomes is also constant.

The total amount of chromosome material in

a snake cell

total area suggests

the total amount

lhe speeiation of

D r y a d o p h i s .

same hui lhe chromosome area of

inammal. The constancy of lhe

ments. involving liltle or no loss

to have played an importanl role in I

S p i lo l <’ s , /' h i I o d r y a s .

the different species is about the

only half of that of a placental

p-sts tliat structural rearrange-

of genetic material, seems

lliose animais.

D r y m ar ch o n and

Chironius of lhe family COLUBRIDAE, the sex chromosomes are of the

same size. differing only by the centromere which is suhmedian in Z and sub-

terminal in W. This as|iecl suggests that a pericentric inversion had occurred

pair of chromosomes, crossing-over between the

reduced. This mechanism of isolation resulted

of sex determining genes in lhe heteroehromo-

the relative smaller size of the W might have

of rhromosomal material.

in one element of the fourth

homologues heing considerahly

in a differential accumulation

somes. In the CROTALIDAE,

hnppencd through elimination

SciELO

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7QK CONSTITUIÇÃO CROMOSSÔMICA E MECANISMO DE DETERMINAÇÃO DO

1 C7 ' J SEXO EM OFÍDIOS SUL-AMERICANOS. II. CROMOSSOMOS SEXUAIS E

EVOLUÇÃO DO CARIÓTIPO

In lhe analysed cells of Iwo species of BOIDAE and CROTALIDAE,

it was not possible lo identify in either sex what could be considered a sex

chromatin body. The somatic and spermatogonia prophases did not reveal lhe

existence of any heteropycnolic macrochromosome. Apparently, a dosage com-

pensation mechanism such as in mammals does not exist in repliles.

The spermatogenesis of Bothrops jararaca was studied. In this and other

snake species, the occurrence of a relative large numher of chiasmas was observed,

suggesling a greater similarily wilh birds than wilh amphibians whieh have a

relative smaller numher of chiasmas. The presence of microchromosomes in

reptiles and birds, and their absence in amphibians, is anolher factor that

suggests a closer relationship between lhe two first groups mentioned.

The microchromosomes of snakes are actually chromosomal slructures with

a pattern similar to the macrochromosomes, from which they differ only by size.

Their individuality is maintained during all phases of cell division and they are

not heteropycnotic, neither in mitosis nor in meiosis. In Bothrops jararaca the

nucleolus is associated to four or five microchromosomes.

Among the snakes studied, the most primitive species are lhe BOIDAE.

Within this family, Corallus caninus with its 44 chromosomes, all of their macro¬

chromosomes heing acrocentrics, would be the most primitive species. This type

of chromosomal constitution may be related lo lhe one that gave origin to lhe

karyotypes found in Boa, Eunectes and Epicrates.

In COLUBRIDAE several species present 36 chromosomes, similar to lhose

of lhe BOIDAE, but they already show heteromorphic sex chromosomes. This

lype of constitution could have originated the one of Xenodon merremii which

lias 2n = 30, and more accentuated heteromorphism of the sex chromosomes.

Clelia occipitoliitea has 2n = 50 and a W-chromosome, which is about twice the

size of the Z; its correlation wilh the other species of COLUBRIDAE is difficult,

perhaps due to a parallel line of evolution.

In the family CROTALIDAE, the most evoluted, the genera Bothrops

and Cro talas have 36 chromosomes with an accentuated heteromorphism of

the sex chromosomes in the female. These genera probably originated from the

COLUBRIDAE with similar numher of chromosomes.

Agradecimentos — Nossos agradecimentos a M. L. Beçak e H. R. S. Nazareth,

pela colaboração na obtenção dos dados utilizados neste trabalho.

Bibliografia

1. BEÇAK, W., Mem. Inst. Butantan, 32, 37-78, 1965.

2. GORDON, M„ J. Heredety, 37, 307-320, 1946.

3. GORDON, M„ Genetics, 32, 8-17, 1947.

4. GORDON, M„ Zoologica, 32, 127-134, 1951.

5. CASTLE, W. E„ Proc. Natl. Acad. Sei. U. S., 22, 679-682, 1936.

6. YAMAMOTO, T„ Genetics, 44, 739-757, 1959.

7. YAMAMOTO, T„ J. exy. Zool., 146, 163-180, 1961.

8. BELLAMY, A. W., and QUEAL, M. L„ Genetics, 36, 93-107, 1951.

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(3): 775-798, 1966

WILLY BEÇAK

797

9. BREIDER, H., Biol. Zenír., 62, 183-195, 1942.

10. MATTHEY, R., Les chromosomes des vértébrés, F. Rouge, Lausanne, 1949.

11. HUMPHREY, R. R., Am. J. Anat., 76, 33-66, 1945.

12. HUMPHREY, R. R., J. exp. Zool., 109, 171-185, 1948.

13. YAO, F. S., e OLSEN, M. W., J. Hered., 46, 133-134, 1955.

14. POOLE, H. K., e OLSEN, M. W., J. Hered., 48, 217-218, 1957.

15. DANTCHAKOFF, V., Arch. Anat. Micr. Morph. Exp., 39, 368-394, 1950.

16. NOGUSA, S., Cytologia (Tokyo), 20. 11-18, 1955.

17. BRINK, J. M. VAN, Chromosoma (Berl.), 10, 1-72, 1959.

18. WITSCHI, E., Z. Zellf. mikr. Anat., 1, 523-561, 1924.

19. YOSIDA, T. H., J. Fac. Sei. S. 6 Hokkaido Univ., 13, 352-358, 1957.

20. CHANG, C. Y., e WITSCHI, E., Proc. Soc. exp. Biol. Med., 89, 150-152, 1955.

21. GALLIEN, L., Buli. Biol. France et Belgique, 90, 163-183, 1956.

22. WEILER, C., e OHNO, S., Cytogenetics, 1, 217-223, 1962.

23. SUZUKI, K., Zool, Mag. (Japan), 42, 358-362, 1930.

24. MILLER, R. A., Anat. Rec., 70, 155-189, 1938.

25. OGUMA, K., Annot. Zool. Japon, 17, 612-622, 1938.

26. YAMASHINA, Y., Cytologia (Tokyo), 13, 270-296, 1944.

27. OHNO, S., Chromosoma (Berl.), 11, 484-498, 1961.

28. MATTHEY, R., e BRINK, J. M. VAN, Evolution, 11, 163-165, 1957.

29. OGUMA, K., Arch. Biol. (Fr.), 45, 27-46, 1934.

30. OGUMA, K., J. Genet., 34, 247, 1937.

31. MAKINO, S., e ASANA, J. J., Chromosoma (Berl.), 3, 208-219, 1948.

32. NAKAMURA, K., La Kromosomo, 5/6, 205-213, 1949.

33. MAKINO, S., Annot. Zool. Japon., 25, 250-257, 1952.

34. SUZUKI, K., Jap. J. Genetics, 25, 222, 1950.

35. MATTHEY, R., Arch. Klaus Stift. Vererb.-Forsch,, 18, 1-16, 1943.

36. MARGOT, A., Rev. Suisse Zool., 53, 555-596, 1946.

37. MATTHEY, R., e BRINK, J. M. VAN, Experientia (Basel), 12, 53, 1956.

38. MATTHEY, R., Rev. Suisse Zool, 64, 709-732, 1957.

39. KOBEL, H. R., Experientia, 18, 173-174, 1962.

40. BEÇAK, W., BEÇAK, M. L., e NAZARETH, H. R. S., Cytogenetics, 1, 305-

313, 1962.

41. OHNO, S„ WEILER, C., e STENIUS, C., Exp. Cell. Res., 25, 498-503, 1961.

SciELO

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Simp. Internac.

33(3):799-808, 1966

N. C. DE ESPIÍÍOZA

799

15. ACCIÓN DEL VENENO DE HAPALOPUS LIMENSIS

N. C. de ESPI5ÍOZA

Museo de Historia Natural “Javier Prado”, Universidad Nacional Mayor

de San Marcos, Lima, Perú

JntroducctÓN

Sieiulo de importância práctica el estúdio dei veneno de las aranas en nuestro

medio se realizo bajo la dirección dei Dr. Vellard, el presente trabajo, llevado

a cabo en los laboratorios dei Instituto Nacional de Salud dei Ministério de

Salud Pública y Asistencia Social.

Pocos son los científicos que se han ocupado dei estúdio de las aranas vene¬

nosas en el Perú. Las principales observaciones fueron realizadas por Escomel, E.;

Cavassa, N.; Leon, A.; Pardo Figueroa, E.; Pesce, H.; Lumbreras, H.; Izu, W.

y Vellard, J., quienes refieren vários accidentes producidos por la picadura de

las aranas, con muchos casos fatales.

Breve resena de las Theraphosides en el Perú

En los aledanos de Lima: San Bartolo, Quebrada de Paehacamac, se ba

encontrado una especie descrita por Vellard, como Hapalopus limensis.

Escomel refiere la existência de una especie de Mygala en la región de

Arequipa dei género vecino Ischnocolus, sin idenficar la especie; no se

ha podido confirmar Ia veracidade de este dato, ya que puede tratarse de una

espécie afín o de la misma Hapalopus. También se han descrito las es-

pecies: Hapalopus pictus Pocock, colectadas en Caraz (Ancash) ; Hemirrhagus

peruvianus , descrita por Chamberlin, procedente de Huadquina, Quimoehaca,

Urubamba (Cuzeo) y Hemirrhagus major dei valle dei Cuzco, descrita por el

mismo autor.

Dentro de la sub-familia Grammostolinae, Chamberlin ha descrito la espe¬

cie Eurypelma aymara, de Aymas (Apurimac).

Dados descriptivos de hapalopus limensis

A continuación me permito proporcionar algunos datos descriptivos dados

por el Dr. Vellard de la especie Hapalopus limensis (Ischnocolinae, THERA-

PHOSIDAE, ORTHOGNATHA ) : El d* adulto alcanza de 50 a 60 mm de

largo total y la 9 adulta hasta 70 mm.

Coloración : el cefalotórax y las patas de un color castano oscuro,

cubiertos con pelos dorados o amarillo claro; abdômen de largos pelos rojizos.

Patela más tibia dei primer par de patas, más o menos iguales al cefalotórax en

SciELO

10 11 12 13 14 15 16

800

ACCIÓN DEL VENENO DE HAPALOPUS LIMENSIS

ambos sexos. Patela más tibia dei cuarto par, mucho más corto que el tórax

en cl maebo y poco más largo que el tórax eu Ia hembra. En ambos sexos el

cuarto par de palas más largo que el primero. Cefalotórax un |>oeo más largo

que ancho, de forma ovalada, con una foseta torácica, procurvada. Grupo ocular

formando un rectángulo, con bordes paralelos, lo que diferencia completamente

a esta espeeie dei género Hemirrhagus. Los ojos anteriores en línea pro-

eurva, los medianos separados entre sí por su diâmetro y un poco más de los

lalerales; los medianos anteriores un tercio mayores que los lalerales anteriores;

los lalerales posteriores iguales a los lalerales anteriores, de los euales están sepa-

rados por menos de su scmi-diámetro; ojos mediano-posteriores alargados, en

forma de coma. Escópulas de los tarsos de las patas dei primer y segundo

par enteras; escópulas dei tercero y cuarto par divididas completamente hasta

su base por una fuerte línea de cerdas, Io que permite incluir esta espeeie en

el grupo de los IsCHNOCOUNAE. La tibia dei primer par de patas dei macho casi

recta, apenas ligeramente encurvada en su parte inferior y un poco más gruesa

en el ápice, con dos espolones ventrales, uno interno casi recto y otro externo,

arqueado, doble dei anterior; (d tarso se dobla sobre (d dorso externo dei espo-

lón exterior. La división de las escópulas de los tarsos posteriores, la forma

dei grupo ocular, la fórmula ocular, permiten incluir esta espeeie en el género

Hapalopus. Se diferencia de Hapalopus piclus, por su grau tamano y por

su colorido distinto.

Aparato v e n <• n o s o

Los bordes dei artículo basal estan provislos de 11 dientes pequeííos en

el adulto.

Las dimensiones medias de los quelíceros son: Largo

ancho máximo dei diente 2 mm, largo de la basal 12 mm

8 mm, largo total de los quelíceros 20 mm.

Las dimensiones medias de los quelíceros son: largo dei

vecinos, son fáciles de descubrir a la disección. Se dividen en

tintas: la glândula propiamente dicha, ligeramente encurvada

media luna, colocada en Ia convexidad de la parte basal y el

que sale dei polo anterior de la glândula, atraviesa después los dientes,

termina cerca a la punta de los quelíceros.

Las glândulas estan rodeadas de poderosos grupos musculares, euya con-

tracción ayuda a vaciar su contenido en el momento de la mordedura. Las glân¬

dulas se encuenlran siempre libres, sin adhereneias con músculos vecinos. La

inoculación dei veneno es voluntária y la arana puede morder sin inyectar su

veneno. Largo de la glândula: 6 a 7 mm.

El veneno se presenta bajo el aspecto de golitas límpidas, más o menos

viscosas, ligeramente refringenles, solubles en (d agua, muy solubles en Ias solu¬

ciones salinas fisiológicas, dejando por evaporación un polvo amarillento. Las

glândulas trituradas con suero fisiológico dan una suspensión opalescente. El

pH dei veneno es ácido (5.4), el dei suero, alcalino (7.8).

dei diente 8 mm,

ancho de la basal

diente 8 mm,

dos partes dis-

en forma de

canal excretor,

dou de

li i o lo g i a

La postura se realiza en los meses de Enero, Febrero y Marzo, enconlrán-

dose durante (d día ejemplares juveniles después de esta época. Su alimcntación

es de pequenos miriápodos, escorpiones, coleópteros, lagartijas y algunas aranas.

SciELO

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Simp. Internar.

33(3):799-80S, 1966

N. C. DE ESPTnOZA

801

Viven debajo de las grandes piedras; nos fué fácil capturarlos en los lugares

pedregosos duranle los meses de Agosto a Noviembre. mienlras eaminaban sobre

las piedras. Sólo conoeeinos las áreas en que liemos realizado nuestras coleccio-

nes, como son: San Bertolo, Quebrada de Pachaeamac y Matucana. Creemos

que puede existir en otros puntos de la costa dei Perú.

Mateiuaí. y métodos

Los ejemplares, 40 Mygalas, de las cuales 30 machos, 4 hembras y 6 juve-

niles, fueron capturados en lugares pedregosos cerca de los alrededores de Lima

como: San Barlolo, Quebrada de Pachaeamac, fueron colocados cada uno en

mi frasco grande de boca ancha, donde permanecieron en reposo durante 15 a

20 dias a fin de que las glândulas se eneontrasen llenas de veneno en el mo¬

mento de extraerlas, porque dehido a la captura pierden cierta cantidad de

veneno al defenderse.

Fué necesario sacrificarias con cloroformo o éter; después de muertas, los

quelíceros fueron seccionados en su base con lijeras muy finas, con la ayuda de

un microscopio estereoscópico. Las glândulas separadas de los quelíceros fueron

secadas rápidamente en un papel filtro, para quitarles el suero; fueron abiertas

en cápsulas de vidrio completamente estériles, y el veneno diluído en solución

fisiológica de Cl Na al 7.5°/ 00 ; se inoculó diferentes soluciones de veneno, utili¬

zando las vias siguientes: intramuscular, intradérmica, endovenosa y empleando

los siguientes animales: cobayos, conejos, perros, palomas. Después de la ino-

culación se procedia a la observación dei animal; sus reacciones sintomatoló-

gieas, si presentaba reacción local. Luego de producida la muerte, se procedia

a la autopsia de cada animal, observándose el lugar de la inoculaeión y los órga-

nos internos afectados; esto se realizaba macroscopicamente y mieroseópieamente.

mediante cortes histológicos.

Se han realizado dos formas de experiencias: por picada directa

inoculaeión de soluciones de veneno.

y por

Los estúdios hislopatológicos de la glândula se han hecho utilizando las

siguientes técnicas: fijación en solución de Zenker, inelusión en parafina, cortes,

eoloración con hematoxilina eosina, tricrómica de Pollak, van Gieson y argéntiea

de Wilder.

Bksui.t mios

Por pica d a directa

Un ratón de 20 g es mordido en el cuerpo sin precisar el punlo de ino-

eulación, |ior un ejemplar grande de llapalopiis Umensis. Kl período de exeila-

eión inicial comienza a los 2"; el animal, con fuertes convulsiones y temblor,

se mueve por todos lados; demuestra eseozor en el hocieo. Este período de ex-

eitacion es seguido 5 después por paresia de los miembros posteriores, acorn-

panada de temblor anterior y perdida de actividad; a los 7’ la paresia alcanza

la parte anterior dei cuerpo; 8’ la parálisis es casi lotai y a los 9 muere el

animal con débiles convulsiones.

Un cobayo de 300 g es mordido en el hocieo por un ejemplar adulto de

Hapalopus Umensis. El período de cxcitación empieza a los 3‘. seguido de agi-

tación locomotora, con fuertes grilos v un poeo de hiper-exeitabilidad. a los 15

SciELO

10 11 12 13 14 15 16

802

ACCMN DEL VENENO DE HAPALOPUS LIMENSIS

comienza Ia paresia cie los miembros posteriores que se acentúa rápidamente;

a los 22’ la parálisis es casi completa; el animal apoya el hocico sobre el suelo,

con las cuatro patas abiertas; la respiración cada vez se hace más lenta; a los 27’,

cesa completamente la respiración y el animal muere a los 28’ con una parálisis

rápida de tipo curare.

Por ino culació n de soluciones de veneno:

Cobayos: Se utilizaron 7 cobayos de diferentes pesos, a los cuales inocula¬

mos soluciones de veneno de distintas dosis por vía intramuscular, conforme el

cuadro n.° 1. Resultados: 1) La inyección de l/f> glândula de veneno de ara¬

rias adultas o juveniles es siempre mortal para el cobayo y la muerte se ba

observado en estos animales entre 35’ y 108’ como promedio; se han presen-

tado casos de 12 y 19 boras, tratándose de ararias juveniles. 2) Con dosis su¬

perior a una glândula, la muerte se produce en pocos minutos. 3) La autopsia

mostro congestión pulmonar intensa, con grandes zonas hemorrágicas, congestión

intensa de Ias vísceras abdominales; líquido hemorrágico en el peritoneo; supra-

rrenales muy congestionadas, sangre fluída que se coagula lentamente al con¬

tacto de los tejidos.

CUADRO I

— Cobayos

inyectados

por via intramuscular

N.° de

serie

Peso

(grs.)

Arana

(edad)

Glândulas

Solución

(CINa)

Resultado

Tiempo

1.0

510

Adulta

l/y

0.5 ml

murió

53'

2°

515

Adulta

1.0 ml

murió

35'

3.°

425

Adulta

1.0 ml

murió

108’

4.°

525

Adulta

1 %

1.0 ml

murió

17’

5.°

525

Juvenil

1.0 ml

murió

12-19 horas

6.°

330

Juvenil

1.0 ml

no murió

—

7“

535

Juvenil

0.5 ml

murió

6-12 horas

Conejos: Se han empleado 6 conejos de distintos pesos, a los cuales inyecta-

mos diferentes dosis de veneno, por Ias vias intradérmica (en la oreja) y endo¬

venosa (vena marginal). La vía intradérmica se ha utilizado en 4 animales

para ver la reacción local (cuadro n.° II). Resultados: 1) For vía intradér¬

mica la mínima mortal es de y 2 glândula de veneno para conejos de 2 kilos y

1 glândula de veneno por via endovenosa. 2) Con dosis inferior a la mínima

mortal los sintomas son muy poco marcados. 3) La inyección intradérmica no

produce reacción local. 4) Autopsia: al abrir la cavidad abdominal, todas las

vísceras de color oscuro; los vasos mesentéricos muy dilatados, llenos de sangre

fluída; 20’ después de la muerte, Ia sangre se coagula muy lentamente al con¬

tacto de los tejidos, Un poco de líquido hemorrágico en el peritoneo, vejiga

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(3):799-808, 1966

N. C. DE ESPINOZA

803

QUADRO II — Conejos inyectados

N.° de

serie

Peso

(grs.)

Arana

(edad)

Glândulas

Solución

(CINa)

Via

Resultado

Tiempo

l.o

1300

Adulta

2.0

Intradérmica

no murió

—

2.o

1830

Adulta

2.0

Intradérmica

paresia

60’

3.»

1500

Adulta

l/ 2

0.25

Intradérmica

murió

52’

d.o

1800

Adulta

2.0

Intradérmica

murió

50'

5.o

1390

Adulta

1 L

0.25

Endovenosa

no murió

—

6.o

1S00

Adulta

1 %

1.0

Endovenosa

murió

9’

dilatada llena de orina, liígado rojo oscuro, sangrando mucho al corte, rinón

oscuro, suprarrenal normal, edema pulmonar muy ligero, fuerte congestión me-

níngea.

Perros (cuadro n.° III) : Resultados: 1) Por vía endovenosa la mínima mor¬

tal es de 1 a 2 glândulas de veneno para perros que pesan entre 5 y 8 kilos.

2) A los 3’ vomitos; 6’ paresia de los miembros posteriores; 10’ paresia gene¬

ral; el animal cae de costado, con grandes dificultades al respirar; 12’ con-

sigue levantar la cabeza y mover un poco los miembros anteriores; 15’ fuerte

contractura diafragmática, con parálisis casi total; 30’ parálisis de la respiración,

el corazón late débilmente; 33’ algún movimiento respiratório; 36’ muerte. Tres

horas después de la muerte no hay rigidez cadavérica. Autopsia: congestión

intensa de Iodos los órganos abdominales, con un color vinoso; el hígado y el

vaso oscuros; vejiga vacía; congestión pulmonar intensa; sangre fluída en todos

los vasos.

CUADRO III —• Perros inyectados por via endovenosa

N.° de

serie

Peso

(grs.)

Arafia

(edad)

Glândulas

Solución

(CINa)

Resultado

Tiempo

l.o

5600

Adulta

1 %

1.0 ml

murió

36’

2,o

6000

Adulta

1.0 ml

murió

9’

3.°

8500

Adulta

1.0 ml

no murió

—

4.o

6200

Adulta

1.0 ml

murió

11’

Palomas: Hemos empleado cuatro palomas a las cuales inyectamos solucio¬

nes de veneno por vía endovenosa (cuadro n.° IV). Resultados: 1) Por vía

endovenosa la dosis mínima mortal es de *% de glândula de veneno. 2) Con

dosis inferior a la mínima mortal los sintomas son bien marcados, pero el animal

SciELO

10 11 12 13 14 15 16

SC4

ACCIÕN DEI, VENENO DE HAPALOPUS LIMENSIS

se restablece. 3) A los 2’ parálisis de las patas, cayéndose al suelo, 5’ caída

de las alas, la cabeza cae sobre el suelo por parálisis de la nuca, los ojos cerra¬

dos; el animal trata de abrir el pico para respirar mejor, muerte a los 121

Autopsia: congestión intensa de las vísceras abdominales y torácicas; sangre par¬

cialmente coagulada en el corazón.

CUADRO IV — Palomas inyectadas por via endovenosa

N.° de

serie

Peso

(grs.)

Arana

(edad)

Glândulas

Solución

(CINa)

Resultado

Tiempo

1.0

400

Adulta

2.0 ml

murió

12’

2.°

375

Adulta

2.0 ml

murió

30’

3.o

300

Adulta

2.0 ml

no murió

—

4.°

350

Juvenil

1.0 ml

murió

5’

Con los animales de sangre fria no se hizo mayores experiencias, por ba-

berse utilizado sólo dos especies de animales, un sa|>o, Bufo spinulosus Umensis,

al que se inyectó 2 glândulas em 2 ml., con resultados negativos y una lagartija

Tropidurus peruvianus , al que se inyectó 1/4 glândula en 0.5 ml, sintoma: ligera

paresia, restableciéndose a los 15’.

Desde el punto de vista de su acción sobre el sistema nervioso, el veneno

de Hapalopus Umensis, posee los caracteres generales de los venenos neurotóxi-

eos. La sintomatologia es uniforme en todos los animales, es una curarización

típica, iniciándose por paresia, seguida de parálisis rápida y una muerte por

parálisis respiratória; el corazón después de la parálisis respiratória continua

latiendo uno o vários minutos. Cualquiera que sea la via de inoculación, la

muerte es siempre rápida.

11 ISTOI.OGÍ \

l.\ GI.AXDt l.\

Se advierten los cortes una cubierta muscular de forma espiralada estriada,

fácilmeute pereeptible, de regular grosor, netamente separada, sobre la cual se

observa el epitelio constituído por una hilera de células basales de las que des¬

prendeu estrias eitoplasmáticas, que, a manera de largos filamentos, avanzan

bacia la luz de la glândula.

Kn Ia base de estas prolongaciones loman asiento núcleos que tieneu el

mismo aspecto de los epileliales. La presencia de una membrana basal es nela.

En algunas zonas próximas a la membrana basal se advierte una aeumulación

circunscrita de grânulos muy finos y que pareceu corresponder a los de la

secreción.

Descripción de las células epileliales: Se las ba visto consliluyendo una sim-

ple hilera celular (epitelio monoestrali ficado), núcleo ovalado con eromatina

densa. A la hematoxilina cl carácter nuclear cromalínieo podría calificar-se de

tipo cromalínieo “pulverulento”.

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(3) :799-808, 19(i(i

N. C. DE ESPINOZA

805

El protoplasma cie bordes imprecisos que nos permite deslindar los limites

intercelulares, toma el aspecto general de condensaciones fibrilares que en forma

de largos filamentos se proyecla bacia la luz de la glândula.

La capa muscular es oblícua con fascículos de diversas direcciones.

La estructura glandular se acerca al li|>o merocrino, donde las traveras per-

sisten aprisionando en sus mallas al contenido de la secreción glandular y la

destrueción celular sólo alcanza a las células centrales de Ia glândula.

Anatomia Patológica

Todos los animales muestran congestión intensa de las vísceras.

Los estúdios histopatológicos se ban heeho en eobayos. conejos y perros.

COBAYO N.° 5: Hígado — congestión, especialmente marcada en los gran¬

des vasos (estasis). Piei — en algunos sítios, junto al punto de inoculación,

pérdida de la sustancia córnea y dei epitelio germinativo. Testículos — con

imagen funcional de reposo. Se observan los canalículos seminíferos completa-

mente llenos de células germinales y de espermatozóides. Corteza cerebral

ligera congestión vascular. Pancreas — este órgano conserva sus caracteres mor¬

fológicos normales en lo que respecta a su posieión exoerina, así como en su

posieión insular. Pulmones — congestión vascular especialmente marcada en los

pequenos vasos; muchas cavidades alveolares repletas de células de aspecto exu-

dativo. li azo — Conserva sus caracteres histológicos propios.

COBAYO N.° 3: Rinon — la congestión vascular es intensa, exibiendo en

ciertos segmentos dei órgano (zona medular) focos hemorrágicos. Cerebro -

caracteres normales. Pulmones — intensa congestión, marcado edema de las

paredes alveolares.

CONEJO N.° 4: Rinon — Se nota en los glomérulos aumento dei número

de núcleos; histológicamente, se observa edema en las paredes de los tubos. Al-

gunas de las pequenas áreas de la zona medular se muestran con infiltración

celular junto a los tubos dei asa de Henle. Igualmente, algunos tubos contor-

neados vecinos a algunos glomérulos se observan infiltrados. Ln la zona medu¬

lar las infiltraeiones celulares pareceu ser periféricas a veces sanguíneas de

mediano calibre. Pulmón — eonservaeión de los caracteres histológicos norma¬

les. Cerebelo — normal. Hígado — se observa en el hígado pequenos focos

de infiltración en las proximidades de los vasos peri-lobulillares.

PhRRO N.° I: Rinon — congestión de algunos eapilares glandulares e in¬

cremento dei número de núcleos en los corpúsculos de Malpighi. Ln la zona

medular de las pirâmides se nota moderada congestión vascular. Algunos tubos

contorneados proximales se presentan con su epitelio de aspecto hidrópico. Hí-

gado - ligera infiltración alrededor de los vasos cenlro-lobulillares, los mismos

que se ofrecen con aspecto congestivo y con discreto edema de sus paredes. La

infiltración corresponde a elementos celulares dei tipo linfóeilo. Capsula suprar-

renal conserva sus caracteres histológicos normales.

Como resultado de la observación de los órganos pertenecientes a los ani-

niales de experimentación, puede deeirse que los afectados son el rinón. el hígado

y el pulmón con lesiones de tipo inflamatório agudo, representadas principal-

mente en forma de transtornos congestivos vasculares, sin câmbios degenerativos

en los respectivos parénquimas. Órganos como e! sistema nervioso central, cére¬

bro, cerebelo v cl ba/.o no exiben câmbios oslcnsibles.

SciELO

10 11 12 13 14 15 16

806

ACCIÓN DEL VENENO DE HAPALOPUS LIUENS1S

Fig. 1 — Hapalopus Umensis — Hembra.

F)g. 2 — Hapalopus hmensis

Fig. 3 — Hapalopus Hmensis

Posicion de la glandula de veneno dentro

dei quellcero.

Corte de la glandula de veneno (aum. de

500 X aproximadamente).

SciELO

Mem. Inst. Butanlan

Simp. Internac.

33(3):799-808, 1966

N. C. DE ESPInOZA

807

SlJMMARY

Experimental injections vvith lhe venom of Hapalopus limensis in guinea-pigs,

rahhits, dogs, pigeons, toacls {Unjo spinulosus limensis ) and lizards (Tropidurus

peruvianus) showed the following results:

1. The venom is, principally, of neurotoxic action.

2. The first symptom ohserved is paresis, followed by death cansed by respi-

ratory paralysis, ohserved in all laboratory animais.

3. In the hot-blooded animais, death occurs in a short-time, generally less than

an hour, in any inoculation ronte.

4. In the cold-blooded animais, when the venom is injected in the ventral lym-

phatic saek of the toad and intraperitoneally in the small lizard, the action

is transitory.

5. lhe minimum lethal doses are: for 400-500 gr. guinea-pigs, intramuscularly,

0.5 gland; for two kilo rahhits, intradermically, 0,5 gland and endove-

nously, 1 gland; for 5 to 8 kilo dogs, endovenously, 1.5 to 2 glands; for

375 to 400 gr pigeons, endovenously, 0,75 to 1 gland.

6. Intensive visceral congestions and degenerative lesions in the liver, kidneys

and Itings can be ohserved in the autopsy and histo-pathological studies.

7. The venom produces neither irnmediate nor late local reaction.

Agradecimientos — Dejo constância la ayuda recibida por los Drs. Jorge Díaz

Encinas y Aristides Herrer, ex-Jefes de los Deptos. de Anatomia Patológica y En¬

tomologia respectivamente dei Instituto Nacional de Salud dei Ministério de Salud

Pública y Asistencia Social.

Bibliografía

1. ESCOMEL, E., Obras Científicas, 1, 603-616, 1921.

2. ESCOMEL, E., Obras Científicas, 1, 581-599, 1929.

3. ESCOMEL, E., Estúdios Científicos, 34-37, 1940.

4. CAVASSA, N., La Crón. Méd., 298-301, 1921.

5. CHAMBERLAIN, R„ Buli. Mus. Comp. Zool, Harvard CoU.. 60, 177-299, 1916.

6. IZU, W., Contribución al estúdio dei araneísmo en el Perú, Tesis de Bachiller

en Medicina, Lima, 1953, pp. 1-167.

7. LEON, A„ An. Univer. Perú, 19, 129-152, 1953.

8. PARDO FIGUEROA, E., La Crón. Méd,, Lima, 13, 148, 1896.

9. POCOCK, R„ Ann. Mag. Nat. Hist., 11, 81-115, 1903.

10. PESCE, H., y LUMBRERAS, H„ Rev. méd. per., 25 (301), 3-18, 1954.

11. VELLARD, J., Cuatro Conferencias sobre Animales Venenosos, Instituto de La

Universidad de Paris en Buenos Aires, Talleres Gráficos “Atlas”,, pp. 21-34,

1944.

12. VELLARD, J„ An. Acad, bras. Cien., 26 (1), 179-185, 1947.

13. VELLARD, J., Reu. Viernes méd., 7, 150-163, 1956.

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(3) :809-820, 1966

AVELINO BARRIO y ADALBERTO IBARR A-GRASSO

809

16. CASOS DE LOXOSCEL1SMO GRAVE OCURRIDOS EN LA CIUDAD

DE RUENOS AIRES Y ALREDEDORES EN LOS ÚLTIMOS ANOS

AVELINO BARRIO y ADALBERTO IBARRA-GRASSO

Instituto Nacional de Microbiología “Caídos G. Malbrán”,

Buenos Aires, Argentina

Los accidentes ocasionados por Loxosceles laeta fNicolet) en la ciudad de

Buenos Aires y alrededores se observan con nnicho más frecuencia en los meses

de calor que en el invierno. La mayor parte de los casos que nos llegan en

consulta siguen una evolución favorable limitandose, a los sumo, a la aparición

de doior local seguido de aureola congestiva y a veces de algunas ampollas.

El diagnóstico de esta forma de araneísmo domiciliário debe hacerse la

mayor parte de las veces, en base al cuadro que presentan los enfermos, puesto

que en muy contadas ocasiones intentan o logran capturar a la araria.

Concordando con Schenone (1), consideramos que dado el conocimiento

actual que se posee de la sintomatologia determinada por la picadura de Loxos¬

celes laeta , no es indispensable su captura y subsiguiente identificación para

hacer el diagnóstico de loxoscelismo. En efecto, el diagnóstico diferencial de la

lesión cutânea ofrece relativamente pocas dificultades puesto que debe hacerse

principalmente con relación a otras dos entidades nosológicas, el carbunco y la

erisipela gangrenosa. En cuanto al compromiso sistémico, habrá que tener en

cuenta otras etiologias determinantes de insuficiência renal aguda, entre las que

seguramente ésta es la única que puede asociarse a la típica lesión cutânea y

al antecedente de una picadura por artrópodo.

Las pequenas dimensiones y la poca agresividad de esta especie han origi¬

nado dificultades y contribuído a demorar la identificación de la verdadera

enlidad responsable de tales accidentes. De ahí, que a pesar de tratarse de un

lipo de araneísmo vastamente extendido en América y conocido desde muchos

anos atrás, recién en 1935 Escudero (2) sospecha, por la investigación experi¬

mental, que sea atribuible a Loxosceles laeta (Nicolet) un caso mortal de ara¬

neísmo cutâneo; en 1937 Macchiavello (3) la identifica en forma indubitable

como causante dei araneísmo necrótico-cutáneo y posteriormente en 1947 el

mismo autor (4) la responsabiliza también dei araneísmo hemolítico, lo que

confirman Mackinnon y Witkind (5) en 1953 para el mismo tipo de accidentes

registrados en el Uruguay.

Damos a conocer a continuación cuatro casos en los que nos cupo actuar

y que presentaron evolución más seria, dos coresponden a Ia forma cutâneo

necrólica hemolítica y los oiros dos a la forma cutâneo necrótica simple.

Caso I

M. S. O., nina de 6 anos, argentina. Procedência: Baradero (Província

de Buenos Aires).

El 15 de febrero de 1954: es picada a las 17.30 hs, por un insecto (?)

en región supraclavicular izquierda, en su domicilio.

í, | SciELO

«10 CASOS DE LOXOSCELISMO GRAVE OCURRIDOS EN LA CIUDAD

DE BUENOS AIRES Y ALREDEDORES EN LOS ÚLTIMOS ANOS

2.° día: A la manana presentaba edema blanco con puntillado rojizo, irre¬

gular en la zona supraclavicular izquierda.

3.° día: Aparece orina

150 cm 3 de sangre total.

hemoglobinúrica. Más tarde se le transfunden

4.° día: Continua la hemoglobinuria y se presenta tinte subictérico en piei

y mucosas. Ante el cuadro de anemia (3.000.000 de hematíes por mm 3 ) se le

transfunden 250 cm 3 de sangre total. Hay colapso circulatório superficial. Está

formada la placa livedoide, que abarca el hemitórax superior izquierdo, cuello

y brazo dei rnismo lado hasta la región deltoidea (Fig. 1). Se administra Cor-

lone y Henadryl inyectable. Posteriormenle se transfunden 300 cm 3 de sangre

total. Hay hipertermia de 39°C.

it.

mg ®

Fig. 1 — Caso I. Se observa el lugar de la picadura

y extensión de la mancha equimótica.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internae.

33(3): 809-820, 1966

AVELINO BARRIO y ADALBERTO IBARR A-GRASSO

811

5. " día: Enferma en anuria, que se prolongará hasta su niuerte; se trans-

funden 200 cm :i de plasma y, por sondeo vesical, se recogen unos 20 cm 3 de

orina color pardusco. La placa livedoide se presenta dura, de color violáceo y

de limites más precisos. Enferma febril, oscilando entre 37°C y 39°C.

6. ° día: Se comienza la diálisis gástrica, y continua el tratamiento con

Cortone y Benadryl. Enfermita lúcida; una deposición abundante.

7. " día: Enferma indiferente y deshidratada, la placa marmórea comienza

a involucionar, disminuyendo el edema y la intensidad de la coloración. Una

deposición diarreica.

d." día: Actitud indiferente y quejosa. Una deposición diarreica.

9. " día: Enfermita adinámica e indiferente, dos deposiciones diarreicas.

Hematocrito 33%.

10. ° día: A las 13 hs, aparecen nistagmus y movimientos convulsivos, no

responde a las solicitaciones. Deposición diarreica. Vomita reiteradamente. La

mancha livedoide con franca tendencia a desaparecer.

77." día: Se retira la diálisis gástrica. Enfermita no responde, continúan

nistagmus y convulsiones. Frecuencia de pulso 110. Presión arterial: Mx 110,

Mn 50. Fallece durante el acto operatorio al intentar efetuársele una exanguineo

transfusión.

En la figura n.° 2 se sintetiza en un gráfico la evolución dei cuadro humo-

ral, senalándose además la duración de la hemoglobinuria y de la anuria.

DIÁLISIS GASTRICA

60-

40 -

20 '

4 -

2 -

Fig. 2 — Caso I. Evolución dei cuadro humoral

SciELO

10 11 12 13 14 15 16

812

CASOS DK LOXOSCELISMO GRAVE OCURRIDOS EN LA CIUDAD

DE BUENOS AIRES Y ALREDEDORES EN LOS ÚLTIMOS ANOS

Caso lí

J. K. de A., argentina. 38 anos, casada, con 3 liijos varones. y embarazo

de 3 meses. Procedência: San Andrés de Giles (província de Buenos Aires).

A continuación hacemos un resumen dei caso ya publicado (6).

El 24 de enero de 1962, aproximadamente a las 15.00 hs, luego de tomar

un bano, al cubrirse con la bata, que había dejado colgada en la pared, siente

una picadura en la región subescapular izquierda. AI buscar la causa, encuen-

tra una araiía a la que arroja, restándole Ioda importância.

Unas 3 boras después comienza un dolor persistente, que va aumentando

en intensidad. A las 4 horas dei accidente hay además sensación de tumefacción

en el hombro y región pectoral.

2. ° día: A las 11.00 bs aparece intensa hematúria, que es confundida con

una metrorragia, lo que agregado al malestar general, rnotivó su internación en

un hospital de la zona. Se nota coloraciôn amarillenta dei tegumento. Hay

ligera epistaxis y mucho dolor de cabeza.

3. " día: Sigue desmejorando y queda establecida la anuria. Sospechando

el médico tratante la posible etiologia dei proceso, se pone en comunicación con

nosotros. Aconsejamos el inmediato traslado a Buenos Aires. A las 55 horas

de la picadura se le inyectan 4 ampollas de suero anti -Loxosceles (en total 200

unidades antinecrosantes).

4. ° día: Estado actual — Enferma lúcida, ictérica, anémica, disneica y con

signos de deshidratación. Tensión arterial Mx 100, Mn 65; con 100 pulsos por

minuto. Abdômen distendido y depresible. Espleno y bepatomegalia dolorosa

a la palpación. El resto sin particularidades.

La picadura tiene forma numular y diâmetro de 2.5 cm con su borde con¬

gestivo, y se baila en el centro de la zona de edema equimótico, elástico y dolo¬

roso (placa marmórea). Hay infartos ganglionares subaxilar y supraelavicular.

Los datos de laboratorio son los siguientes: Hematocrito: 24%. Hemoglo-

binemia: 316 mg. Leueocitosis: 20.500 con neutrofilia. Urea: 1.70 g/litro. Bi-

lirrubina directa, indirecta y reacciones de floculación dentro de limites norma-

le.s. Cl: 102 mEq/1. Bicarbonato: 16 mEq/1. Sodio: 144 mEq/1. Potásio:

5.2 mEq/1.

Evolución: La paciente alravesó una fase oligo-anúrica que duró 22 dias,

oscilando su diuresis entre 30 y 180 cm 5 . Durante esta etapa reflejó mediante

una severa anemia el resultado de su importante hemólisis, la que fue corregida

cuando sus hematocritos deseendían dei 25%, con transfusiones de glóbulos des-

plasmatizados. La potasemia Uivo tendencia a ascender siempre a cifras patoló¬

gicas, llegando algunas veces a 6.8 mEq/litro a pesar de administrarse diaria¬

mente 50 gramos de resinas. El bicarbonato luvo descensos graduales y para¬

lelos a la elevación de la urea y prolongación de la anuria. La creatinina se

mantuvo alrededor de los 15 mg.

Durante estos 22 dias de oligo-anuria, la paciente fue sometida a una exan-

guino-transfusión y dos diálisis (Fig. 4).

No fue posible evidenciar toxinas hemolíticas en las muestras de sangre

extraída con ese objeto.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internar.

33(3):809-820, 1966

AVELINO BARRIO y ADALBERTO IBARR A-GRASSO

813

La paciente fue dada de alta a los 35 dias, hallándose en la etapa de

poliuria compensadora. Durante el tiempo de su internación no hubieron indi-

cios sugestivos de interrupción de embarazo y a su alta un urocitograma no

aclaro el cuadro y una reacción de Galli-Mainini dió resultado positivo.

A los cuatro meses y medio de embarazo el útero presentaba la altura

correspondiente y la madre percibía movimeintos activos dei feto. Los controles

efectuados permilieron valorar la buena evolución de la paciente.

Fig. 3 _ Caso II. Muestra el lugar de la picadura y

extensión de la placa livedoide.

El parto se produce a los 8 meses justos, espontâneo; resultando un feto fe-

menino hipotrófico, de 1.850 g de peso.

La nina ha evolucionado normalmente hasta el presente.

La escara final resultó notablemente pequena, alcanzando apenas a 1 cm de

largo al tiempo de desprenderse.

í, | SciELO

DIAS

814

CASOS DE LOXOSCELISMO GRAVE OCURRIDOS EN LA CIUDAD

DE BUENOS AIRES Y ALREDEDORES EN LOS ÜLTIMOS ANOS

cn o co

o o o

o o o o

r 'o r\j

o co

o o

o o —» rsj d í» oi (Ti

n 1 1 1 I n 1 1 1 i

i i r

IXANGUI NO - TRAHSFUSIDN

l â DIALIS15

! \

2 a 0IAUSI5

J / \ I

/ ! I

/ í

\ / i

\ I

i í

\ 1 I I

Fig. 4 — Caso II. Evolución dei cuadro humoral.

SciELO

10 11 12 13 14 15 16

(Pypjp jp pjnppiid) Pli Ji| 0 ujiy -jsod v d I

Mem. Xnst. Butantan

Simp. Internac.

33(31:809-820, 1966

AVELINO BARRIO y ADALBERTO IBARRA-GRASSO

815

Dias después de la picadura fueron traídos 7 ejemplares de ararias capturadas

en el cuarlo de haiio de la casa (que era de construcción reciente), resultando

todas corresponder a una única especie: Loxosceles laela.

Caso III

V. N. T., argentina, 2 anos. Procedência: Villa Dominico (Avellaneda),

provincia de Buenos Aires.

El 18 de octulire de 1964 a la manana experimenta dolor y aparece una

pequena manchita por arriba de la rodilla iszquierda.

2. ° día: Hipertermia moderada, excitación; la lesión local toma el aspecto

de una pustulita.

3. ° día: Aparición de una gran mancha marmórea en la parte interna dei

muslo. Al desprenderse la costra de la ampolla pustulosa íluye un líquido se¬

roso. Se administran antibióticos.

8. ° día: La mancha se extiende aún más hasta la ingle, asentándose sobre

un edema duro. Continua tratamiento con antibióticos.

9. ° día: Sin mayores modiíicaciones. Continua el tratamiento con anti¬

bióticos. Es internada en un Servieio de Pediatria con el diagnóstico presuntivo

de tromboílebitis de la vena femoral. La mancha equimótica toma la parte

interna y se extiende hacia la cara anterior dei muslo izquierdo y a través de

la ingle sobre el púbis I Fig. 5). Todo ello se asienta sobre un edema duro que

Mtet,

, Hw*

Fig. 5 — Caso III. Día 10.°. Mancha equimótica.

se extiende desde el bajo vientre hasta el muslo y en menor proporción a la

pierna y tobillo dei mismo lado. Aparece ligero tinte subictérico y orinas apa¬

rentemente no hematúricas. Continua tratamiento con antibióticos y antihista-

mínicos. Se diagnostica loxoscelismo y se le administran 2 ampollas (100 uni¬

dades antinecrosantes) de suero anti -Loxosceles (Instituto Butantan). Hi-

perleucocitosis: 37.000. Hematocrito: 35%. Bilirrubinemia: 0.30 g%. Uremia:

0.40 g%. Potasio: 4.5 mEq/1. Sodio: 120 mEq/1.

SciELO

10 11 12 13 14 15 16

Fig. 6 — Caso III. Día 28.°. Lábios evertidos en la

lesiOn principal.

Posteriormente ingresa a un Servicio de Cirurgia Plástica, adonde ]>revio

desprendimiento de los restos de la escara (quedando la aponeurosis expuesta)

y toilette de la herida, se le hacen injertos en tira según técnica de Thierch. Es

dada de alta a los dos meses.

Durante gran parte de su evolución fue tratada con antibióticos y mientras

estuvo internada no se observaron alteraciones en la orina ni disminución notoria

de la diuresis.

Clfi CASOS DE LOXOSCELISMO GRAVE OCURRIDOS EN LA CIUDAD

u DE BUENOS AIRES Y ALREDEDORES EN LOS ÚLTIMOS ANOS

10. ° día: Enfermita quejosa. Lesión local sin mayores modificaciones. He-

matocrilo: 28%. Bilirrubinemia: 0.90 g%. Uremia: 0.38 g%. Polasio: 5.4

mEq/1. Sodio: 113 mEq/1. Cloro: 92.5 mEq/l.

11. " día: Más animada, comienza a alimentarse espontáneamente, se le si-

gue administrando suero por fleboclisis, y antibióticos. Hielo local.

12. ° día: Buen estado general. Comienza a aparecer una línea oscura que

bordea la placa violácea. Hematocrito: 28%. Bilirrubinemia: 0.40 g%. Pota-

sio: 4.1 mEq/1. Sódio: 143 mEq/1. Cloro: 98 mEq/1.

13.°

día :

Continua

buen estado general.

e acentua la demarcación se-

nalada.

22.°

día:

Comienza

el desprendimiento

los bordes de la

escara dei

muslo y

biótieos.

dei

pubis. Ha

habido hipertermia

40°C. Se continua

con anti-

23.°

día:

Buen estado general. Continua

desprendimiento de

los bordes

de la escara. Hipertermia de 38°C. Continua antibióticos.

28." día: Buen estado general. El desprendimiento de los bordes de la

escara incluye piei y celular subcutâneo, presentándose los lábios evertidos, mien¬

tras la escara principal permanece firme en ei centro (Fig. 6).

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3):809-820. 1906

AVELINO BARRIO y ADALBERTO IB AR R A-GRASSO

817

Caso IV

A. I?.. argentina, 25 anos. Procedência: Capital Federal. Si liien este caso

no adquirió Ia gravedad de los anteriores, lo damos a conocer porque se pudo

seguir sn evolnción práctieamente desde el momento de la picadura.

El 16 de noviembre de 1965 a las 02.00 bs, en una confitería dei centro de

la ciudad de Buenos Aires, al apoyar ei brazo sobre el tapado dejado sobre una

silla, siente un íuerte pinchazo en mitad dei brazo izquierdo, lado inferior, que

le hace levantar el brazo bruscamente, cayendo al suelo una pequena araria.

Cuatro horas después hay fuerte dolor en el brazo, y poco más tarde observa

una pequena ampollita con contenido seroso. El dolor aumenta y a las 17.00

llega hasta el pectoral. Concurre a un servieio hospitalario, donde le administran

un antialérgico. Apenas durmió por la noehe. No observo fiebre ni orina roja.

2. ° dia: Es vista por nosotros. Presenta lodo el brazo edematizado y

rojizo. En el lado interno inferior se destaca una mancha blanquecina, de unos

12 X 6 cm, de bordes irregulares. No hay indícios de ampollas, y en el lugar

de la picadura apenas se nota una pequena mancha más oscura, de unos 2 mm.

Dolor quemante en todo el brazo y especialmente donde está la mancha blan¬

quecina. Estado general sin nada de particular. Se aplican dos inyecciones

anti-E o x o s c e le s (Instituto Butantan; 100 unidades anti-necrosantes).

3. ° y 4.° dias: Se vuelven a administrar 4 ampollas más, es decir que en

lotai se aplicaron 6 ampollas o 500 unidades.

5. ” dia: El dolor sigue muy intenso. No hay temperatura, ni albumina, ni

sehales de hemoglobinuria.

6. ° dia: La mancha rojiza se extiende hasta la muneca por el lado externo

y rodea casi el brazo a la altura dei codo. Sobre la mancha blanquecina (live-

doide) se va formando una zona azulada. Todo el brazo edematizado (Fig. 7).

El dolor sigue intenso y continuo. Apirexia y orina normal. Se palpan gânglios

axilares dolorosos.

Fig. 7 — Caso IV. Dia 6.°. Formaclón de ia

mancha livedoide.

SciELO

10 11 12 13 14 15 16

SIS CASOS DE LOXOSCELISMO GRAVE OCURRIDOS EN LA CIUDAD

DE BUENOS AIRES Y ALREDEDORES EN LOS ÚLTIMOS ANOS

9.° día: Brazo totalmente deshinchado; ha cesado el dolor, salvo en el

borde de la zona livedoide; la parte central de ésta es insensible.

33." día: Còmienzan a levantarse los bordes más delgados de la escara de

9.5 cm X 5.5 cm (Fig. 8).

Fig. 8 — Caso IV. Dia 33.°. Placa necrótica.

40.° día: A la larde eomienza a separarse la escara ya bastante disminuída

de tamano. Hay dolor (Fig. 9).

Fig.

- Caso IV. Dia 40.°. Comlenza el desprendi-

mlento de la escara.

55.° día: En un servicio hospitalario le desprenden la escara que apenas

estaba unida por el centro, quedando la lesión limpia, granulosa. Es aconscja-

da la plástica.

Se deja constância que a pesar dei tratamiento precoz y aparentemente sufi¬

ciente (6 ampollas en total) la evolución fue lenta y termino en una lesión

necrótica.

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(3):809-820, 1966

AVELINO BARRIO y ADALBERTO IBARRA-GRASSO

SüMMARY

819

Four human cases of loxoscelism, studied from lhe clinicai and therapeu-

tical point of view, are reported. Two were of lhe simple cutaneous-necrotic type

and lhe other two had a more severe evolulion, death ensuing in one of them,

and were of the cutaneous-necrotic-haemolytic type.

Bibliografia

1. SCHENONE, H., Boi. chil. Parasit., 14 (1), 7, 1959.

2. ESCUDERO, F., Rev. chil. Hist. Nat., 39, 339, 1935.

3. MACCHIAVELLO, A., Rev. chil. Hist. Nat., 41, 11, 1937.

4. MACCHIAVELLO, A., Puerto Rico J. Publ. Health, 22, 467, 1947.

5. MACKINNON, J. E„ y WITKIND, J„ An. Fac. Med. Montevideo, 38, 75, 1953.

6. MARTINEZ, C. R. J„ LORENZINO, G„ BARRIO, A., IBARRA-GRASSO, A., y

RUBIANES, C„ Rev. Asoc. med. argent., 71 (12), 634, 1963.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

38(3): 821-828, 1966

EDY DE LELLO

821

17. BEE VENOM: GLANDS, INTOXICATION, ACCIDENTS

EDY DE LELLO

Faculdade de Ciências Médicas e Biológicas. Botucatu, São Paulo. Brasil

Primitive bees and wasps are solitary animais. Infra social or social be-

haviour is a relatively recenl evolutionary step paralleled by morj)bological and

])hysioIogical adaptative changes.

Onr purpose in íbis study is to discuss some of lhe changes in the sting

apparatus as well as to comment on the bee venom and its action.

Bees are HYMENOPTERA from the super family APOIDEA. Michener (1)

proposed an evolutionary laxonomic tree for this super family (Fig. 1).

The majority of the families systematized are composed of solitary or infra

social bees. In the family APIDAE there are three social tribes: Bombini,

Aimni and Meliponini. Bombini are the “bumblebees”; among the Apini are

the honey-bees and the Meliponini are stingless bees.

The sting apparatus of Bombini and Apini was studied by Dufour (2) and

Bordas (3). Il is located in the posterior part of the abdômen betvveen the

rectum and the ovaries. Rectum, ovaries and lhe sting apparatus share a com-

mon opening. Tvvo glands were described: one spindle shaped, was called

‘iiasic”, by Dufour. The other, called “acid”, is bifid in its distai part, and

thread shaped; the two distai segments imite in a common, long, convoluted.

thread like tubule, that opens in a pear shaped reservoir, leading through its

proximal, slender segment, in lhe sting chamber (Fig. 2). In Bombas ( Fervi -

dobombus) atraías Franklin, the acid gland is more ramified and lhe common

tubule, before opening in the reservoir, is very short.

Meliponini cannot sting, but still have a vestigial sting provided with a

glandular sac, the size of vvhich, changes from very large, occupying 2/3 of lhe

abdominal cavity in some species, to very small. vestigial structures, in others (4).

The loss of lhe sting may be interpreted as a useful evolutionary step, since

its sheding, as it occurs in lhe Apis genera, after lhe action of stinging, is followed

by dealh of the insecl. The inability lo sting preserves lhe insect and maintains

the eolony.

Stingless bees are not defenseless bees: many defense mechanisms have

been described (5, 6) as the development of powerful mandibles; the ability lo

hinder flying or walking performances of the enemies through mixtures of wax

and vegetable gums that are stieked to lheir bodies or moving apendices; the

emission of bad smelling or bad tasting secretions; massive altack to repel an

intruder, and many others.

The appearance of lhese defense mechanisms lead to the obsolescence of lhe

sting and its eventual atrophy (4).

SciELO

10 11 12 13 14 15 16

822

BEE VENOM: GLANDS, INTOXICATION, ACCIDENTS

£//croC/ni

Bbo ^hymin/

Fp^o/o/d/ni

P/o^epeoZ/n/

^eo/om/j/

Tt>wn $enç/,e//ini

B/Ost/m

Feopos/Z/m

Fm mo bafO/d)'ni\

Pmmo6of/n/

C oenoprosopiúín/

Fo/vodZni

F/deÁ/^oe.

Oosypü c//no e

/Ve ///drjQè

docrop /d/r?o e

C tenop/ec. b/nOG.

Dvfoc/re/nQÇL

do/ZcZ/noe

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ZZyZoe/noe.

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F/pÒQg/ossin .

Sfenotnf/tfQ.

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PQ/ocoZZoZ, ; n/

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Fn //) op/? 0/7/7/

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£mphor/O/

£/y cg r/n/

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Fig. 1 — Taxonomic tree o£ APOIDEA according to Michener, 1944.

1 , | SciELO

824

BEE VENOM: GLANDS, INTOXICATION, ACCIDENTS

The sting apparatus ok Apini

Morphologists, pharmacologists and hiochemists have been interested in the

sling apparatus of Apini. The first studied ils anatomy and hislology. The

latter, lhe poison and ils consequences.

We hegan our studjes on morphology (4) and could not agree willi Du-

four (2), Carlet (7) and Bordas (3). They descrihed that, liolh lhe aeid and

Basic glands, hefore opening in the sling chamher, were nnited in a commoii

duct. Trojan (8). however, descrihed lhe Iwo glands as opening independently

into lhe chamher. Our ohservations agree with tliis last author. In lliis work

he suggests that, in the queen hees, the hasic glatid secretion, pouring over the

eggs, forms a layer around them that serves lhe double purpose of protecting

them and sticking them into the cell vvalls.

According to Trojan (8) and our ovvn ohservations, the bee venom is the

secretion of the acid gland only and not a mixlure of the secretions of the two

glands.

Biochemists and pharmacologists studied the poison as eliminated from the

sting and not the properties of the secretions of each gland.

The chemistry of the venom

Langer in 1897 (9) considered lhe bee poison as an essential hasic alcaloid.

Phisalix in 1922(10) descrihed three components of the poison; one histiotoxic,

one convulsive and the last paralising, each with different physicochemical cha-

racteristics.

Reinert íll) identified a protein, rich in triptophane, that could he dialysed

into two fractions, one with hemolytic activity, the other neurotoxic. He also

said that the inflammatory exudate of the sling contained 1 to 1.5% of hista-

mine. Ackermann and Mauer(12) were of lhe opinion that histamine was not

found in the poison itself hut was part of the liost reactions.

Feldherg and Kellaway had shown previously (13) the histamine releasing

properties of the bee poison.

More recently a dehydrogenase inlnhitor and one hyaluronidase have been

isolated from the venom (reviewed by Hodgson, 1955) (14).

1 ii 1954, Neumann and Hahermann (15) isolated and characterized a hasic

protein, mellitin, of marked cytolitic activity over mast cells and red hlood cells.

Phospholipase A, isolated by Híigberg and Uvniis (16, 17) acts also degra-

nulating rat mast cells.

In 1965, Rothschild (18) studied the release of histamine in rats by phospho-

lipase A and mellitin from the bee venom.

The symptoms and consequences of bee sting

Symptoms and signs of bee sting are very well known by the lay men all

over the world. In temperate and cold climales, hee stinging is much more

frequent than most of the other poisonous hites or stings by snakes, spiders or

scorpions. Despite this, the medicai literalure on the suhject is scarse.

Symptoms may vary from case to case; they usually are more severe when

the patient receives numerous stings; they also tend to he more severe after

many exposilions of the same patient lo the poison.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3):821-828, 1966

EDY DE LELLO

825

The first symptom is acute, local pain, described as “burning”, followed

after a few minutes hy local swelling and itching; the area shows a small. clear,

round, central zone, surrounded hy a red halo.

In the case of A pis mellifera (honeyhee) lhe sting itself is found in the

clear, central zone; it pierces the epidermis and may penetrate 2 to 3 mm in it.

Wasps and the majority of the solitary bees, however, do not lose lhe sting.

The local reaction subsides in a few hours and all the symptoms and signs

have usually disappeared after 24 hours.

In a few instances, the consequences are more severe and Thompson (19)

ranked them in lhe following order:

1. Abnormal swelling and irritation, lasting an abnormal time.

2. Massive urticaria and oedema supervening.

3. Shock and collapse or even loss of consciousness with hypotonia and

tachycardia, followed hy mild pyrexia and sometimes diarrhoea and

polyuria.

4. True dyspnoea soon afler sting.

5. Sudden severe general symptoms:

a) hot flush over the whole body;

b) severe dyspnoea;

c) wheezing and coughing, almost choking;

d) general trembling;

e) great anxiety, sometimes followed by coma. In women this may

be accompanied hy uterine contractions. This severe condition may

last up lo three or four days.

6. Death.

Death due to bee sting

The available data about death following hee stinging are not relyable.

Swinny (20) reported on seven deaths, in lhe State of Texas, in 1949; he sug-

gesled that this is a conservative estimation, the actual, correct figure being close

to 20 to 30 per year in the area studied. Parrish (21) reviewing the morta-

lity due to insect bites or stings in 1950-1954 in the U.S.A. found 84 cases

(review of death certificates) ; from these, 50% were caused hy Apis mellifera

ligustica; at the same time only 71 deaths were ascribed to snake bites.

In Denmarck hotween 1951 and 1959 bees were responsable for 2/3 of all

the deaths ascribed to poisonous animais (22).

All the cited authors were of the opinion that their figures are conserva¬

tive. Doctors, in general, are very cautious to ascribe death to such a simple

and common accident as a bee sting: they ralher look for some other more

usual cause, as acute cardiovascular disease. Indeed, many authors think that

one can better estimate lhe frequency of deaths after hee sting by reviewing lhe

newspapers than by reviewing death certificates.

SciELO

10 11 12 13 14 15 16

826

BEE VENOM: GLANDS, INTOXICATION, ACCIDENTS

Jensen (22) studied 34 cases of deallis due lo bee slinging. Fig. 3 and 4

summarize, graphically, his findings. Fig. 3 shows his 22 cases of inales and

12 cases of females, by age. Death is rare up lo 30 years and the majority

of lhe cases died between 30 and 50 years.

i o

20 30 40 50 60 70

Fig. 3 — Death caused by bee and wasp sting in the U.S.A. according

to the age. Data from Jensen, 1961.

Delpech (23) came to the conclusion, lhat death by bee sting is miich more

common in adults. From lhe 34 cases of Parrish (21) only 4 (11,7%) were

nnder 30 years of age; all but five of the cases in his series were perfectly

heallhy before the accident. The age distrihution seems peculiar since children

are usually more exposed to bees.

Fig. 4 shows a correlation hetween death by bee stinging and previous

allergic com])lainls in Jensen cases. In 16 of his 34 cases there was past history

of allergic disorders, five had been previously stinged and one had never been

stinged before.

The hypolhesis that anaphylaxis might be the pathogenetic mechanism of

death is atractive. Indeed, death supervenes within one hour of the accident in

the great majority of the reported cases. The symptoms are also compatible

with the hypolhesis being of the same type observed on other types of human

anaphylactic shock.

The age distrihution of the cases also agrees with the suggested hypolhesis.

As said above, children are much more exposed to bees than adults; being smaller

they should be more susceptible to the poisou if one thinks in term of dosage

by kg of weight.

Death by other poisonous animais as snakes, spiders or scorpions is more

common helow 20 years of age (40% hetween 0 and 19 years). Only 7% of

the deaths ascribed lo bee sting oceur below 20 years of age. In the case of

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3) :.S21-828, 1966

EDY DE LELLO

827

Fig. 4 — Past history of allergy and mortality by bee sting. Data from Jensen, 1961.

snakes, scorpions and spiders lhere is a definite correlation between dosage per

kg of body weighl and tlie severity of the symploms. We know of 4 beekeepers

who have been ex]>osed, in the same day, to 600 to 1500 bee stings who survived,

nicely, after the usual local signs and syin])toms.

Death by bee sting in Brazil

Up to 1956 our beehives were populated by Apis mellifera ligustica, the

Italian honeyhee. At that time Dr. W. E. Kerr, a geneticist from the “Escola

Superior de Agricultura Luiz de Queiroz in São Paulo, made a trip to África

sponsored by the State Department of Agriculture, the University of São Paulo

and the Brazilian Association of Beekeepers. His objective was to study the

possibility of amelioration of the honey yield of our beehives through imbreed-

ing of our Italian honeybees with the more productive Apis mellifera adansonii,

an African bee. The “African” bee produces about 400% more honey then the

“Italian” bee.

Since last year, newspapers in São Paulo have callcd the attention to an

apparent increase in death due lo bee stings and the proposed explanation was

the agressivity of the “African” bees. At this moment we are collecting data

ou the subject and our impression is that the facts are not as serious as to advo-

cate the destruction of all African colonies, an action that lias been proposed and

discussed.

It is true that the African bees are more agressive but the data that we

have up to now gathered, register cases of death among chicken and swines.

Actual danger to men seems to he exagerated in the press.

In the State of São Paulo, witli 16 million inhabitants lhere were 6 certain

human deaths between 1964 and 1965. Two of those cases were men over 70

vears of a^e and one was a small child that was abandoned by his sister at the

door of a beehive.

In the same period of one year, in the State of Texas, U.S.A., with a

population, at that time, of 8 million inhabitants there were 7 definite deaths

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10 11 12 13 14 15 16

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BEE VENOM: GLANDS, INTOXICATION, ACCIDENTS

caused by l>ee stings. The bees in Texas are only of Italian or Caucasian

origin: no African bees had been imported.

There appears lo be no difference in lhe nature of lhe poison produced by

Apis mellifera ligustica and Apis mellifera adansonii.

Once lhe industry of honey develops in any country lhere is an increase in

lhe number of morlal accidents and this is enlirely independent of lhe cultivated

species.

In conclusion, we do not believe lhal lhe introduction of African bees in

onr country is a serious danger.

There is no elear indication that it has increased lhe risk of a serious

damage of men or animais beyond lhe expected increase of risk in consequence

of lhe rapid developmenl of beekeeping as a profitable industry.

References

1. MICHENER, C. D., Buli. Amer. Mus. Nat. Hist., 82, 151-326, 1944.

2. DUFOUR, apud BORDAS, M. L., Ann. Sei. Nat. Zool., 19, 1-362, 1841.

3. BORDAS, M. L„ Ann. Sei. Nat. Zool, 19, 1-362, 1895.

4. KERR, W. E., and LELLO, E„ J. N. Y. Entomol. Soc., 70, 190-214, 1962.

5. KERR, W. E„ Evolution, 13 (3), 386-388, 1950.

6. KERR, W. E„ An. Esc. sup. Agr. “Luiz de Queiroz ”, 8, 219-354, 1951.

7. CARLET, G., Ann. Sei. Nat. Zool., 9 (7), 1-17, 1890.

8. TROJAN, E., Z. Morphol. õkol. Tiere, 19, 678-685, 1930.

9. LANGER, J., Arch. exp. Path. Pharmak., 38, 712, 1897.

10. PHISALIX, M., Animaux et venins, Masson, Paris, 1922.

11. REINERT, M., Zur Kenntniss des Bienengiftes, Basel, Festschrift Emil Barell,

1936.

12. ACKERMANN, D., and MAUER, H., Pflügers Arch. ges. Physiol., 247, 623, 1944.

13. FELDBERG, W., and KELLAWAY, C. H., Aust. J. exp. Biol. med. Sei., 15,

461-473, 1937.

14. HODGSON, N. B., Bee World, 36, 217, 1955.

15. NEUMANN, W., und HABERMANN, E., Naunyn-Schmiedeberg’s Arch, exp.

Path. Pharmak., 222, 367-387, 1954.

16. HõGBERG, B.. and UVNÃS, B„ Acta physiol. scand., 41, 345-369, 1957.

17. HÜGBERG, B., and UVNÃS, B., Acta physiol. scand., 48, 133-145, 1960.

18. ROTHSCHILD, A. M., Brit. J. Pharmacol., 25, 59-66, 1965.

19. THOMPSON, F., The Luncet, 446, 1933.

20. SWINNY, B., Texas State J. Med., 46, 639, 1950.

21. PARRISH, H. M., Arch. int. Med., 104, 198, 1959.

22. JENSEN, O. M., Acta path. microbiol. scand., 54 (1), 9-29, 1961.

23. DELPECH, A., Ann. Hyg. publ. (Paris), 3, 289, 1880.

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Mem. Inst. Butantan

Simp. Internac.

33(3):829-834, 1966

HUGO PESCE y ÁLVARO DELGADO

829

18. LEPIDOPTERISMO Y ERUCISMO. EP1DEMI0L0GÍA Y ASPECTOS

CLÍNICOS EN EL PERÚ

HUGO PESCE y ÁLVARO DELGADO

Facultad de Medicina, Universidad de San Marcos, Lima, Perú

Aceptamos la convención de distinguir los accidentes tóxicos, locales o

generales, causados eu humanos por lepidópteros adultos con el nombre de

LEPIDOPTERISMO y los causados por orugas (en latiu “eruca”), con el nom-

bre de ERUCISMO.

LEPIDOPTERISMO

Casi todas las observaciones conciernen a Sud América. Casos aislados han

sido observados en la Guayana Francesa, en LIruguay, en Argentina; pequenos

brotes mayores en el Brasil (Amapá) y en el Perú.

Epidemiología

Los agentes causantes más comunes son lepidópteros de Ia familia HEMI-

LEUCIDAE, género H y l e s i a. En el Perú la especie causante más común es

H. volvex Dyar, mariposa de dimensión mediana, cm 5 a 6, color pizarroso,

que vive sobre varias plantas y las abandona raras veces.

Las armas vulnerantes son espinas barbadas o ‘‘flechitas” de 150 micra,

implantadas sobre el abdômen de las bembras, impregnadas de veneno hidro-

soluble.

La única región dei Perú en donde se ha registrado accidentes masivos es

la amazônica. En Tingomaría, pequena ciudad en la hoya dei alto Huallaga,

durante anos, H. F. Allard observo invasión de mariposas adultas después de

terminada la estación de las grandes lluvias, de Abril a Junio: en 1952 registro

la invasión mayor, que duró 7 semanas, con apogéo en Mayo. Los vuelos eran

vespertinos y nocturnos; millares de mariposas envolvían los focos elécticos exterio¬

res y frecuentemente penetraban en las casas. El pulvísculo de setas desprendidas

era muy abundante. Por lo menos un 70% de los habitantes, o sea más de 3000

personas sufrieron los efeetos dei envenenamiento; muchos acudieron al Hospital.

En 1966 A. Tejada confirmo estos datos, registro 514 casos en 15 locali¬

dades de la hoya dei Huallaga, y además estableció lo siguiente: a) en el ano

existen 2 invasiones (abril-mayo; diciembre-enero) y a veces una tercera (agos-

to-septiembre) ; b) se han producido exacerbaciones periódicas por invasiones

masivas cada 4 ó 5 anos (1947. 1952, 1957. 1962, 1966).

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QQn LEPIDOPTERISMO Y ERUCISMO. EPIDEMIOLOGIA Y ASPECTOS

CLÍNICOS EN EL PERÜ

El mecanismo habitual cie producción de los accidentes humanos colectivos

ha sido “indirecto” por desprendimiento de las setas ponzonosas cpie flotaban en

la atmosfera, depositándose luego sohre ventanas, muehles y objetos, y de allí

a la piei.

Aspectos clínicos

Sintomatologia

a) Los accidentes espontâneos se manifiestan como una dermitis que ocupa el

cuello, la cara de flexión dei eodo, y otras zonas de piei delicada. Allí

aparece una zona eritematosa, pruriginosa, varias pápulas, y algunas vesí¬

culas, que suelen declinar en 6-7 dias. La repetición de los accidentes en

cada noche agrava estos sintomas y puede mantenerlos por varias semanas.

b) Experiências efectuadas (1952) en la región sobre pobladores que antes no

habían padecido de estos accidentes, mediante la frotación cutânea con el

cuerpo de estas mariposas, determinaron en lodos a los 15-20 minutos la

aparición local de un eritema pruriginoso.

c) Experiência análoga (1952) con estas mariposas remitidas un mes después

al extranjero determino eritema a las 2 horas; a las 4 horas calor local,

pápulas edematosas, prurito; a los 2 dias una vesícula y varias petequias.

La dermitis aguda declino a los 7 dias y las petequias desaparecieron a

los 13 dias.

d) A. Tejada (1966) experimento en 16 personas, en la región y fuera de

ella, con resultado análogo.

e) El manejo de mariposas muertas determino (1966) accidentes extensos en

dos entomólogos.

El cuadro clínico observado concuerda con una etiologia tóxica por pro-

ductos dei grupo histamínico. Por otra parte la tendencia crónica que acompana

la repetición diaria de los accidentes sugiere algún grado de sensibilización.

El diagnóstico se efectúa por el dato epidemiológico y por la dermitis típica.

El no haber tenido en cuenta el primer dato ha dado lugar a falsos diagnósticos

de brote de rubeola.

T ratamiento

El uso en la primera media hora de una loción de hiposulfito de sodio al

50% parece neutralizar el veneno “in situ” pues desaparecen las lesiones y el

prurito; dentro de 1 hora hay alguna eficacia; después ninguna.

El uso precoz de antihistamínicos sintéticos por via oral atenua rápidamente

la erupción cutânea y suprime el prurito; su uso tardio actúa bien sobre el

prurito y el consiguiente insomnio.

El uso de corticosteroides ha sido favorahle en algunos casos.

Prevención

Durante las invasiones, mantener apagadas las luces desde el ocaso hasta la

hora dei sueno.

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Mem. Inst. Butantan

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33(3):829-834, 196fi

HUGO PESCE y ÁLVARO DELGADO

831

ERUCISMO

Las orugas fanerotóxicas peruanas que hemos observado pertenecen en su

mayor parte a la família MEGALOPYGIDAE.

Tenemos registrados 1383 casos de erucismo individual, directo.

Epidemiología

Las Orugas Ponzonosas dei P e r ú

Las especies predominantes y su frecuencia son:

a) Una Megalopyge sp. (“cuy dorado”) . 28,9%

b) Una Megalopyge sp. ("uteu balluca ) 16,8%

c) Una Podalia sp. ("sarohomé cremoso ) 10,2%

d) Una Megalopyge. sp. ("‘cuy de fuego’ ) 6,2%

Especies de menor difusión pertenecen a SATURNIDAE, a HEMILEUCIDAE

y a SPHINGIDAE. (La descripción morfológica de las orugas, asi como el estúdio

macro y microscópico de su aparato venenoso los hemo efectuado en otro trabajo

ya entregado para publicacíon.)

Sinecología

La correlación entre el habitat y el modo de vida de las orugas y los hábitos

regionales dei hombre tiene dos aspectos:

a) Ecologia de las Orugas

En el Perú, como en casi toda la región Neotropical, la ecologia de las

orugas es opuesta a la de las regiones Paleártica y Neártica. El ciclo de vida

de los lepidópteros es con frecuencia semestral y aún trimestral. Su habitat

preferente es la selva densa con escasos vientos y población sumamente rala. La

perdida de setas, absorbidas por la selva, no reviste importância. Por lo tanto

no hay accidentes colectivos: todos son individuales y directos.

La frecuencia cronológica de las orugas puede deducirse dei número de

accidentes que provocam La primera serie de 635 casos (1958-64) estudiados

en 34 distritos (de 9 províncias) se distribuyen en los meses dei ano con una

proporción promedia mensual dei 7%, salvo en el bimestre marzo-abril suce-

sivo a la “grán” estación lluviosa en que asciende al 12.5% mensual, y en el

bimestre septiembre-octubre sucesivo a las “pequenas lluvias , con 9,6% men¬

sual, totalizando ambos períodos 44,2%.

b) Ecologia humana

El “hinterland” de los escasos pobladores es un retazo arrancado a la selva

virgen en el que las plantas cultivadas producen todo el ano. Los accidentes

observados en la citada serie de 635 casos son individuales y se produjeron:

cultivando y cosec bando 59,7%, talando 12.8%, cargando 9,9%. Las plantas

infestadas ban sido: alimenticias 42.0% y comerciales (cafeto, banana, algodón,

tabaco) 35,0%. El accidente directo, en estas zonas, es obligado producto dei

trabajo agrícola individual. Es casi inevitable: 42,5% de los casos sufrieron

de 2 a 5 accidentes y 15,8% de 6 a 15.

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832

LEPIDOPTERISMO Y ERUCISMO. EPIDEMIOLOGIA Y ASPECTOS

CLÍNICOS EN EL PERO

Etiopatogenia

El critério ecológico aplicado a la etiopatogenia nos permite distinguir vá¬

rios mecanismos:

a) Erucismo verdadero directo — Es por contacto directo de una oruga peluda,

viva con el tegumento humano de un indivíduo. En el Perú es la regia.

b) Erucismo verdadero indirecto — Las setas de las orugas peludas despren¬

didas, en el foliaje o en el tronco o en el suelo son las responsables de

accidentes. En el Perú es la excepción: Burstein y col. (1957) observaron

casos alrededor de un árbol “maio".

c) Para-erucismo — Es producido por orugas desnudas criptotóxicas, sin apa¬

rato vulnerante. No lo hemos observado.

d) Meta-enicismo — Las setas lanarias tóxicas son transmitidas a la cocona

o hasta al insecto adulto. Lo llamamos erucismo “por encargo”. En el

Perú se han dado algunos casos.

Aspectos clínicos

Nuestras observaciones de erucismo individual directo se refieren a 2 series:

la l. a (Pesce y Delgado, 1958-64) con 635 casos; la 2. a (Pesce y Tejada, 1966)

con 748 casos; totalizan 1383 casos.

La primera serie, ya tabulada, con 635 casos comprende 1939 accidentes

cpie se distribuyen así: a) 265 accidentes en 265 personas con accidente único;

h) 1674 accidentes en 370 personas con accidentes repetidos. En la tabulación

sintomatológica solamente hemos considerado los 635 accidentes cada uno de los

cuales ha motivado una historia clínica.

Cuadro clínico

La dermitis local aguda es el componente obligado de todos los accidentes.

Si bien en un 40% de los casos ella es el único sindrome y evoluciona en pocas

horas (2 a 12), en un 60% de los casos se agrega extensión regional dei pro¬

cesso con neuritis difusa (24-36 h), y en un 20-25% de los casos se presentan

también signos generales, que en nifios y mujeres pueden revestir gravedad.

La sintomatologia, sumamente rica y variada, merece por lo menos el si-

guiente análisis somero:

Sintomatologia local y regional

a) Debemos destacar el sindrome dérmico de tipo histamínico o histaminoide

con erilema (97,1%), edema (65,0%), pápula-habón (36,4%), petequias

(27,9%), observando que el prurito aparenta una cifra haja (14,5%) por

ser enmascarado por el dolor urente difuso (56,8’%).

h) Entre los signos inflamatórios de origen tóxico merecen relevarse el dolor

ganglionar (32,9%) y el infarto ganglionar (29,0%).

c) El compromiso neural es alto: dolor urente (56,8%) casi siempre difuso, a

veces acompanado por neuralgias; dolor articular (2,7%).

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Mem. Inst. Butantan

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33(3) :829-834, 1966

HUGO PESCE y ALVARO DELGADO

833

d) Efectos necróticos superficiales: flictenas (14,2%).

e) Una secuela muy frecuente es la hipercromia residual (64,0%); a veces

huellas cicatriciales (2.0%), resíduo de íliclenas.

Sintomatologia general

Su notable frecuencia observada en la selva dei Perú contrasta con lo refe¬

rido por otros autores.

al Entre los signos generales, hay malestar (21,6%), esealofrío (9,9%) y sen-

saeión dei alza térmica (31,9%) aunque en parte falseada por el dolor

urente difuso, a veces casi generalizado.

1>) Hay signos tóxicos compatibles con el tipo histamínico: nauseas (7.1%),

vómitos (2,7%), diarreas (2.0%), erupción urticariana (1,4%).

c) Signos circulatórios atribuibles a la misma causa son: cefalea por probable

edema cerebral (17.5%), angustia (5.4%).

d) Signos nerviosos son: confusión (2,7%), dromofilia (1,3%).

e) Colapso en sus distintos grados: adinamia (19.1%) postración (3,1%),

lipotimia (1,6%).

Aspectos inmunológicos

Consideramos para este efecto los 1939 accidentes registrados en la l. a serie

de los cuales 1674 son repetidos.

Entre los 370 indivíduos que han sufrido 1674 accidentes comprobamos que

en 82,5% de los casos el último accidente guarda similitud con los anteriores;

en 10,4% hubo atenuación marcada; en 7,1% el último accidente fue más serio

que los anteriores. Sin embargo 1/3 de estos accidentados atribuyen la mayor

seriedad dei accidente a la diferente especie de la oruga causante y 1/5 la

atribuye a la mayor intensidad dei contacto.

Al parecer el veneno de las orugas peruanas tendría tendencia a producir

cierto grado de inmunidad. No conocemos casos de sensibilización; aparente¬

mente son muv raros.

T ratamiento

En el medio rural más remoto nuestra experiencia nos indica que la apli-

cación de compresas húmedas calientes y la inmersión segmentaria en agua

caliente produce rápida remisión de la dermitis, de la neuritis y de la sintoma¬

tologia general, en lapsos entre 1 y 3 boras según la seriedad dei caso.

Donde ba sido posible, la ingestión de antihistamínicos sintéticos ha dado

resultado excelente en lapsos entre 30 y 80 minutos, aun cuando existia compro-

miso general.

No tuvimos oportunidad de ensayar otras terapêuticas.

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QCM LEPIDOPTERISMO Y ERUCISMO. EPIDEMIOLOGIA Y ASPECTOS

CLÍNICOS EN EL PERÜ

Bibliografía

A — LEPIDOPTERISMO

1. ALLARD, H. F„ and ALLARD, H. A., J. Washington Acad. Sei., 48 (1), 18-21,

1958.

2. DYAR, H. J., Results of the Yale Peruvian Expedition of 1911. LEPIDOPTERA,

Washington, 1913.

3. PESCE, H„ and DELGADO, A., in W. BÜCHERL, V. DEULOFEU, and E.

E. BUCKLEY, (Editors), Venomous Animais and their Venoms, Vol. 3, Acad.

Press, in press.

ERUCISMO

1. BOSO, J. M., La Medie. pop. peruana, Vol. 3, Valdizán y Maldonado, Lima,

1922, pp. 348-388.

2. COBO, B„ Historia dei Nuevo Mundo (1639-1653), Vol. 2, Ed. 1, Sevilla, 1890,

p. 273.

3. DELGADO, A., La fauna ponzonoza dei Valle dei Rimac, Lima, 1963, pp. 28-29.

4. DELGADO, A., y PESCE, H., 1 Congr. Peruano Microbiol. y Parasitai., Are-

quipa, 1964.

5. DELGADO, A., Erucismo por Megalopyge sp. en Lima (inédito), 1965.

6. FERNANDEZ LANCHO, M., Erucismo por “Jashpairo” en la Merced (1950-

1958), Comun. pers., 1963.

7. PESCE, H„ y NORIEGA, T„ Boi. Direcc. Salubr., Lima, 14, 1935.

8. PESCE, H„ y DELGADO, A„ J. Microbiol., Trujillo, 29-30, 1962.

9. PESCE, H„ y DELGADO, A., VII Congr. Internac. Med. Trop., Rio de Janeiro,

1963, Vol. 4, 1964, pp. 211-212.

2 3

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Mem. Inst. Butantan

Simp. Internac.

33(3): 835-844, 1966

A. FAIN

835

19. TOXIC ACTION OF ROVE BEETLES ( COLEOPTERA,

STAPHYLIN1DAE)

A. FAIN

Institut de Médecine Tropicale, Antwerpen, Belgique

Introduction

The vesicant action of beetles helonging lo the genus Paederus

(STAPHYLINIDAE) was recognized for lhe first time hy cia Silva, in 1912 (1),

in Brazil.

The species involved was Paederus columbinus Lap.. This insect is fre-

quently eneountered in the region of Bahia from June lo September, especially

along the borders of the main streams. It appears to be a positive pest for

people working in the fields of potatoes, corn or beans and it is well known to

them under the name of “potó” or “trepa moleque”. When these insects alight

on the bare skin they produce a pruriginous erythema which is followed by a

blister and a slowly healing ulceration. These lesions occur on the legs, the

arms and the neck.

Since the paper of da Silva (1) many other authors have reported similar

observations in different parts of the world and involving various species of

Paederus. A general review of that question appeared in the thesis of

Allard(2), in 1948. This work was completed by Theodorides in 1950 (3) and

more recently by lhe thesis of Gruvel(4). Several other contributions have

been added during the last years. Among them the most important seem to be

those of Pavan and Bo (5, 6) and Pavan (7, 8) on the nature of lhe toxic

factor responsible for the cutaneous or ocular lesions.

The rove beetles are characterized by their narrow body and their short

elytra under which the long membranous posterior wings are folded. They have

beautiful colours. In P. sabaeus the head and the posterior segments of the

abdômen are black. the thorax and the anterior part of the abdômen brownish-

yellow, the elytra melallic blue. Most of the species are small, measuring not

more than 1 cm in length. Some species have the habit of curling the abdômen

over the thorax, and they may therefore easily be mistaken for flying ants.

GeOGRAPHICAL DISTRIBUTION OF THE ROVE BEETLE DERMATITIS

The dermatitis produced by the beetles of the family STAPHYLINIDAE

has been observed on the five continents. So far the vesicant properties have

been recognized as belonging only to the genus Paederus. It is lo be noted,

however, that the species ruficollis which is known as producing experimental

dermatitis, has been removed recently from the genus Paederus and placed

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836

TOXIC ACTION OF IÍOVE BEETLES ( COLEOPTERA, STAPHYLfNIDA K)

in the genus Paederidus Rey íFig. 6). According to Mr. G. Fagel of

Brussels, the vvell-known speeialist in the study of these heetles, some of the Soulh-

American species could, in fact, helong to other genera of the STAPHYLINIDAE.

The genus Paederus has a world wide distrihution and is represented hy

several hundred species among which a small number, not more lhan 30, have

been recorded in relalion with human dermatitis. It seems, however, very pro-

hahle thal the majorily of lhe species of Paederus contain a vesicant fluid.

Specifie identification in that grouj) is very difficull and may he made only hy

an expert.

In South America the rove beetle dermatitis has heen reported for lhe first

time in Bahia, Brazil, hy da Silva in 1912(1) in connection with Paederus

columbinas Laporte.

Soon lhereafter, Gcildi in 1913 (9) ohserved the same loxic action in the

species Paederus goeldi (Wasmann, 1905) in Amazonas, Brazil. A third species,

Paederus amazonicus (Sharp) has been mentioned hy Bequaert (10) as causing

dermatitis, in the same country. Another recorded from Brazil is that of Gor-

don (11) who stated that Paederus amazonicus had heen first encounlered at

Manaus, Amazonas, in January 1921. Froes (12, 13) in Bahia, Brazil, showed

experimentally that Paederus brasiliensis Fr. (Fig. 5) and Paederus rulilicor-

nis Er. are able to produce vesicular dermatitis. More recently Pickel (14) has

mentioned the presence in Brazil of 20 species of Paederus, among which

only 6 are strongly vesicant. He also noted that Paederus feras Er. and P. bra¬

siliensis Er. produce dermatitis in Pernambuco.

In Ecuador epidemic vesicular dermatitis in man was observed by Chapin (15)

in 1920 in connection willi Paederus irritam Chap.. Soon afler, Campos (16)

reported cases produced by 8 different species of Paederus. Earle(17), in

1949 described cases of dermatitis in connection with Paederus ornaticornis

Sharp. In ihis country the disease is known under the name “Fuetazo derma¬

titis”. The name “fuetazo” is a spanish word for whiplash. Il calls attention

to lhe linear aspect of the lesions, especially when they occur on the face.

According to Dallas (18, 19) vesicular dermatitis is produced in Argentina

hy Paederus brasiliensis Er. and P. ferus Er..

Bequaert (20) has shown thal Paederus signaticornis Sharp causes derma¬

titis in Guatemala .

In África the first

Houssiau in 1915 (21)

cases was identified by H. Nolman (in

(Fig. 3). In 1916, Boss(22) showed

nym = P. crebrepunctatus Eppelsheim)

Kenya (Fig. 2).

may also produce a

report of rove heetles dermatitis is thal of Rodhain and

in Leopoldville, Congo. The species involved in these

Bequaert, 10) as Paederus sabaeus Er.

thal Paederus eximius Reiche (syno-

is the agent of a vesicular dermatitis

several parts of Kenya, especially in Nairobi, this species

severe conjunctivitis which is known as “Nairohi-Eye” (see

- 23 — and Roberts and Tonking — 24 —). Other records

by Paederus sabaeus are lhose of Gordon (11)

Sudan and of Denys and Zumpt (26) in South

Symes and Roberti

of dermatitis produced in África

in West África, of Lewis (25) in

West África and in Nyasaland.

In Europe, Sacharow, in 1915 (27) reported cases of dermatitis produced

by Paederus fuscipes Curt (= P. idae Sharp) in Southern Rússia (Fig. 1).

Several other authors have confirmed these observations (Portehinsky, 1915 -—-

28 —, and Pawlowsky and Stein, 1926 — 29 -—•). This species is very common

along the banks of the lower Volga and fishermen and herdsmen are commonly

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A. FAIN

837

1 /

(

Fig. 1-6 — Some harmful Rove-beetles:

Fig. 1 — Paederus fuscipes Curtis

Fig. 2 — Paederus eximius Reiche.

Fig. 3 — Paederus sabaeus Er.

Fig. 4 — Paederus ornaticornis Sharp

Fig. 5 — Paederus brasiliensis Er.

Fig. 6 — Paederidus ruficollis Fabr.

(Photograph made by Institut des Sciences Naturelles

de Belgique, Brussels).

blistered by the crushing of beetles on lhe skin. In 1919, Netolitzky (30), in

Bukovine, was able to produce experimental dermatitis in man witb two other

European species ( Paederus limnophilus Er. and Paederus ruficollis Fab. -—

Fig. 6). Experimental dermatitis in man has also been prodnced in France by

Allard (2) witb Paederus littoralis Crav. and by Theodorides (3) with P. ripa¬

rias L. and P. fuscipes Curt.. It is in Italy lhat the irritant action of Pae¬

derus on the eye has been recognized for the first time by Farina in

1926 (31). These lesions seem to be rather common in that country and Cas-

telli in 1935 (32) recorded numerous cases of conjunctivitis that he called “Oftal-

mozoosi endemica di Paederus . All these ocular lesions were prodnced

by Paederus fuscipes.

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10 11 12 13 14 15 16

838

TOXIC ACTION OF ROVE BEETLES ( COLEOPTERA , STAPHYL1NIDAE)

In Asia lhe vesicular dermalitis has heen reported from severa! countries.

In Judia the disease is known as “Spiderlick” and is produced mainly l>y Pae-

derus fuscipes Curt. (cited hy Strickland — 33 — Strickland and Roy — 34 —

and Isaac — 35 —). Pujatti, in 1947 (36) re|)orts that Paederus melampus Er.

produces dermalitis in the State of Benagolore (índia).

According lo Eysell(37), Paederus peregrinus Er. is able to produce simi¬

lar lesions in Malaysia.

In Indochina epidemic dermalitis is produced by Paederus fuscipes and P.

alternans Walk (see Genevray and col., 38).

Rove beetles dermatitis has also been observed in Japan by Wada in

1926(39). The species involved was Paederus riparias L.. Esaki (40) reports

that the dermatitis produced by P. idae, whioh is a synonym of P. fuscipes Curt.

has been known in Japan, Korea and China since 1590, and Kandu Kanji. in

1935 (41), mentions that severe ocular lesions are produced by this species in

Formosa.

More recently the vesicular dermatitis has been reported from Australia,

by Millard in 1954 (42). The disease is produced by Paederus cruenticollis

Germ.

Biology of the paederus spp.

Many of the authors observing rove beetle dermatitis in man have drawn

attention to the seasonal character of the disease. In Brazil Paederus columbinas

is eneountered from June lo September. In Ecuador the beetles appear with the

first rains in December or January and persist until the onset of lhe dry season

in May or June. In Leopoldville, Central África, Paederus sabaeus is frequent

from April to June, and completely absent at any other time of lhe year (Rodhain

and Houssiau and personal observations). This period corresponds with the end

of the raining season and the beginning of lhe dry season. According to Dr. C.

Rossetti (University Lovanium), lhe beetle population in Leopoldville reaches a

peak every two years (personal comm.). In South West África, Deneys and

Zumpt (26) report cases of dermatitis produced by P. sabaeus during the month

of December. In Freetown, Sierra Leone, P. sabaeus is fairly common during

June. July and August; it disappears during September and October but reappears

in the middle of November (Cordon, IT).

In índia, Isaac (35) has noted that Paederus fuscipes reaches ils grealest

density in May and June, when the vesicular dermatitis become abundant. In

the same country Pujatti (36) made similar observations with Paederus me¬

lampus which oecured mainly from May to July.

These observations show that in tropical regions the beetles are mosl frequent

or al least most active during the rainy and hot season. That a high degree of

humidity is an important condition for the activity of these beetles is shown

by lhe fact that Paederus fuscipes, P. sabaeus, P. eximias and other species are

always found close to river banks or swampy areas. In Europe the most favou-

rable conditions are realized al the end of the Summer.

This periodicity seems, however, not to lie absolute. for in Manaus, Amazonas

(Brazil), Cordon (11) reports that Paederus amazônicas is common in all the

seasons of lhe year.

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A. FAIN

839

Another feature that is to he mentioned in relation with lhe hiology of those

heetles is lhe fact that some species are attraCted to lhe artificial light. It seems

therefore very probable that some cases of dermatitis are contracted during the

night.

PaTHOGENY OF THE ROVE BEETLES

Dermatitis is the most commonly pathological feature observed in relation

with rove heetles; however, in some cases eye lesions may also have been pro-

duced. The latter are generally a result of lhe spread of the irritant with the

fingers and they are therefore secundary to the crushing of the insect on lhe skin.

The skin lesions consist essentially of a vesicular dermatitis and they are

usually seen on lhe exposed parts of the hody. The clinicai evolution of this

dermatitis is rather characteristic. A good description has heen made hy Theo-

dorides (3) who experimented upon himself with Paedems fuscipes. This author

divided the lesions into lliree stages: an erytliematous stage characterized hy a

red patch, a vesicular stage following the preceding one during which the hlisters

appeared, and a squamous or healing stage. The erythematous stage developed

approximately 10 hours after contact with the heetle and it persisted during 48

hours. The macula was not spontaneously painfui hut presented a tickling or

burning sensation. The firsl hlisters appeared 48 hours after lhe beginning of

the erythema. 'I hey enlarged gradually and attained their complete development

wilhin 2 days. During the healing stage the vesicules hecame umbilicate and

dried out. Finally they exfoliated leaving pigmented scars which persisted for

two months. The healing stage began 6 days after the contact with the heetle;

it persisted for about 8 days.

4 he spontaneous dermatitis that I have observed in Leopoldville with Pae-

derus sabaeus presents a similar evolution (Fig. 7). In some cases the maculas

coalesce into large areas up lo 10 cm in diameter. A linear dermatitis. 5-15 cm

long, may appear at lhe place where the heetle has heen dragged along lhe skin

when brushed off hy lhe hand. The lesions are not spontaneously painful;

p l

*•. «

* t

ta**.

mit

f I

£ p*

Fig. 7 — Rove-beetle dermatitis on the arm of an African

in Leopoldville. (Photograph made by Prof. C. Rossetti).

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10 11 12 13 14 15 16

840

TOXIC ACTION OF ROVE BEETLE3 ( COLEOPTERA , STAPHYLINIDAE )

however, a sliglil burning sensation followed by itching may be present. rhe-

vesicules, which are generally very small, contain either a clear or a seropu-

rulent fluid. In some cases lliey coalesce lo lorm a single large blister. The

lesions produced in East África by Paederus eximius (= P. crebrepunctatus) are

more severe and are often accompanied by general symptoms.

When lhe irritant enters into lhe eye, conjunctivitis may occur. Ocular

lesions have been described for lhe first time in Italy. They were produced by

P. fuscipes. These lesions are more frequently encountered and more severe with

Paederus eximius lhan with other species of Paederus. lliey are well known

in Kenya as ‘‘Nairohi-Eye’\

According lo Goldi (9) the hemorrhagic enteritis, well known in lhe Marshall

Islands as "Toddy Disease” is probably produced by swallowing of palm-wine

(called also “toddy”) into which some rove beetles have fallen. So far lhe toxic

action of rove beetles for lhe digestive tract in man or in animais is not esta-

blished wilh cerlainty. In that respect it seems lhat lhese beetles are less

pathogenic lhan those of lhe family MELOIDAE whose irritatíng power for lhe

digestive mucosa is well known in man and in animais.

NaTURE OF THE IRRITANT PRINCIPLE OF THE ROVE BEETLES

Da Silva (1) ibought that the irritant substance responsible for the derma-

tilis was a secretion of the beetle.

Rodhain and Houssiau (21) experimenting on man with Paederus sabaeus

in Leopoldville showed that when the insects were allowed to wander freely over

lhe skin and even when lhey were irritated, no lesions, either immediate or

delayed, were produced. The lesions appearcd only when the beetles were crushed

and rubbed over the skin. Other authors have repeated these experiments wilh

the same results.

Gordon (11) found that lhe reaction occurs on areas smeared with the thorax

and lhe abdômen of lhe beetle but none with lhe head.

Pawlowsky and Stein(43), experimenting wilh /’. fuscipes, have shown that

lhe irritant principie is always present in the genital organs and that it reaches

its highest concentration in the haemolymph.

Some observations have proved lhat the irritant can be absorved through

a non-injured epidermis. It seems probable that the softening of the skin by

an excessivo perspiration or after bathing makes this penetration more easy and

that on lhe contrary a greasy condition of the skin lessens its action.

So far it had been commonly thought that this irritant was cantharidin.

Netolitsky (30) was the first lo surmise that lhe active principie contained in the

rove beetles is not cantharidin. Pawlowsky and Stein, in 1920 expressed the

same opinion. The true nature of this principie lias been shown by Pavan

and I?o(5, 6) and by Pavan (7, 8). These authors succeeded in isolaling and

obtaining in its pure crystalline State lhe active principie of the vesicant substan¬

ce, to which lhey give the name of “pederin”. They also proved lhat pederin

is clearly distinct from cantharidin not only in terms of its biological but also

in terms of its physical and chemical properties.

DiAGNOSIS OF THE ROVE BEETLE DERM ATITIS

The diagnosis of the disease is generally not difficult. It should be based

on the following characteristics:

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A. FAIN

841

1. The sudden appearance of the lesions.

2. The ahsence of bilateral symmetry of the lesions. Generally only one

region of the body is affected.

3. The aspect of the lesions consisting often of a group of small vesicles

or of a single larger vesicle. The linear grouping of the vesicles is

characteristic.

4. The seasonal and epidemic character of the disease.

The long delay (2 to 3 days) between the contact with the beetle and the

appearance of the vesicles may render the diagnosis difficult in absence of an

epidemy.

It may be difficult lo distinguish the rove beetle dermatitis from the

cantharid dermatitis which is produced by beetles of the family MELOIDAE,

especially the genera Ly tt a, Epicauta and Mylabris. The best known

of thern is lhe bright metallic green “Spanish fly”, Lytta vesicatória.

According to Theodorides(44) the vesicle produced by cantharidin develops

on the normal skin while that produced by the Paederus sp. is always preceded

by an erythema.

Bo and Vancurone (45) have summarized in a table their experiences on

rnan with either cantharidin and pederin (see Table 1).

TABLE I — DIFFERENCES BETWEEN THE EXPERIMENTAL DERMATITIS

PRODUCED IN MAN BY CANTHARIDIN AND THAT PRODUCED BY PEDERIN.

(AFTER BO AND VALCURONE, 1958).

Lesion produced by eatharidin

(beetles of the family

MELOIDAE)

Lesion produced by pederin

(beetles of the family

STAPHYLINIDAE)

Delay between the

contact of the ir-

ritant and the on-

set of the erythema

18 — 24 hours.

36 — 72 hours.

Character of

erythema .

the

Mild, without any subjective

symptoms.

Well developed and painful.

Character of

the

Small bullae coalescing quickly

into a single voluminous blis-

ter. This blister contains a

clear fluid and is situated on

a superficial base.

Subjective symptoms almost

absent.

Small, or very small, vesicles

containing clear fluid trans-

forming into larger pustules

(with purulent fluid) which

tend to coalesce. These pustu¬

les are situated on a deep base.

Subjective symptoms, very mar-

ked, consisting of pruritis and

burning sensation.

Healing .

Either by resorption or bursting

of the blister.

By formation of a squamous

crust.

Residual lesion

Pigmentation, if present, is

light and disappears rapidly.

Pigmentation well developed

and persisting. Pruritis may

be present.

1, | SciELO

842

TOXIC ACTION OF ROVE BEETLES ( COLEOPTERA, STAPHYLINIDAE)

TkEATMENT OF THE ROVE BEETLE DERMATITIS

Roberts and Tonking (46) have treated the lilislers with Magnesium Sulfate

compresses.

Earle (17) recornmends a formula containing, among other drugs, Butesin

jiicrate.

Deneys and Zumpt (26) found that eortisone and antihistaminic prepara-

tions had no beneficiai effect and may even be hannful by favouring secondary

infection. They recommend to treat lhe more severe lesions by protection and

the application of an antibiotic powder.

In Leopoldville, on the advice of Professor C. Rossetti (University Lova-

nium), we have treated our cases with the water paster of Darier (equal parts

of Tale, Zinc Oxyde, Glycerine, Water) with good results.

Acknowledgments —- I am indebted to Prof. C. Rossetti, University Lovanium,

Leopoldville, Republique of Congo, who furnished unpublished information and

photograph on rove-beetie dermatitis at Leopoldville.

I also wish to thank Mr. G. Fagel, Institut des Pares Nationaux du Congo,

Brusseis, who reviewed the taxonomic part of the manuscript and supplied the

material for illustration of some Paederus sp.

References

1. SILVA, P. da, Arcli. Parasitol., 15, 429-430, pi. I, 1912.

2. ALLARD, V., Les Staphylinides vésicants du genre Paederus. Thèse. Imprim.

R. Fouion, Paris, 1948, pp. 1-54.

3. THEODORIDES, J„ Buli. Soc. Path. exot., 43, 100-113, 2 pi., 1950 b.

4. GRUVEL, J., Les Coléoptères vésicants. Thèse. Impr. R. Fouion, Paris, 1957,

pp. 1-63.

5. PAVAN, M., and BO, G., Meni. Soc. entomol. ital., 31, 67-82, 1952.

6. PAVAN, M„ and BO, G., Fisiol. Comp. Oecol., 3 (2-3), 307-312, 1953.

7. PAVAN, M., Atti Acc. Nuz. It. Entomol., Rendiconti, 1», 119-124, 1962.

8. PAVAN, M., Ricerche biologiche mediche su pederina e su estratti purificati

di Paederus fuscipes Curt., Impr. Mario Ponzo, Pavia, 1963, pp. 1-93.

9. GOLDI, E. A., Die sanitarisch-pathologische Bedeutung der Insekten, 1913.

10. BEQUAERT, J„ Ann. Soc. belge Méd. trop., 1 (2), 227-229, 1921.

11. GORDON, M. R., Ann. trop. Med. Parasit., 19 (1), 47-52, pl. I, 1925.

12. FROES, H. P., Rev. med. Bahia, 9, 290-292, 1934.

13. FROES, H. P., J. Parasitol., 21 (2), 124, 1935.

14. PICKEL, D. B„ Rev. Entomol., Rio de Janeiro, 11, 775-793, 1940.

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A. FAIN

843

15. CHAPIN, E. A., Arch. Schiffs-u. Tropenhyg., 30, 369-372, 1926.

16. CAMPOS, R. F., Rev. Col. Rocafuerte, Guayaquil., N.°s 27-28, 1927.

17. EARLE, K. V., J. trop. Med. Hyg., 53, 183-187, 1949.

18. DALLAS, E. D., Soc. argent. Patol. Reg. Norte, Buenos Aires, 1163-1167, 1930.

19. DALLAS, E. D., Rev. chil. Hist. Nat., 39, 219-224, 1935.

20. BEQUAERT, J„ Buli. Brookl. entomol. Soc., 37, 107-112, 1932.

21. RODHAIN, J„ et HOUSSIAU, J., Buli. Soc. Path. exot., 8, 587-591, 1915.

22. ROSS, P. H., J. trop. Med. Hyg., 19, 202, 1916.

23. SYMES, C. B., and ROBERTS, J. I., Rep. med. Res. Kenya, Natrobi, (1932),

21-27, 1934.

24. ROBERTS, J. I-, and TONKING, H. D., Ann. trop. Med., 29. 415-420, 1935.

25. LEWIS, D. J., Proc. royal entomol. Soc. London, 33, 37-42, 1958.

26. DENEYS, J. B„ and ZUMPT, F., S. A. med. J., 37, 1284-1285, 1963.

27. SACHAROW, N., The injurious insects noticed in the Govt. of Astrachan.

from 1912 to 1914. From the report of the Station for 1914. Publ. of the

Entom. Station of Astrachan, 1915.

28. PORTCHINSKY, I. A., Friend of Nature, Petrograd (in Russian), 12, 364-366.

1915.

29. PAWLOWSKI, E. N., und STEIN, A. K., Med. Obozr. Nizn. Povolz., N>s 7-8

Astrakhan (in Russian), 1926 a. Trans. royal Soc. Trop. Med. Hyg., 20. 450-451.

1926 b.

30. NETOLITZKY, F„ Z. angevo. Entomol., 5, 252-257, 1919.

31. FARINA, F„ Lett. Oftalm., 10, 1926.

32. CASTELLI, A., Ann. Ottal., 63 (3-4), 204-228, tav. I-VI, 1935.

33. STRICKLAND, C., Indian med, Gaz., 8. 1924.

34. STRICKLAND, C., and ROY, D. N., Indian med. Gaz., 74. 285, 1939.

35. ISAAC, P. V., Agr. Live-Stock índia, 3, 33-36, 1934.

36. PUJATTI, D., Mem. Soc. entomol. ital., 26, 5-11, 1947.

37. EYSELL, A., Handbuch der Tropenkrankheiten, Vol. 1, 1913, p. 247.

38. GENEVRAY, J., GASCHEN. H., AUTRET, M. et DODERO, J., Arch. Inst.

Pasteur Indochine, 19, 313-329, 1934.

39 WADA, H„ Jap. J. Dermatol., 26 (11), 68-70, 1044-1060, 1 pl., 1926.

40 ESAKI, T„ Honzo, Tokio, 11, 27-31, 1933.

41. KANDU KANJI, Taiwan Igak. Zass, 34. 1013-1014, 1935.

42. MILLARD, P. T„ Med. J. Aust., 1 (20), 741-744, 1954.

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FINDLAY E. RUSSELL

845

20. METRONIDAZOLE IN SNAKE VENOM POISONING

FINDLAY E. RUSSELL

University of Southern Califórnia, School of Medicine, County General Hospital,

Los Angeles, Califórnia, U.S.A.

The use of metronidazole [1 (/?-hydroxymethyl) -2-methyl-5-nitroimidazole] in

the treatment of 43 patients having 67 indolent, vascular lesions of diverse

origins has heen noted by Taylor(l). In her series of cases, improvement of

the lesions as demonstrated by decreased vasculitis and perivascular infiltration

was noted in 42 patients (63 lesions). One of these patients was bitten by

an adult prairie rattlesnake, Crotalus hórridas, and treated initially by the

present autbor. Following hospitalization for the acute stages of the poisoning,

the patient was referred to Dr. Taylor for metronidazole therapy. Four addi-

tional patients were subsequently treated witb this drug following the acute stages

of their envenomations. The present report treats of the observations on three

of these patients.

Case 1 — A 12-year-old hoy was bitten on the right middle finger by a

134 cm Southern Pacific rattlesnake Crotalus viridis helleri. The finger was

immediately placed in ice and the patient taken to a hospital. He was given

one vial of Antivenin (Polyvalent) CROTALIDAE and an intramuscular corti-

costeroid. The finger was kept in ice for five days. On the sixth day, the ice

was discontinued and the patient transferred to a second hospital for possihle

amputation of the injured finger (Fig. 1). The. author was called on con-

sultation.

Under anesthesia, the necrotic areas were surglcally debrided. A program

of physical therapy was initiated on day seven, and the patient placed on metro¬

nidazole, 100 mg four times daily for eighl days. Healing appeared to be more

rapid than generally expected in such cases and lhe patient was discharged frorn

the hospital 16 days following the accident. A slight contracture of the finger

was subsequently corrected. Figure 2 shows the finger two months following

the bite.

Case 2 — A 9-year-old boy was bitten on the left foot in the region of the

medial malleolus by a large red diamond rattlesnake Crotalus ruber ruber. Lon¬

gitudinal cuts were made through lhe fang marks approximately 45 minutes after

the bite. Suction was not applied. Because of the chikFs known sensitivity to

horse serum, he was referred to the author for further medicai care.

The patient was first seen by the author eight hours following the accident

(Fig. 3). In spite of lhe lapse of time, 2.1 gm of C. ruber ruber antivenin,

prepared in goats after the method of Criley (2), was injected intravenously over

a 30-minute period. Further therapeutic measures, as previously described (3),

were carried out. At day three the skin lesions extended to the knee (Fig. 4).

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10 11 12 13 14 15 16

METRONIDAZOI.E IN SNAKE VENOM POISONING

846

Fig. 1

Fig. 2

On day foiir the hemorrhagic vesicles and necrosis were debrided by surgical

excision under anesthesia. On day five physical therapy was initiated, and the

patient placed on metronidazole. 125 mg four limes daily; lliis was continued

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Mem. Inst. Butantan

SirriD. Internac.

33(3):845-850, 1966

FINDLAY E. RUSSELL

847

Fig. 3

for one vveek. The

the accident. There

in" the bite.

Fig. 4

patient was discharged from the hospital 10 days following

was no residual. Figure 5 shows the foot one month follow-

Case 3 — A 44-year-old reptile handler was bitten on the dorsum of the

left thumb hy an extremely large (170 cm I timber rattlesnake Crotalus horridus

hórridas. lt was suspected that one fang pierced a blood vessel as there was

excessive bleeding from one of the puncture wounds. The patient was rushed to

the hospital, arriving in a cyanotic and comatose State (B.P. 50/0). Emergency

measures consisted of fresh whole blood, intravenous antivenin (including poly-

SciELO

g48 METRONIDAZOLE IN SNAKE VENOM POISONING

Fig. 7

Fig. 8

SciELO

Fig. 5

Fig. 6

Mem. Inst. Butantan

Simp. Internac.

33(3): 845-850, 1966

FINDLAY E. RUSSELL

849

valent Crotalus antivenin prepared in goats), oxygen vasopressor agents,

localized infillration of the wounds with calcium disodium edetate (EDTA) and

measures previously described (3). The palient’s condition remained criticai for

four days, during which time he received 10 pints of blood.

Figure 6 shows the left hand and forearm four days following lhe enveno-

mation. At day seven the vesicles and necrotic areas were surgieally debrided

under anesthesia (Fig. 7), and physical therapy was instituted. Subsequently,

the patient was placed on metronidazole, 250 mg four times daily for two weeks.

Healing appeared to he rapid (Fig. 8).

Au infection over the base of the first metacarpal phalangeal joint ne-

cessitated surgical drainage. The incision exposed the extensor tendons and

bone, and a subsequent chronic osteomyelitis of the joint developed. The com-

plication lead to some loss of bone and soft tissues. Orthopedic surgery corrected

most of the defect so that lhe patient now has some use of the thumb.

The lesions produced by the venoms of most of the North American rattle-

snakes are troublesome in that they are usually multiple and often times large,

and tinis particularly susceptible to infection. They are also painfnl and very

slow to heal. The present limited series of cases indicates that the use of metro¬

nidazole following the acute stages of lhe poisoning should he given further

clinicai trial. The drug appeared to increase healing and perhaps reduce pain

over that expected in similar cases of envenomation by rattlesnakes.

There would not seem lo he any contraindication for the use of the drug,

although administration should he limited to the period following the acute stages

of the poisoning, which generally persists for 4 to 6 days following the Lite. It

should not be used in the presence of serious systemic poisoning.

References

1. TAYLOR, J. T., Trans. west. Pharmacol. Ass., 9, 37, 1966.

2. CRILEY, B. R., in E. E. BUCKLEY and N. PORGES (Editors), Venoms,

Amer. Ass. Advanc. Sei., Washington, 1956, p. 373.

3. RUSSELL, F. E., in G. M. PIERSOL (Editor), Cyclopedia of Medicine, Sur¬

gery and the Syecialties, Vol. 2, Philadelphia, 1962, p. 197.

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F. MARKWARDT

851

21. THE RELEASE OF BIOGENIC AMINES FROM BLOOD FLATELETS

UNDER THE INFLUENCE OF CROTALUS D. TERR1F1CUS VENOM

F. MARKWARDT

Institute of Pharmacology, Medicai Academy, Erfurt, Germany

In previous studies ou the action of certain snake venoms and of bee venom

on blood platelets we found, lhat the venom of the Brazilian rattlesnake (Cro-

talus d. terríficas ) causes in even very small concentrations a release of l)iogenic

amines and of ATP from these cells(l). The venom component responsible

for this effect is of special interest with regard to an analysis of both the action

of the venom and the mechanism of amine release. Therefore, we endeavoured

in the isolation and characterization of the amine releasing component of the

venom and in clarifying its mechanism of action.

In order to isolate the active amine releasing principie, Crotalus venom

was disassemhled into several components by gelfiltration on columns of Sephadex

G 100. The experimental results are summarized in Fig. 1. A separation of

the amine releasing component from clotting and phospholipase activities and

other ballast proteins could be estahlished. The fractions active on blood pla¬

telets were collected and freed from buffer salts hy dialysis against destilled

water. The specific activity of the solution obtained wms 20 times higher than

that of the crude venom measured hy the serotonin release. The yield of active

siihstance averaged 60-65 per cent. The factor gives the reactions of a high

molecular protein.

Furthermore it was studied, whether the amine releasing component posses¬

sos other biochemical activities, known to be present in the whole venom. Resides

its action on blood platelets. Il was shown, that the isolated component is related

to one of the activities, which conld be demonstrated and investigated with whole

venom. with the exception of only a small caseinolytic activity. Moreover. phar-

macological reactions caused hy whole venom as well as general toxicity were

found not to be connected with it.

Serotonin release induced hy the amine liberating component proceeds with

high speed. Under the experimental conditions used more than 50 per cent

of the slored amines were released during 5 min hy only 0.01 pg of lhe isolated

component per ml. Amine liberation by lhe venom factor can be inhibited by

omilling of calcium and hy blocking of the platelet metabolism (Fig. 2). After

incubation of platelets with the amine releasing component in the absence of

calcium the eells release lheir serotonin already by resuspending them in a

médium containing calcium. Amine liberation hy the active venom component

is also possible in blood. In the presence of plasma its effectiveness is only

slightly diminished. Therefore, we also investigated the action of the isolated

component in the circulating blood by injecting il intravenously into rabbits

under urethan narcosis. Intravenous injection of the component was followed

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10 11 12 13 14 15 16

released

852

THE RELEASE OF BIOGENIC AMINES FROM BLOOD PLATELETS

UNDER THE INFLUENCE OF CROTALUS D. TERRIFICUS VENOM

E 280

Tube number

Fig. 1 — Gelfiltration of C. d. terrificus venom on Sephadex G 100.

Fig. 2 — Release of serotonin from

rabbit platelets by C. d. terrificus venom.

a) in Tyrode-solution; b) without glu¬

cose; c) with 10 — 3 m monoiodoacetic

acid; d) with EDTA.

SciELO

10 11 12 13 14 15 16

Activity m °/ c

Mem. Inst. Butantan

Simp. Internac.

33(3) :S51-854, 1966

F. MARKWARDT

853

by a fali of the hlood pressure, which in the same intensity could be produced

also by injectioii of a serotonin quantity equivalent to the amount contained

in blood platelets. A second injection di cl not result in a hlood pressure respon¬

se (Fig. 3). The number of thrombocytes was found to be lowered to 50 per

cent afler the injection. Platelets isolated from the hlood after administration

of the venom factor contained only 20 per cent of its original serotonin levei.

Fig. 3 — Effect of the isolated amine liberating component of C. d. terrificus venom

(AfK) on blood pressure.

Conclusion — In lhe venom of the Brazilian rattlesnake í ('nilahts d. ter¬

rificus) lhere exists a relatively high molecular protein component, which is

able to Iiberate stored biogenic amines from blood platelets. The factor is active

in platelet suspensions in vitro as well as in the circulating blood. 1 his compo¬

nent is not identical with other biochemical and pharmacological active princi¬

pies of the whole venom. The induced release-reaction seems to proceed in two

steps. In the first step the amine releasing venom factor induces a change in the

permeahilily of the platelet membrane, and in a subsequent reaction amines be-

came liberated.

References

1. MARKWARDT, F., BARTHEL, W., GLUSA, E., and HOFFMANN, A., Naunyn-

Schimiedebergs Arch. exp. Pathol. Pharmak., 225, 297, 1966.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3):855-856, 1966

JOSEPH G. GENNARO and NEWTON C. McCOLLOUGH

855

22. FURTHER OBSERVATIONS ON CORAL SNAKE RITES IN THE

UNITED STATES: SYMPTOMS AND THERAPY

JOSEPH G. GENNARO and NEWTON C. McCOLLOUGH

University of Florida, College of Medicine, Florida, U.S.A.

Since our first ohservations on the Dite of a North American coral snake

were published ( Journal oj the Florida Medicai Assoe., 49, 968, 1963), our

work lias continued along parallel experimental and clinicai lines. The snakes

used in these experiments are pari of a colony of over two hundred animais,

primarilv of Micrurus fulvius , from whieh venom has been collected for toxico-

logical study.

Histological studies of lhe venom apparatus of lhe North American coral

snake indicate that lhe venom gland is a combined serous and nuicus secreting

organ. The mucus portion of the gland is somewhat more extensive in pro-

portion lo the lotai glandular strueture than lhat which has been described in tbe

pit viper (Gennaro et ai, New York Academy of Sciences) and other vipers

(Kochva & Gans, American Journal of Anatomy). Since it has been shown that

the mucus secreting portion may be responsible for spreading and enhancing the

toxicity of the serous secretion, the increase in relative size of the mucus se-

creling portion of the gland may be responsible, in the coral snake, for the

high levei of toxicity manifested by the venom.

The lyophilized venom of Micrurus fulvius used in the experiments here was

titer for lethality in mice and dogs. The L.D.,„ in the albino mouse (20 grams)

is 0.23 microgram per gram of mouse or 0.23 milligrams per kilo. The L.D. 30

value in dogs is 0.3 microgram per grani or 0.3 milligrams per kilo. Though

these values appear to be similar, il is easy to see that the amount of venom

injected in the dog is really much greater than that lethal in a mouse, ft is

interesting to note that the dog exhibited very little variation in sensitivity to

this venom as they commonly do to the venom of the Crotalus snakes.

The symptoms tnost frequently occurring in cotai snake envenomation in

dogs followed two general patterns.

In sublethal doses the initial aspect (first hour) was characterized by a

drowsiness and favoring of lhe injected leg. The total course of envenomation

in the suhlethally injected dog proceeded wilh various body tremors, some respi-

ratory difficulty, and dizziness .possibly associated wilh bloody urine.

In lethally injected animais the course of symptoms presenting in the first

hour included vomiting and a general parasympathetic-stimulation picture which

included salivation and defecation. Death in these animais usually followed

paralysis, respiratory difficulty, continued salivation, and repeated vomiting, some

impairment of the blink reflex, and fixation of the pupils centrally. No retinal

hemorrhage is indicated although hematúria is common and blood can a]ipear

from most of the body orifices.

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856

FURTHER OBSERVATIONS ON CORAL SNAKE BITES IN THE

UNITED STATES: SYMPTOMS AND THERAPY

In cases of actual snake bite lhe character of lhe Lite itself, and its duration,

was estahlished to be more painful than has been recorded in lhe literature and

mucli more quickly occurring. Some pain, favoring, and tenderness alvvays seem

lo accompany the Lite vvhich lasts from four or five seconds to too short a

period to measure using a stop watch; all bites of snfficienl duration do produce

a lethal effect.

In most oi the liuman cases recorded in the literature deatli occurs within

twenty-four liours or not at all. In dogs, lhe L.D.50 dose produces survival in

the range of 91/í> hours. When this dose is increased hy íive times the sur¬

vival is shortened but lhere is considerahle variation in the survival period which

can be as little as fifty-eight minutes or as long as íive and one-half hours at

this dose.

The L.D .50 dose is completely reversihle hy lhe administralion of antiserum

(Butantan anticoral or lhe rabbit anticoral made in our lahoratory). The first

effect of the antiserum is to lengthen the survival time. Doses as high as three

times the L.D . 50 may be survived if the antiserum is given in a dose sufficient

to provide 2 ^ milliliters for every milligram of venom injected as soon as tvvo

hours after the bite (intravenously). The importance of the treatment time is

illustrated hy the fact that 3.3 milliliters of antiserum per milligram of venom is

valueless if the treatment time is delayed until five hours after the bite. On

lhe other hand the L.D . 50 dose may be neutralized and lhe animal may survive

with only 1.4 ml of antiserum per milligram of venom if it is given within the

liour after the bite.

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10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3):857-860, 1966

JOSÉ MONROY VELASCO

857

23. INYECCIÓN IN SITU DE ANTISUERO EN EL TRATAMIENTO DE

LA INTOXICACIÓN POR MORDEDURA DE SERPIENTE

JOSÉ MONROY VELASCO

Laboratorio de Control Biológico, Laboratorios “MYN”, México

Presento ante la ilustrada eonsideración de usledes un procedimiento senoillo,

pero lógico, para neutralizar el veneno de serpiente en el mismo lugar en donde

lo deposito el animal al producir la mordedura.

El veneno de las serpientes, al ser inoculado, causa sintomas graves: gene-

rales tóxicos y locales en el mismo lugar de la mordedura.

La acción local sobre los tejidos se ejerce por medio de enzimas. Tiene

una rápida acción difusora por la hialuronidasa que contiene, que bidrolisa el

ácido hialurónico dei lejido conjuntivo.

Además de esta acción de difusión, el veneno, principalmente el de B o -

t h r o p s , produce lesiones en los vasos sanguíneos, alterando las células endo-

teliales y provocando la disolución de sus paredes, por medio de lecitinasas y

proteinasas. Consecuentemente, provoca extravasación de eritrocitos hemolisados

e de suero en los tejidos. El resultado es un edema hemorrágico muy extenso.

Las propiedades digestivas de los venenos, más acentuadas en el de las

Bothrops, unido a la acción destructiva sobre los vasos sanguíneos, provoca grandes

necrosis de los tejidos, a tal grado, que estos llegan a desprenderse, dejando los

huesos al descubierto.

Se atribuye a las reacciones enzimáticas la producción dei choque. Y se

ha achacado a la histamina el descenso de la presión sanguínea, histamina que

ha sido liberada en las lesiones celulares causadas por la lecitinasa.

Como es tan sabido, desde 1897, los sueros se producen usandose como anlí-

genos venenos de serpientes Bothrops, Crotalus, etc. que provocan la

formación de anticuerpos. Los anticuerpos se caracterizan por su acción electiva

sobre los antígenos que intervinieron en su producción. Para que los anticuer¬

pos puedan ejercer su acción neutralizante, deben ponerse en contacto con el

antígeno homólogo.

En la titulación dei antiveneno se hacen mezclas de antiveneno y veneno,

para determinar la dosis de veneno que es neutralizada poi un ml de suero.

El problema — Neutralizar la acción tóxica dei veneno, antes de que

sea absorbido por el organismo.

Criando ocurre un accidente ofídico, la primera preocupación que se tiene,

es la eliminación dei veneno, para lo cual se ha recurrido hasta a la amputación

dei miembro lesionado. Pero lo que más se usa es la desbridación amplia dei

sitio mordido, para succionar el veneno, sea con la boca directamente o mediante

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10 11 12 13 14 15 16

858

INYECCIÓN IN SITU DE ANTISUERO EN EL TRATAM1ENTO DE

LA INTOXICACIÓN POR MORDEDURA DE SERPIENTE

el uso de ventosas. Procedimiento que tiene inconvenientes por provocar he¬

morragias e infecciones y la fácil contaminación de la herida por el Clostridium

letani.

El antisuero homólogo, en inyección intramuscular, neutraliza al veneno ya

absorbido que se encuentra en la sangre circulante. Pero no llega a evitar las

graves lesiones locales que el veneno a una alta concentración en los tejidos,

produce mediante sus enzimas.

El problema es eliminar la acción local y la general dei veneno, tal como se

ha pretendido por la amputación y la deshridación eon succión.

HesoluciÓn — Antes hemos mencionado que el suero se titula según la

dosis de veneno que neutraliza un nd “in vitro”, por lo tanto, se supone que

también pueda ser neutralizado el veneno por el antisuero homólogo en el mismo

sitio en el que la serpiente lo deposito, en los tejidos de la víctima.

Bajo estas premisas procedimos a la experimentación.

Experimentación

Veneno empleado — Veneno deshidratado por medio de liofi 1 ización

de Bothrops atrox, en solución salina al 3% (cada mililitro corresponde a

30 mg de veneno).

Suero — Proporcionado por los Laboratorios "MYN”, S.A. de México,

eon título neutralizante de 12.5 mg de veneno deshidratado por un mililitro.

Animal usado — Conejos de aproximadamente 3 kg. de peso corporal.

Lugar de inoculación dei veneno — Parte superior de uno de

los muslos, previamente depilado.

V í a u s a d a — Subcutânea.

Se emplearon 3 grupos de 5 animales cada uno:

Al grupo A se le inyectó un mililitro de la solución (30 mg de veneno).

Al grupo B se le inyectó la misma dosis de veneno y en seguida se le

inyectó 4 ml de suero Antibothropico, por via intramuscular, en el oiro muslo.

Al grupo C se inyectó eon la misma dosis de veneno (1 ml = 30 mg) e

inmediatamente después 4 ml de suero “in situ’’, de tal manera que se mezclaron.

Resultados

Los conejos dei grupo A, a quienes solamente se les inyectó el veneno,

murieron Iodos en un promedio de una hora y media después.

Los conejos dei grupo B, a quienes además dei veneno recibieron 4 ml de

suero por via intramuscular, sobrevivieron, pero se les produjo, en el lugar de

la inyección dei veneno, un extenso edema, a las 24 horas, eon formación de

escara a las 48 hs. Los conejos dei grupo C, a quienes se les inyectó el suero

en el mismo lugar de la inyección dei veneno, solamente se observo, en el lugar

de la inyección, un pequeno edema que desapareció después de 4 dias. No se

formó escara.

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3)-.857-860, 1966

JOSÉ MONROY VELASCO

859

Técnica de la inyección dei antisuero — Deve hacerse en

la región mordida una verdadera infiltración dei antisuero de tal manera que

aún que el veneno haya emigrado a la parte declive dei miembro lesionado.

Ilegue a mezclarse con el veneno y lo neutralice.

Los conejos que recibieron la inyección dei antisuero por via intramuscular

y sobrevivieron, los dei grupo B, la escara no se formó precisamente en el lugar

de la inyección dei veneno, sino en la parte declive dei muslo. Tal vez la

acción difusora dei veneno al hidrolizar el ácido hialurónico dei tejido conjun¬

tivo facilita su migración. Por lo tanto debe tomarse en cuenta esta mobili-

zación dei veneno para la inyección dei antisuero, de modo que lleguen a mez¬

clarse.

Caso clínico — En la práctica hemos tenido la ocasión de observar

a uno de nuestros empleados quien tuvo tres accidentes por mordedura de Cro-

talus d. terríficas. En el primer accidente se le inyectó el suero “in situ” y además,

por via intramuscular, una dosis de suero. Localmente no presentó más tarde

ninguna lesión, y los sintomas generales fueron sin importância. En la zona

mordida solamente se observo ligero edema que desapareció a las 48 horas.

El segundo accidente consistió en una mordida en el índice izquierdo y fué

una lesión superficial, por tal motivo y tomando en consideración que debía de

haber una sensibilización al suero y a petición de la víctima, no se le inyectó

suero. Después de 2 horas, se tuvo que inyectar de emergencia debido a los

graves sintomas de intoxicación que se desencadenaron. Salvó la vida, pero la

lesión que se produjo en el índice tardó en cicatrizar más de un mes con peligro

de perder la falangeta.

El tercer accidente fué la mordedura en el dorso de la mano derecha por una

Crotalus. Inmediatamente se procedió a inyectarle “in situ” el suero homó¬

logo (Anticrotálico). En esta ocasión no se produjo ninguna acción destructiva

y no se presentaron sintomas de intoxicación ya que además recibió una inyec¬

ción intramuscular de suero.

C o ti d ició n “ sine qu a no n ” — Para que este procedimiento de neu-

tralización dei veneno por el antisuero sea útil, debe aplicarse tan pronto como

se produce el accidente ofídico. Con tal objeto debe encontrarse el suero allí

en donde precisamente se necesita, acompanado de una jeringa estéril y algodón

con un antiséptico. Ciertamente que el suero líquido tolera por un ano la tem¬

peratura ambiente, pero pierde el 50% de su actividad durante un ano a 30°C.

En cambio el suero liofilizado tolera esa temperatura durante un máximo de

5 anos. El suero cuyas proteínas han sido modificadas por digestión enzimática

conserva su actividad por más liempo que el suero líquido.

El suero liofilizado debe formar parte dei botiquín de emergencia que

deben usar nuestros campesinos. Además debe capacitarse a las personas res-

pousables para aplicar el suero inmediatamente. De tal manera que de una tre¬

menda emergencia como es la mordedura de serpientes venenosas pasa a ser un

accidente sin importância con sólo inyectar in situ el sueio homólogo. 5 a

es tiempo de evitar la muerte de nuestros hombres dei campo por mordedura

de serpiente. Cuando la víctima es transportada al hospital más cercano, o

fallece en el camino o si se salva sufre la acción local dei veneno, con la forma-

ción de esfacelos, que tardan vários meses para cicatrizar y que muchas veces

terminan por amputaeión dei miembro lesionado, después de permanecer hospi-

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10 11 12 13 14 15 16

CfiO INYECCIÓN IN SITU DE ANTISUERO EN EL TRATAMIENTO DE

ouw LA INTOXICACIÓN ROR MORDEDURA DE SERPIENTE

talizado bastante tiempo. Hospitalización que además de ser costosa impide al

enfermo trahajar y por lo tanto deja de producir, para más tarde convertir-se

en un lidado.

Este procedimiento que, como una comunicación previa fué publicado en ia

Revista Mexicana de Ciências Médicas y Biológicas en et número correspondiente

a los meses de enero-febrero de 1952, lo presento ahora ante la docta consideración

de ustedes como un homenaje a Vital Brazil, el fundador no sólo dei Instituto

Butantan, sino de ia seroterapia antiofidica de nuestras Américas.

SuMMARY

The venom of Bothrops snakes, generally produce local necrosis in the

tissues, due lo its intense proteolytic action. In this paper an injection melhod

“in situ” of the homologous antiserum was proposed, to avoid those lesions,

neutralizing the venom at lhe same site of the hite, thus preventing for the most

part lhe general and local activities of the venom.

The results are effective to such extend, that the employement of this pro-

cedures is proposed and at the same time the adequate instruction for the rural

population to start a rapid Ireatment of lhe accidents hy venomous snakes.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3):861-864, 1966

E. R. TRETHEWIE

861

24. PHARMACOLOGICAL EFFECTS OF THE VENOM OF

HAPALOCHLAENA MACULOSA

E. R. TRETHEWIE

Department of Physiology, University of Melbourne, Australia

The octopus Hapalochlaena maculosa, banded oetopus, was pinned out on

cork and the brain pierced vvilh a sharp instrument. The venom gland on each

side was dissected. The gland was weighed and ground up with silica in Tyrode.

Initial studies have heen carried out with this Tyrode exlract and more specia-

lized chromatography is heing done on an acid acetone extract.

• .

Fig. 1

Dissected venom glands removed from the Octopus.

In Australia fatal cases of poisoning from handling this octopus have occured

and some near fatal cases. The beak Iike process allows venom injection. The

most obvious physical feature is respiratory paralysis and prolonged artificial

respiration can be expected to end in survival.

The venom gland is the posterior salivary gland and the handing on the

octopus is not unlike that on the cone shell and lhe tiger snake which also pro-

duce neurotoxin.

The isolated jejunum of the guinea j)ig was tested for activity and the res¬

ponses are similar lo those produced by snake venom. They differ in that this

venom greatly reduces the sensivity of the gut lo histamine and contraction to

the venom is immediate. Following the immediate contraction tliere is a typical

delayed S.R.S. reaction such as we have in anaphylaxis and with snake venom

We are now engaged in purification studies to dissociate these responses.

SciELO

10 11 12 13 14 15 16

Beak like process attached to the terminal ducts

of the gland.

View of beak like process inclicating the solicllty

of the beak allowing skin penetration.

View of Octopus showing bamling on tentacle.

SciELO

10 11 12

Mem. Inst. Butantan

Simp. Internac

33(3) : 861-864, 1966

E. R. TRETHEWIE

863

Fig. 4 — Responses of Isolated jejunum of guinea-plg

O.V. 4.5 mg Venom extract showing immediate eon-

traction followed by delayed relaxation. Unlettered con-

tractions to 0.1 ng histamine. Halving of responses after

venom injection.

When we perfuse the isolated heart of the cat we find that venom in doses

quite effective on the gut produce very little effect on the heart. The amplitude

of contraction falis off oídy slightly more rapidly than that of control hearts.

The eoronary flow is also little affected even when the venom is recirculated

with lhe perfusion. The rate of the heart is slowed immediately following in-

jeclion of venom hut lliis recovers rapidly. The ECG under the Langendorf

type perfusion with Tyrode normally soon shows bloek in contra distinction to

that of the blood perfused heart which is normal. We were not able therefore

lo determine with this technique whether heart bloek could occur from a direct

effect of the venom and its appearance in the intact animal follows probably

from anoxia.

Fig. 5 — Contraction of the isolated perfused heart of the eat. At arrow, intra-

coronary injection of venom (18 mg). There is little fali off of amplitude over

40 minutes. Time S = 1 min, R = 1 sec, M = 10 sec intervals.

In lhe intact cat following the injection of gland extract intravenously lhe

respiration slows and the hlood pressure declines slightly. Sometimes respiration

stops, returns temporarily, and finally ceases a few minutes laler. Large doses

of gland extract lower the hlood pressure significantly and this is coincidental

with failure of respiration. Artificial respiration and cardiac massage have not

been adequate to restore the animal in acute experiments.

The ral diaphragm preparation is very sensitive to venom extract. The

response to indirect stimulation is rapidly abolished. At a later stage the res¬

ponse to indirect stimulation fails and recovery on repeatedly washing out the

bath occurs in reverse order; hut lhe response to nerve stimulation remains im-

paired for a very long time.

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10 11 12 13 14 15 16

864

PHARMACOLOGICAL EFFECTS OF THE VENOM OF

HA PA LOCHLA ENA M ACU LOS A

r 2

f —---

I' 20mm Hg

T-" ——^

Fig. 6 — Upper tracing — respiration. Lower tracing — blood pressure, in the intact

cat. At the arrows, venom extract injected, 18,50 mg gland extract. Respiration after

temporary increase soon fails. Blood pressure slovvly declines with temporary depression

from extract. Respiration fails completely before heart beat fails.

D 2%o

N D

5 5

Fig. 7 — Responses of rat diaphragm to (D) direct and (N) indirect (phrenic nerve)

stimulation. Failure to direct stimulation is not complete nor as severe as that to

indirect stimulation, and recovers much more rapidly.

It is apparent lhat lhe main effect of the venom is respiratory paralysis.

Cardiac effects are largely secondary, prohably due lo anoxia, and appear in lhe

inlact animal but lo no marked degree in the isolated heart. Respiration fails

before the blood pressure falis signifieanlly. One wonld expect the blood pres-

sure lo rise with the failure of respiration from asphyxia unless lhe venom lias

a similar effect on the vaso-motor centre or peripheral arteriolar inervation as il

has on lhe rat diaphragm. Il may well be that there is a cenlral effect on

respiration also and we shall investigale this possibility later. The temporary

slowing of the heart but without impaired amplitude of beal suggests some

direct cardiac effect similar to that of lhe venom on the muscle of the diaphragm

and the gut.

The isolated diaphragm progressively shows failure of transmission of the

nerve impulse as well as less evident direct muscle depression and since this

effect is rapid and marked in relation to quantity of gland compared with any

cardiac effect it wonld appear that the main effect of the venom is in blocking

nerve eonduction and so causing death. It wonld therefore appear advisable in

man in addition lo applying artificial respiration lo administer prostigmin, and

lhereby enhaneing transmission.

Chromatographic studies indicate there are two muscular toxins.

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac

33(3): 865-868, 1966

AVELINO BARRIO

865

25. DIFERENCIAS INMUNOLÓGICAS ENTRE ENTIDADES SIMPÁTRIDAS

DE ARANAS DEL GÉNERO LATRODECTUS WALCKENAER

AVELINO BARRIO

Instituto Nacional de Microbiología “Carlos G. Malbrán”,

Buenos Aires, Argentina

La sistemática de Ias ararias dei género Latrodectus resulta suma-

mente compleja, especialmente en relación ai grupo mactans.

Ln Ia actualidad parece haberse llegado a un punto en el que la utilización

exclusiva de los caracteres somáticos no aportaria más elementos de juicio para

dilucidar este problema.

Así, mientras ciertos aracnólogos como Schiapelli y Pikelind, 2) sostienen

que en la Argentina solamente existe una especie, L. mactans (Fabricius), 1775.

tal como fuera establecido en la tesis de Sampayo de 1942 (3) y en trabajos

posteriores, otros, por ei contrario, como Levi (4) y luego Abalos (5) opinan

que en nuestro país se eneuentran varias especies afines (gemelas o crípticas)

a L. mactans. Este ultimo autor utiliza la designación provisória de Latrodectus

A ." 1 , N.° 2, N.° 3 y N.° 4 para las 4 formas dei grupo mactans, según él de

jerarquia específica, encontradas en la província de Santiago dei Estero. Fun¬

damenta su diferenciación en su ecologia y principalmente en las características

constantes de las ootecas.

Dadas las diferentes opiniones y ante la posibilidad de obtener apreciables

cantidades de ejemplares de estas ararias, resolvimos encarar el problema desde

el punto de vista inmunológico, con el fin de obtener nueva información en

torno a esta controvérsia. En este sentido y con similares propósitos merece

eitarse la reciente contribución de Mc Crone y Netzloff (6) sobre las propieda-

des inmunológicas y electroforéticas de los venenos de las especies norteameri-

canas dei género Latrodectus.

Material y métodos

Se investigaron las precipitinas por el método de la doble difusión en agar,

según Outcherlony (7) y Grasset y colab. (8) entre otros, por su sencillez, posi-

bilidades de trabajar con pequenas cantidades de material y por la reproducti-

bilidade de sus resultados.

Se emplearon como antígeno exlractos acuosos de aparatos venenosos de ejem¬

plares hembras (100 aparatos por ml de solución salina) de Ires entidades que

conviveu en la ciudad de Santiago dei Estero y aledaiios, a saber: Latrodectus

geomelricus, especie perfectamente identificable y sobre cuya clasificación no

existe problema alguno; Latrodectus N.° 1 y Latrodectus N.° 2 (de Abalos), enti¬

dades sobre cuyo “status” existem discrepâncias (debido a su menor abundan-

eia en esa zona no bemos podido investigar, por ahora, Latrodectus N.° 3 y

SciELO

10 11 12 13 14 15 16

866

DIFERENCIAS INMUNOLÓGICAS ENTRE ENTIDADES SIMPÁTRIDAS

DE ARASAS DEL GÊNERO LATRODECTUS WALCKENAER

Latrodectus N.° 4). Por los motivos que se expondrán más adelante, y a los

efectos de fundamentar nuestras conclusiones, consideramos de capital importân¬

cia que lodos los ejemplares de Latrodectus N.° 1 y Latrodectus N.° 2 proce-

dieran de la misma área, vale decir que fueran simpátridas.

Como anticuerpos se utilizaron los obtenidos en suero de caballo inmuni-

zado con cantidades no determinadas de aparatos venenosos de Latrodectus N.° 1

y N.° 2, puesto que durante el proceso de inmunización no se tuvo en cuenta

la posihilidad de que se tratara de dos entidades diferentes. El título dei suero

es tal que 1 ml protege contra 150 DMM ratón (1 DMM = 0.005 ml de una

solución que contiene 32 ap. ven./ml) por via endovenosa, posleriormente ésle

fue diluído al doble en solución fisiológica para su utilización en estas expe¬

riências.

Resultados

Los espectros de las líneas de precipitinas de la figura 1 evidencian que

si bien las tres entidades poseen ciertos antígenos idênticos, existen otros no

comunes a L. geometricus por un lado y a L. N.° 1 y L. N.° 2 por otro; esto

es bien notorio por los entrecruzamientos observables en las zonas de contacto.

Igualmcnte se verifican diferencias antigénicas, aunque de grado menor,

entre Latrodectus N1 y N.° 2. En la figura 2 se observan las líneas prin-

cipales A, B, C y D, en general desdobladas, que corresponden a los antígenos

similares comunes y la línea B’ característica de Latrodectus N.° 2 y que no

existe en Latrodectus N.° 1, como puede apreciarse en los tres lugares senalados.

Además debemos hacer notar que existen otras líneas de menor intensidad

que aparecen más próximas a la cúpula dei suero que tampoco corresponden a

antígenos idênticos de ambos extractos.

Fig. 1 — Espectros <le bandas cie preci-

pltinas de aparatos venenosos de La¬

trodectus. 1: ( mactans) N.° 1; 2:

( mactans ) N.° 2; G: geometricus ;

S: suero antilatrodectus.

Finalmente seíialaremos que, en lo que se refiere a la formación dei espectro

existen significativas diferencias en el orden cronológico y en la intensidad con

que aparecen las líneas en ambos espectros. Este liecho babla a favor de ciertas

diferencias cuantitativas y de velocidad de difusión existentes aún en los antí¬

genos comparables y considerados como similares de ambos extractos.

SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(3):865-868, 1966

AVELINO BARRIO

867

Fig- 2 — Espectros de bandas de precipitinas de aparatos venenosos de Latro-

dectus. 1, 2 y S igual a Fig. 1. A, B. C y D: bandas eorrespondientes a

antigenos comunes. B’: banda correspondxente a un antigeno no común.

Comentário

De lo anteriormente expuesto surge la no lotai identidad antigénica entre

las entidades Latrodectus N.° 1 y N.° 2, sobre las que tratamos de aportar

nuevos elementos de juicio relativos al esclarecimento de su “status”.

Ahora bien, de acuerdo a las investigaciones con venenos ofídicos de Schen-

berg (9, 10) y nuestras (11, 12), se ba podido comprobar que diferencias anli-

genicas de la magnitud senalada son registrables dentro de una misma especie

al comparar nniestras procedentes de diferentes poblaciones separadas geográfi¬

camente, vale deeir alopátridas.

Teniendo en cuenta estos antecedentes es que tuvimos especial cuidado en

tomar, para nuestras investigaciones, material de indivíduos de Latrodectus N.° 1

J N.° 2 de la misma procedência, vale decir que fueran simpátridas, o más

exactamente microparapátridas, según la reciente denominación propuesta por

Hobart M. Smith (13). A este respecto es objetable que en el trabajo de

McCrone (op. cit.), no se baga mención con exactitud a la procedência dei

material estudiado, puesto que no tendrían validez específica "per se” Ias dife¬

rencias inmunológicas bailadas en ejemplares alopátridos. Como en este tipo

de relacion espacial el intercâmbio genético es posible, de no producirse éste,

debe admitirse forzosamente la existência de un aislamiento en su reproducción.

En efecto, esto es lo que pensamos que acontece por haber comprobado que

ambas entidades mantienen conslantemente diferenciados sus componentes anti-

génicos a pesar de la proximidad de sus nichos ecológicos.

En síntesis, somos de opinión que, a las distintas preferencias ecológicas y

de comportamiento mencionadas por Abalos (op. cit.) deben sumarse las que

ahora sefialamos en relación a la diferente composición antigénica de suas res¬

pectivos aparatos venenosos. Estos hechos deben ser indudablemente referidos

a la constitución desigual dei genoma respectivo y son prueba de la existência

de mecanismos de aislamiento pre y/o postnupciales que permiten suponer que

SciELO

868

DIFERENCIAS INMUNOLOGICAS ENTRE ENTIDADES SIMPATRIDAS

DE ARARAS DEL GÉNERO LA TRODECTUS WALCKENAER

estas dos entidades en su diferenciación ya han alcanzado un rango específico.

Cuando se prosigan las investigaciones en otros lugares se estará en condiciones

de apreciar cual es la amplitud de la dispersión geográfica de cada una de estas

dos formas y cuan extensa es su área de simpatría.

SUMMARY

15y means of double-diffusion-tests llie homogenates of the venom from fe-

inale Latrodectus spiders, belonging lo the species geometricus Koch and

L. N. n 1 and N.° 2 (mactans group of Abalos) whose status is questioned, were

studied. These spiders eame from Santiago dei Estero city and surroundings.

The precipitin spectrum shows that the three entities possess several common and

certain uncommon antigenic components. The immunochemical differences

between lhe two sympatric (or microparapatric) entities Latrodectus N° 1 and

Latrodectus N.° 2 and their ecological and ethological characteristics prove the

existence of isolating mechanism. These facts allow us to conclude that these

entities could have to be raised to spècific levei.

Agradecimiento — Quedamos muy reconocidos al Dr. Jorge W. Abalos por

la provisión de numerosos ejemplares vivos de las diferentes entidades de Latro¬

dectus empleadas en este trabajo, así como de sus aparatos venenosos ya

desecados.

Bibliograkía

1. GERSCHMAN, B. S., y SCHIAPELLI, R. D.. XVI International Congress of

Zoology, Washington Proc., 1, 194, 1963.

2. GERSCHMAN DE PIKELIN, B. S., y SCHIAPELLI, R. D., Rev. Soc. entomol.

argent., 27 (1-4), 51, 1965.

3. GERSCHMAN, B. S., y SCHIAPELLI, R. D., in SAMPAYO, Latrodectus

mactans y Latrodectismo, Tese de Concurso, Universidade Nacional de Buenos

Aires, Argentina, 1942, cap. 2.

4. LEVI, H., Amer. micros. Soc., 78 (1), 194, 1959.

5. ABALOS, J. W., Psyche, 6» (4), 268, 1962.

6. Mc CRONE, J. D., and NETZLOFF, M. L„ Toxicon, 3, 107, 1965.

7. OUTCHERLONY, O., Acta path. microbiol. scand., 25, 187, 1948.

8. GRASSET, E„ PONGRATZ, E., et BRECHBUHLER, T„ Ann. Inst. Pasteur,

91 (2), 162, 1956.

9. SCHENBERG, S., Ciência e Cultura, 13 (4), 225, 1961.

10. SCHENBERG, S„ Toxicon, 1, 67. 1963.

11. BARRIO, A., y MIRANDA, E., Mem. Inst. Butantan, Simp. Internac., 33 (3),

887, 1966.

12. BARRIO, A., y MIRANDA, E., Mem. Inst. Butantan, Simp. Internac., 33 (3),

869, 1966.

13. SMITH, H. M., System. Zool, 14 (1), 57, 1965.

SciELO

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33(3):869-880, 1966

AVELINO BARRIO y MARTA E. MIRANDA

869

26. ESTÚDIO COMPARATIVO MORFOLÓGICO E INMUNOLÓGICO ENTRE

LAS DIFERENTES ENTIDADES DEL GÉNERO MIC RU RU S WACLER

( ORHIDIA, EL ARI D AE) DE LA ARGENTINA

AVELINO BARRIO y MARTA E. MIRANDA

Instituto Nacional de Microbiotogia “Carlos G. Malbrán”,

Buenos Aires, Argentina.

En relación a las corales dei género M i c r u r u s diversos autores han in¬

tentado elucidar el problema sistemático de las formas dei sur dei continente,

a saber: Schmidt(l), Amaral (2), Vanzolini (3), Shreve (4) y Pradèl Aze¬

vedo (5).

Es obvia la conveniência de que el enfoque de dicho tema sea hecho en

forma integral tratando comparativamente todas las entidades dei continente con

el objeto de poder apreciar debidamente su dispersión geográfica, sus áreas de

contacto, de simpatría y de intergradación; sus afinidades morfológicas, proba-

bles relaciones filogenéticas, etc.

Este critério, el más acertado en relación a este grupo, es seguido con

promisorios resultados, según nuestra información, por Janis Roze, actualmente

en V ashington. No obstante, pensamos que, a veces, ciertos aspectos parciales

dei problema, restringidos a zonas determinadas pueden tratarse independiente-

mente con resultados valederos y con posibilidades de ser luego incorporados ai

tonsiderarse el género en su totalidad. Es por este motivo que hemos encarado

cl asiinto encuadrándolo dentro de los limites de nuestro país, de acuerdo a las

ac i i cia des de obtención de material fidedigno y en series apreciables como para

peTmitir el tratamiento estadístico de su morfologia. Pero, la razón fundamen-

la que nos restringió a la consideración exclusiva de las entidades que habitan

en la Argentina fue nuestra intención de extender comparativamente su estúdio

al análisis inmunológico de sus respectivos venenos. Para ello era menester

contar con muestras de ponzoíía originarias de ejemplares ciertamente individua¬

lizados, tanto desde el punto de vista de sus caracteres ectosomáticos, como de

su procedência. Además, con el objeto de que nuestros datos puedan ser ulte-

riormente confrontados con los proporcionados por olras muestras de venenos,

siguicndo las mismas técnicas, hemos empleado en el sistema antígeno-anticuerpo

mi suero ya standardizado, de uso terapeútico y por eonsiguiente de fácil obtención.

Existen en la Argentina cuatro entidades dei género Mi cr urus. El

area mayor corresponde a la forma que preferimos llamar, por los motivos que

más adelante se expondrán, Micrurus frontalis pyrrhocryptus (Cope), que ocupa

la totalidad de las províncias situadas al oeste de los rios Paraguay y Paraná y

al norte dei paralelo 36. En las províncias de Entre Rios y Corrientes situadas

al este dei rio Paraná y al oeste dei rio Uruguay se distribuye una nueva sub-

especie que hemos denominado Micrurus frontalis mesopotamicus. Einalmente,

en la procincia de Misiones, situada en el confín noreste dei país y también entre

SciELO

10 11 12 13 14 15 16

870

ESTÚDIO COMPARATIVO MORFOLÓGICO E INMUNOLÓGICO ENTRE

LAS DIFERENTES ENTIDADES DEL GÉNERO MICRURUS WAGLER

( OPHIDIA, ELAPIDAE ) DE LA ARGENTINA

los rios precilados, conviveu Micrurus jronlulis allirostris (Copo) y Micrurus

corallinus (Wied). Por lo que antecede, se pone liien en evidencia que los

rios Paraguay y Paraná y en parte el Lruguay constituyen efectivos lindes que

delimitan netamente cada una de las formas, debiéndose a esta causa la inexis¬

tência dc intergradaciones, las que juslamentc se producen en las zonas de con¬

tacto entre M. j. allirostris y M. j. mcsopotamicus donde non existen dichas

harreras (Fig. 8).

Material y métodos

El material básico de nuestro estúdio lo constituyeron ejemplares dei género

Micrurus que llegaron vivos a! Instituto Nacional

G. Malbránprocedentes de distintas localidades dei

lidos a extracciones periódicas de veneno; luego de

meses fueron sacrificados y fijados, e ingresaron

(CHINM). Además se examino cl material existente

tológicas dcl Musco Argentino de Ciências Naturales

Buenos Aires (MACN), dei Instituto Butantan

grandense de Ciências Naturais de Pôrto Alegre

de Microbiología “Carlos

país. Estos fueron some-

lapso de unos cuatro

nuestras coleccioncs

en las coleccioncs herpe-

Bernardino Rivadavia” de

de São Paulo, dei Museu Rio-

(MRCN), y dei Museo de His-

Hoge

Montevideo (MHNM) a cuyos respectivos jefes, Jorge Cranwell,

Thales de Lema y B. Orejas Miranda quedamos muy reco-

toria Natural

Alphonse lí.

nocidos.

Se investigaron las prccipilinas por el método de la doble difusión en agar

según OutcJierlony (6), Grassei y col. (7) y otros, y microdifusión utilizado por

Schenberg (8, 9).

Las muestras de veneno extraído de cada cjemplar fueron desecadas y con¬

servadas por separado. Se utilizo una solución que contenía 5 mg de veneno

en 0.75 ml de solución fisiológica.

Se crnpleó el suero antielapídico elaborado por el Instituto Butantan contra

veneno de Micrurus frontalis y Micrurus corallinus, con un título lai que 1 ml

neutraliza 3 D.L.M. de veneno elapídico; en este caso fue diluído al 50 c /c en

solución fisiológica.

Resultados

A u álisis iu m u nológico

LI método dc la doble difusión en agar nos permilió poner en evidencia

que existen entre los venenos de las cuatro entidades de M i c r u r u s conside¬

radas. algunos antígenos no comunes, mientras que se aprecian otros que exiben

una total o parcial identidad entre si. Según puede observarse en las Figs. 1,

2 y 8 hay un cierlo número de bandas de prccipilinas comunes a las tres sub¬

espécies de Micrurus jronlulis ( pyrrhocryptus, mesopotamicus y allirostris) , a

la par que tal identidad no parece ser tan absoluta entre dichas subespecies y

M. corallinus. También es dable observar la presencia de anchas bandas inter¬

nas (véase Figs. 1. 2 y 3) peculiares y exclusivas de cada una de las entida¬

des. tanto en lo que rcspecla a cada subespecie de Micrurus jronlulis como en

M. corallinus.

Esta última comprobaeión no debe extranar, puesto que ya Schenberg (8, 9)

halló variaciones de esta naturãleza, aunque no de esta magnitud entre diferentes

subespecies de Bothrops neuiciedi e inclusive entre poblaciones diferentes de Bo-

tlirops jararaca.

SciELO

10 11 12 13 14 15

Mem, Inst. Butantan AVELINO BARRIO y MARTA E. MIRANDA

Simp. rnternac.

33(3):S69-880, 1966

Fig. 1 — Bandas de precipitinas. A: M.

corallinus (proc. Puerto Esperanza, Mi-

siones), B: M. /. pyrrhocryptus (proc. La

Posta, Córdoba), C: M. f. mesopotamicus

(proc. El Cimarrón, Entre Rios), D: M.

f. altirostris (proc. Puerto Mineral, Mi-

slones), S: suero antielapídieo Inst. Bu¬

tantan. 1: bandas comunes a los vene¬

nos Dy B, 2: banda exclusiva veneno B,

3: banda exclusiva veneno C, 4: bandas

exclusivas veneno A. 5 y 6: bandas co¬

munes a B, C y D.

Fig. 2 — Bandas de precipitinas. A: M.

corallinus (proc. Puerto Esperanza, Misio-

nes), B: M. f. pyrrhocryptus (proc. Ave-

rias, Santiago dei Estero), C: M. f. me¬

sopotamicus (proc. El Cimarrón, Entre

Rios), D: M. f. altirostris (proc. Puerto

Mineral, Misiones), S: suero antielapídieo

Inst. Butantan. 1: banda exclusiva dei

veneno D, 2: banda exclusiva dei vene¬

no B, 3; banda exclusiva dei veneno C,

4: bandas exclusivas dei veneno A, 5 y 6:

bandas comunes a B, C y D.

Fig. 3 — Bandas de precipitinas. B: M.

f. pyrrhocryptus (proc. A verias, Santiago

dei Estero), C: M. f. mesopotamicus

(proc. El Cimarrón, Entre Rios), D: M. f.

altirostris (proc. Oberá, Misiones), S: sue¬

ro antielapídieo Inst. Butantan. 1: ban¬

das exclusivas dei veneno D, 2: bandas

exclusivas dei veneno B, 3: banda exclu¬

siva dei veneno C, 5: bandas comunes a

los venenos C, D y B, 6: bandas comu¬

nes a los venenos D, B y C aunque poco

visible en este último.

En resumen, nos parece que la divergência inmunológica de los venenos de

las entidades de M. frontdis entre sí. es de menor grado que la comprobada

entre cilas y M. corallinus. Estos datos, como se verá por lo que sigue, sou

concordantes con los dalos morfológicos estudiados.

Debemos hacer notar que se comprobó una significativa homogeneidad inmu¬

nológica entre las diversas muestras de venenos pertenecientes a una misma enli-

dad, a pesar de que la existência de algunas débiles líneas de precipitinas no fue

constante en todas las muestras procedentes de distintas localidades.

SciELO

872

ESTÚDIO COMPARATIVO MORFOLÓGICO E INMUNOLÓGICO ENTRE

LAS DIFERENTES ENTIDADES DEL GÊNERO MICKURUS WAGLER

( OPHIDIA, ELAPIDAE) DE LA ARGENTINA

A nálisis morfológico

Micrurus frontalis pyrrhocryptus (Cope) 1862

Denominamos así a esta enlidad de acuerdo al critério sustentado por

Shreve (4), que concuerda perfectamente con nuestras propias conclusiones, tanto

desde el punto de vista de la morfologia como dc la composición de su veneno.

Vale decir que, en primer lugar, no compartimos la posición de Amaral (2) en

cl sentido dc que esta enlidad deba considerarse una subespécie de M. lemnis-

catus y, cn segundo lugar pensamos que, aunque con características propias bas¬

tante peculiares, no ba aleanzado aún el nivel suficiente como para separaria dc

frontalis , máxime que hasta el momento no hemos podido comprohar Ia simpa-

tría de ambas formas. Pero, lo que nos condujo a definimos más categórica-

mente en este sentido, fue el hallazgo de la subespecie M. j. mesopotamicus (juc

constiluye, como se verá, una raza con caracteres intermédios entre /!/. /. pyrrho-

cryptus y M. f. altirostris.

Como se aprecia en los gráficos adjuntos (Figs. 4 y

posee los siguientes caracteres en sua escutelaeión:

' ' 214-240, ? 9 218-238; subcaudales, J cf 25-30, ,

número dc triadas dei cucrpo 5-11.

5) M. f. pyrrhocryptus

dorsales 15; ventrales,

V 21-29; nucales 4-11;

Distribucióii geográfica. — Comprende las siguientes provincias argentinas:

Formosa, Chaco, Santa Fe, Córdoha, San Luis, Mendoza, La Pampa, San Juan,

La Rioja, Catamarea, Tucumán, Santiago dei Estero, Salta y Jujuy, extendién-

dose al Chaco paraguayo, sudeste de Rolivia y sur de Mato Grosso (Brasil).

Material estudiado — CHINM: N.°

18-X11-53; X.° 704, La Verde, Chaco,

Córdoha, 22-1V-57; N.° 708, Chalacea.

29-11-56; X." 1354. Los Juríes, Santiago dei Estero.

tencia, Chaco, 29-VI-62; N.° 1479, Naré. Santa Fe

Avispas, Santa Fe, 29-X-62; N." 1513, Rio Piedras,

Pirané, Formosa, 20-V-63; N.° 1660, Padre Lozano,

La Banda, Santiago dei Estero, 18-V-64; N.° 1713,

N.° 1736, llaase, Santiago dei Estero, 18-V-64; N.° 1738, Lucila, Santa Fe,

14-X-64; N.° 1782, Padre Lozano, Salta, 9-X-64; X.° 1786, Lucila, Santa Fe,

2-XII-64; N.° 1840, Formosa, Formosa. 19-V11-65; N.' 1 1815, Embarcación, Salta,

2-VIII-65; N.° 1853, Chorotis, Chaco, 24-V-65; N.° 1866, Formosa, Formosa,

13-VIIf-65; N." 1878, La Posta, Córdoha, 14-V-65; X.» 1907. Chilecilo, La

Rioja, 1-11-65; N.° 1982, Siján. Catamarea, 26-1V-66; N." 1997, Formosa, For¬

mosa, 20-IV-66.

703. Estanislao dcl Cani|)o. Formosa,

29-1-54; N.° 706, Canada de Luque.

Córdoha; N.° 725, Quitilipí, Chaco,

29-111-61; N.° 1472, Resis-

, 8-VII1-62; N.° 1180, Las

Salta. 22-111-63; X.» 1553,

Salta, 23-1V-64; N.° 1667.

Atahona, Tucumán, 9-X-61;

MACN: X." 1699, Sur de la Sierra, San Luis, l

5149, Catamarea. 16-111-1928; N.° 6897. La Rioja,

Paz, Mendoza, 22-X-1941; X." 8863, Santa Rosa, La

I-VI1-1925; X." 5117-5148-

23-X-1931; X." 7738, La

Pampa, 22-XI1-16.

Micrurus frontalis mesopotamicus

n. ssp.

Por su dispcrsión geográfica nela, así como por los caracteres morfológicos

e inmunológicos de su veneno, constiluye una entidade hien definida. Como se

dijo precedentemente exhibe rasgos intermédios entre la forma con la que eolinda

hacia el oeste ( M. f. pyrrhocryptus) y con la que contacta al norte y al este

(M. f. altirostris).

SciELO

10 11 12 13 14 15

Meni. Inst. Butantan

Simp. Internac.

33(3):869-880, 1966

AVELINO BARRIO y MARTA E. MIRANDA

873

Descripción dei hololipo — CHINM 1\.° 1823, macho adulto. Longitud

total 82.6 cm. Villa Federal, Entre Rios. Argentina, 28-1V-65; J. Vermeersch leg.

Coloración: Cabeza negra, una ancha franja blanquecina transversal que

coniprende las escamas prefrontales, loreales y mitad posterior de las nasales;

una angosta línea blanca que bordea la parte anterior de las parietales y se

continua hasta las labiales superiores; parietales negras con el extremo posterior

rojo. Labiales y región guiar blanquecina con pequenas manchas oscuras irre¬

gularmente distribuídas.

Escamas dorsales 15; ventrales 218; subcaudales 24; nucales 3; número de

triadas dei cuerpo 13; el anillo negro central es aproximadamente dos veccs

más ancho que los laterales y casi igual ai espacio rojo intermédio.

Alotipo CHINM N.° 1512, hembra adulta. Longitud total 73,5 cm, El

Cimarrón, Entre Rios. Argentina, 26-IV-63.

Las diferencias de coloración con el holotipo son las siguientes: escamas

parietales lotalmente negras, la línea blanquecina característica dei borde anterior

de los parietales se presenta discontínua. Escamas ventrales 225; subcaudales

22. nucales 3, número de triadas dei cuerpo 12.

Paratipos — ' ' CHINM N.° 1783, 1861, 1871. 1952, 1983, 2009;

9 9 CHINM N.° 1525, 1627, 1720, 1994, 2006. 2008. Semejantes al bolo y

alotipo presentan variaciones eu la nitidez de la línea branquecina dei borde an¬

terior de los parietales, número de ventrales, subcaudales, nucales, número de tria¬

das dei cuerpo y la relación entre el ancho de sus anillos negros centrales y

laterales, con valores que oscilan dentro de los limites seííalados en los gráficos

correspondientes (véase Figs. N.° 4 y 5).

En un principio pensamos que la relación entre el ancho

central y los laterales era menor en esta subespecie que en ,17.

el análisis estadístico demostro que

pero

dei anillo negro

/. pyrrhocryplus,

dieha diferencia no es significativa.

Distribución geográfica — Provindas de Entre Rios. Corrientes y sudoeste

de Misiones (Argentina). En las zonas limítrofes (NE de Corrientes y SO de

Misiones) aparecen esporádicamente intergradaciones.

Material estudiado — CHINM: N.° 740. Saladas, Corrientes; N.° 743, San

Ignacio, Misiones, 26-IV-48; N. ü 1512. El Cimarrón, Entre Rios, 26-1V-63:

N.° 1521, Villa Federal, E. Rios, 31-1-62; N.° 1522. Paraná, E. Rios, 31-1-62;

N.° 1523, El Cimarrón, E. Rios, 11-1-62; N.° 1524, Santa Elena, E. Rios, 17-IV-62;

N.° 1525, El Cimarrón, E. Rios, 2-IV-62; N.° 1526. EI Cimarrón, E. Rios,

31-1-62; N.° 1627, Chajarí, E. Rios, 20-V-64; N.° 1720, Santa Lucía, Corrientes,

15-IV-64; N.° 1783, El Cimarrón, E. Rios. 17-XI1-64; N.° 1823, Villa Federal.

E. Rios, 28-1 V-65; N.° 1861. San Roque, Corrientes, 24-V-65; N.° 1871, Villa

Bovril, E. Rios, 30-111-65; N.° 1952-1967-1969, El Cimarrón, E. Rios, 9-111-66;

N.° 1983, El Cimarrón, Entre Rios, 25-11-66; N.° 1994, El Cimarrón, E. Rios,

27-1-66; N. ü 2003, San Roque, Corrientes, 2-11-62; N.° 2004. San Roque. Cor¬

rientes, 12-1-62; N.° 2005, Santo Tomé, Corrientes, 30-X-61; N.° 2006. El Ci¬

marrón, E. Rios, 9-111-65; N.° 2007, El Cimarrón, E. Rios. 15-11-66; N.° 2008,

Santa Lucía, Corrientes, 4-V-66; N.° 2009, El Cimarrón, E. Rios, ll-IV-66.

MACN: N.° 6427. Corrientes, 1923; N.° 6575. Santa Elena, E. Rios, 1933;

N.° 8629, Paraná, E. Rios, 1946.

SciELO

10 11 12 13 14 15 16

874

ESTUDTO COMPARATIVO MORFOLÓGICO E TNMUNOLÓGICO ENTRE

LAS DIFERENTES ENTIDADES DEL GÉNERO MICRURUS WAGLER

( OPHIDIA, ELAPIDAE ) DE LA ARGENTINA

Escamas ventrales

totat

200 210 ;

Micrurus frontalis pyrrhocryptus

12 _

Micrurus frontalis mesopotamicus

Hicrurus frontalis altirostris

20 n^n

200 2 IO 220 230 240 200 210 220 210 240 200 210 220 230 2*0

4 a -

-4 11

30_i ~^ —i

3o_ □r.i-

33 _ Q

4 1

.o“ rr

Escamas subcaudales

£ ? total

~ZÕ Ts 30 IO is 20 25 To 1 5 is 20 25 To

Micrurus frontalis pyrrhocryptus

12 ry

30 _Ijpi

Micrurus frontalis mesopotamicus

1 5 _C^] _

30 r^T;

Micrurus frontalis altirostris

?o_r^i _

3 o ri"

Fig. 4 — Análisls estadístico. La línea horizontal corresponde al rango, la vertical

a la media, el rectángulo negro a dos errores standard y el rectángulo blanco a

una desviación standard.

Escamas nucales

total

5 IO IS 2Ò

5 10 IS 20

O S 10 IS

M'crurus frontalis pyrrhocryptus

-4 o -

30 r^n

Micrurus frontalis mesopotamicus

is a

3 0 í|l

Micrurus frontalis altirostris

20.

10l|

30 j|l

N °

de tríadas

total

S 10 IS 20

S IO IS 20

O S IO is 2<

Micrurus frontalis pyrrhocryptus

u r^i

4 1 -

30 njn

Micrurus frontalis mesopotamicus

15 J^l_

15 J^L

Micrurus frontalis altirostris

20 r^i

10 J^L

30 _

Fig. 5 — Anâlisis estadístico. Referencias Igual a Fig. 4.

SciELO

10 11 12 13

Mem. Inst. Butantan

Simp. Internac.

33(3):869-880, 1966

AVELINO BARRIO y MARTA E. MIRANDA

875

Micrurus frontalis altirostris (Cope) 1859

La delerminación de esta forma ofreció algunas dificullades puesto que

entorpeció nuestra larea la existência de una subespecie posterior de Amaral

(M. f. multicinclus) 1944, que presenta evidentemente muchas afinidades y áreas

de superposición. El examen dei tipo. paratipo y otros ejemplares, realizado

en el Instituto Butantan, así como material de la colección dei Museu Hiogran-

dense de Ciências Naturais, nos decidió a no aceptar la validez de esta forma.

Compartimos el critério adoplado por Shreve (4) en el sentido de que esta enlidad

exhibe rasgos de j. altirostris y f. frontalis. En efecto, muchos ejemplares que

observamos son intergradaciones entre Ias precitadas subespecies, de allí que el

material proveniente de localidades más meridionales se acerque a /. altirostris

y el de más al norte a /. frontalis. Todo ello hace imprecisa su caracteriza-

ción y definición. El estúdio comparativo con el material procedente dei Museo

de Historia Natural de Montevideo, Uruguay, nos permitió sin lugar a dudas

clasificar nuestra forma como perteneciente a M. f. altirostris, dada la total

coincidência en la escutelación y en los patrones de diseno, aunque debemos

consignar que los ejemplares misioneros son algo menos melánicos que los dei

1 fruguay.

Sus vinculaciones con M. j. frontalis son evidentes, pero puede ser separada

de ésta por el número de escamas ventrales que varían entre: cf cf 216-236,

9 9 221-242 en M. f. frontalis y entre: o' c? 201-219, 9 9 210-221 en M. f.

altirostris y las triadas dei euerpo que oseilan entre 9 y 15 en la primera y

entre 13-19 en la segunda (véase Eigs. 4 y 5).

En cuanto a su colorido debemos mencionar que contrariamente a lo que

sucede en M. f. frontalis , donde las parietales son negras y sólo por exeepción

Para

t u*

t’F°

Fi«. fi

Micrurus frontalis mesopotamicus. Paratipo n.° 1627.

SciELO

10 11 12 13 14 15 16

876

ESTÚDIO COMPARATIVO MORFOLÓGICO E INMUNOLÓGICO ENTRE

LAS DIFERENTES ENTIDADES DEL GÉNERO MICRURUS WAGLER

( OPHIDIA, ELAPIDAE) DE LA ARGENTINA

es de color claro, en

constante una ancha zona anterior clara.

M. j. altirostris presentan en forma

Los restantes caracteres cie escutelación cie M. f. altirostris son los siguien-

tcs: suhcauclales, cT cT 16-25, ? 9 18-24, nucales 1-3.

Distribución geográfica —- P

araguay y

los

rovincia cie Misiones

Estados brasilenos de Rio Grande do Sul

(Argentina), llruguay,

y Paraná.

Material estudiado — Argentina. CHINM: N.° 727, Puerto Esperanza, Mi¬

siones, 21-1-49; N. u 1361, Eldorado, Misiones, 26-IX-60; N. u 1605, Posadas,

Misiones, 30-IX-63; N.° 1670, Eldorado, Misiones 7-V11-64; N." 1689, Posadas,

Misiones, 19-X-64; N.° 1802, Puerto Paranaí. Misiones, 23-11-65; N.° 1817, Puer¬

to Mineral, Misiones, 13-X-65; N.° 1920, Puerto Mineral, Misiones, 13-X-65;

N.° 1928, Puerto Azara, Misiones, 29-X-65; N.° 1940, Oberá, Misiones, 8-X-62;

N.° 1941, Oberá, Misiones, 29-X1-65; N.° 1942-1943, Oberá, Misiones, 10-X11-65;

N.° 1946, Oberá, Misiones, 3-1-66; N.° 1953, Oberá, Misiones, 15-1V-66;

N.° 1980, Leandro N. Alem, Misiones, 3-1-66; N.° 1981, Oberá, Misiones, 5-IV-66;

N.° 1984, L. N. Alem. Misiones, 26-1V-66; N.° 1992, Oberá, Misiones, 18-1-60;

N.° 1999, Posadas, Misiones, 18-X1I-61; N.° 2000, Puerto Esperanza, Misiones.

26-IX-61; N.° 2001, Puerto Esperanza, Misiones, ll-X-61; N.® 2002, Eldorado,

Misiones 3-IV-62. MACN: N.° 14a (2 ej.), San Ignaeio. Misiones, 1918;

N.° 1602, Misiones, 1902; N.° 2447, San Ignaeio, Misiones, 22-XI1-1913;

N.° 7785, San Ignaeio,

Misiones, 20-IV-1942; N.° 9486, Puerto Londera,

nes, IX-1947; N.° 12647, Puerto Remberg, Misiones, 11-1950.

Misio-

Uruguay. MHNM: N.° 0977, San Gregorio, Puntas Arroyo Chamizo, San

José, 29J-1965; N.° 0979, Tambores, Tacuaremhó, 11-66; N.° 1116, Catalán

Chico, Artigas, 13-111-65.

Material estudiado de Micmrus frontalis multicinctus (Amaral) : Inst. Bu-

tantan, Tipo N.° 8877, Teixeira Soares, Paraná, Brasil; Paratipo N.° 7874, Iratí.

Paraná, Brasil; MRCN N.° 1789, Viamão, Rio Grande do Sul. Brasil; N.° 2656.

prob. Viamão, Brasil; N.° 2658, Estrada de Taquara, Brasil.

Micrurus corallinus ( W ied) 1820

Es ésta la más pequena de las formas que hahitan nuestro país. No se

baila estrechamente relacionada con las precedentes, tal se desprende dei estúdio

de su morfologia y de las propiedades inmunológicas de su veneno. Sus anillos

negros simples y bordeados de blaneo amarillenlo se presentan en iin número

que oscila entre 16 y 20. Escamas dorsales en 15 filas, el número de esca¬

mas ventrales y subcaudales varia notablemente según el sexo: ventrales A ,7

203-205, 9 9 216-220; subcaudales ' 44-46, 9 r 29-31.

Distribución geográfica

sur dei Brasil.

Provincia de Misiones (Argentina), Paraguay,

Material estudiado —- C11INM N.° 697-698, Puerto Esperanza, Misiones.

21-1-49; N.° 699, Puerto Esperanza, Misiones, 6-X-49; N.“ 700. Puerto Tabay,

Misiones, 19-X-55; N.° 1273, Posadas, Misiones, 20-X-61; N.° 1432, Puerto Es¬

peranza, Misiones, 30-1-62; N.° 1530, Posadas, Misiones, 29-VII-63; N.° 1911,

SciELO

10 11 12 13 14 15

878

ESTÚDIO COMPARATIVO MORFOLÓGICO E INMUNOLÓGICO ENTRE

LAS DIFERENTES ENTIDADES DEL GÉNERO MICRURUS WAGLER

( OPHID1A , ELAPIDAE ) DE LA ARGENTINA

Fig. 8 — Mapa de la distrlbucibn de las cuatro entidades dei género

M ter urus en Ia Argentina.

2 3 4

6 SciELO 10 11 12 13 14 15

Mcm. Inst. Butantan

Simp. Internac.

33(3) :869-880, 1966

AVELINO BARRIO y MARTA E. MIRANDA

879

Colonia Belgrano, Misiones, 111-65; N.° 2010, Puerto Esperanza, Misiones, 4-IX-61.

MACN: N.° 10058 al 10045, Capitán Meza, Alto Paraná, Paraguay, 1988;

N.° 12627-12628, Rio Uruguay, a 20 km Puerto Bemberg, Misiones, 1X-49;

N.° 12645-12646, Rio Uruguay, a 20 km Puerto Bemberg, Misiones, 111-50.

SlIMMARY

The morphology of the four entities of the genus M i c rur u s from Argen¬

tina are statistically analyzed and their respective venoms studied from the

immunological point of view. One of them belongs to the non questioned species

M. corallinus and the other three are considered as subspecies of M. frontalis.

The widest area is occupied by M. f. pyrrhocryptus which occurs west of the

rivers Paraguay and Paraná; to the northeast, in Misiones Province, M. corallinus

and M. frontalis altirostris overtap. Our new subspecies which we have called

M. f. mesopotamicus inhabits an intermediate area with natural boundaries given

by the rivers Uruguay and Paraná. Morphological characters are also interme¬

diate to those of the other two subspecies, M. f. pyrrhocryptus and M. f. altirostris.

From the immunological point of view each entity shows common and non common

precipitins bands to the others, being the divergency more apparent arnong M.

corallinus and M. frontalis group than arnong each entity of the latter.

References

1. SCHMIDT, K. P„ Field. Mus. Nat. Hist. Zool, Ser. 20, 189, 1936.

2. AMARAL, A. do, Pap. av. Dept. Zool., S. Paulo, 5 (11), 83, 1944.

3. VANZOLINI, P. E„ Rev. bras. Biol., 8 (3), 377, 1948.

4. SHREVE, B., Breviora. Mus. Comp. Zool., (160), 1, 1953.

5. PRADEL AZEVEDO, A. C., Iheringia Zool,, (14), 5, 1960.

6. OUTCHERLONY, O., Acta path. microbiol. scand., 25, 187, 1948.

7. GRASSET, E., PONGRATZ, E„ et BRECHBUHLER, T., Ann. Inst. Pasteur, 91

(2), 162, 1956.

8. SCHENBERG, S„ Ciência e Cultura, 13 (4), 225, 1961.

9. SCHENBERG, S„ Toxicou, 1 , 67, 1963.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internac.

38(3): 881-886, 1966

PINYA COIIEN

881

27. IN VIVO NEUTRALIZATION OF CORAL SNAKE VENOM

PINYA COHEN

National Institutes of Health, Bethesda, Maryland, U.S.A.

Inthoduction

Treatment of persons bitten by venomous snakes with antivenin is generally

eonsidered one of lhe most effective forms of therapy. The potency of the par¬

ticular antivenin usually is based on an in vitro neutralization test where venom

and antiserum are mixed in a tube, incubated and injected inlo mice. The

residis of ihis type of test largely reflect the degree of neutralization that occurs

in lhe tube. However, in the case of snakebite, and in the subsequent anti¬

venin therapy, the true xneasure of potency and efficacy of antiserum is more

logically based on its capacity to neutralize venom in vivo.

The present sludies were designed to evaluate the effectiveness, in vivo, of

coral snake antiserum in neutralizing the lethal effects of coral snake venom.

Similar tests were also conducted with other venoms and antisera.

IN VIVO NEUTRALIZATION OF MICRURUS FULVIUS VENOM IN MICE BY

HOMOLOGOUS ANTISERUM PRODUCED IN GOATS

Neutralization experiments were carried out with a randomly bred strain of

albino, male, mice, weighing 16-18 grams. The Micrurus fulvius venom used

was a freeze-dried pool of numerous milkings of a large number of snakes. The

antiserum was a pool of two hyperimmune goat sera (1) specific for M. fulvius

venom.

The in vivo neutralization tests were carried out by injecting groups of

mice with venom. Each group of envenomed mice was then injected with goat

antiserum at a different time interval. Control mice received normal goat serum

following envenoming. Different combinations of routes of injections were used

for lhe venom and antiserum.

The initial group of experiments was conducted with M. fulvius venom and

anti-A/. fulvius goat serum. The residis of these experiments are shown in

Table I. The intraperitoneal ED.-,o for the venom was 13 jig. All mice received

2 LD 50 of venom. The antiserum dose was contained in 0.25 ml, and had an

in vitro neutralization potency of 13.6 mouse LD 50 per dose. When venom was

given inlraperitoneally and antiserum was given intravenously, mice were com-

pletely protected by antiserum given immediately after the venom and some of

lhe mice were protected when it was given after 30 minutes. Mice were not

protected when antiserum was given after 1 hour. However, the time of death

for these mice was prolonged 3 to 5 hours beyond the Controls. Control mice

which received normal serum died between 4 to 6 hours.

SciELO

10 11 12 13 14 15

882

IN VIVO NEUTRALIZATION OF CORAL SNAKE VENOM

When 2 LD.-, 0 of vcnoiri were given subcutaneously and antiserum inlrave-

nously, mice were nol prolecled nor were death times prolonged as compareci to

Controls. The subcutaneous LD 50 of the venom was 32.5 p,g which inclicated

greater tolerance by mice lo venom administered by íbis route llian by the inlra-

periloncal route. Tbis suggests tbat even ibough the mouse is more resistant to

venom deposited subcutaneously, neutralization of ihis venom by antiserum given

intravenously is not as effective as when the venom was deposited intraperito-

neally. To further test this phenomenon, venom was again given subcutaneously

but the same antiserum dose was given intraperitoneally. The antiserum failed

lo proteet the mice further confirming the observation that the route of venom

injection — in this case subcutaneously — is a factor in the ability of a given

dose of antiserum to proteet mice in vivo. In addition to the route of venom

injection, lhe ability lo bring about in vivo neutralization by antiserum should

depend on the potency and volume of antiserum used. This is illustrated by the

lasl experiment reporled in Tahle 1 which ineluded antiserum over lliree times

more potent. Proteetion was obtained when lhe stronger antiserum was given

immediately and some of the mice were proteeted when it was given after 30

minutes. These results suggest that the routes of envenomation determine lhe

dose of antiserum required for in vivo neutralization of the venom.

TABLE I

IN VIVO NEUTRALIZATION OF M. FULVIUS VENOM IN MICE BY

MOMOLOGOUS GOAT ANTISERUM

Route venom

injected

Route antise¬

rum injected

Survivors as a funetion of

antiserum given

time

Prolongation

of death

time by

antiserum

(Hours )

Time

Alive

Dead

Intraperitoneal

Intravenous

0 min

Control

Antiserum *

30 min

Control

Antiserum

3 to 5

1 hr

Control

Antiserum

3 to 5

Subcutaneous

Intravenous

0 min

Control

Antiserum ,f

Subcutaneous

Intraperitoneal

0 min

Control

Antiserum *

3 to 5

Antiserum

30 min

Control

Antiserum *

Antiserum **

Antiserum dose neutralized 13.fi mouse LD W in vilro.

Antiserum dose neutralized 48.0 mouse LD_„ in vilro.

IN VIVO NEUTRALIZATION OF M. FULVIUS VENOM IN MICE BY

SÔRO ANTIELAP1DICO

A similar experiment using Sôro Antielapídieo, a purified antivenin pro-

duced in horses at the Instituto Butantan, was condueted. Although this serum

is produeed with venoms frotn South American coral snakes, it is more than

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3) :881-SSfi, 19(56

PINYA COHEN

883

90% effective in neutralizing M. julviiis venom. The results of this experimenl

are summarized in Table II. The same M. julvius venom dosage and roules

of injection used previously were employed. Protection of most mice was obtained

if antiserum was given within 30 minutes of envenomation and the time to death

was prolonged for several hours when the antiserum was given as late as 1 hour

afler envenomation. These results are in general agreement with the data from

experiments with goal antiserum.

TABLE II

IN VIVO NEUTRALIZATION OF M. FULVIUS VENOM IN MICE BY

SÔRO ANTIELAPIDICO

Route venom

injected

Route antise-

rum injected

Survivors as a function of

antiserum given

time

Prolongation

of death

time hy

antiserum

(Hours)

Time

Alive

Dead

Intraperitoneal

Intravenous

0 min

Control

Antiserum *

30 min

Control

Antiserum

6 to 8

1 hr

Control

Antiserum

1 to 3

2 hr

Control

Antiserum

* Antiserum dose neutralized 16.2 mouse LD.„ in vitro.

IN VIVO NEUTRALIZATION OF M. FULVIUS VENOM IN GUINEA PIGS

BY HOMOLOGOUS GOAT ANTISERUM

An in vivo neutralization experiment was conducted in guinea pigs. The

LD., U for Hartley, female, 120-130 gram guinea pigs was found to he 23.3 pg

hy the subcutaneous route, as compared to 32.5 /ig for this roule in 16-18 gram

mice, 30% less venom was required lo achieve an LD 5 „. This indicated that

these guinea pigs were considerably more sensilive to lhe venom than the mice

and for these two species a toxicity relationship based on a unit of hody weight

is not valid. The guinea pigs received 2 LD ; „, of venom subcutaneously and

the goat antiserum intraperitoneally. I he antiserum had a neutralizing potency

in vitro , of 40.8 mouse LD,„ per dose. The results of this experiment are shown

in Table III. Complete protection was obtained when antiserum was given 30

minutes after envenomation. Antiserum given after 1 hour did not preveni death;

however, the time of death was prolonged by as much as 24 hours. Antiserum

'dven as late as 2 hours after envenomation still prolonged the time of death

several hours. Control guinea pigs which received normal serum died afler

4 hours.

In experiments with both guinea pigs and mice, antiserum gave complete

protection only when il was injected within 30 minutes of envenomation. Howe¬

ver, the time of death for guinea pigs was prolonged nearly 24 hours longer

than in mice even though lhey both received the same dose of antiserum.

SciELO

10 11 12 13 14 15

884

IN VIVO NEUTRALIZATION OF CORAL SNAKE VENOM

TABLE III — IN VIVO NEUTRALIZATION OF M. FULVIUS VENOM IN GUINEA

PIGS BY IlOMOLOGOUS GOAT ANTISERUM

Route venom

injected

Roule antise¬

rum injected

Survivors as a function ot

antiserum given

time

Prolongation

of death

time by

antiserum

( Hours )

Time

Alive

Dead

Subcutaneous

IntraperitoneaJ

0 min

Control

Antiserum *

30 min

Control

Antiserum

1 hr

Control

Antiserum

2 hr

Control

Antiserum

2 to 4

4 Antiserum dose neutralized 40.8 mouse LD,„ in vitro.

IN VIVO NEUTRALIZATION OF NAJA ATRA VENOM IN MICE BY

HOMOLOGOUS ANTISERUM PRODUCED IN ITORSES

Experiments were clesignecl lo determine whether lhe need to administer

antiserum within 30 minutes was a characteristic unique to the coral snake venom

System. For this purpose in vivo neutralizations were conducted with the venom

of Naja atra, lhe Formosan cobra. Crude, liorse, zmú-Naja atra serum was

obtained írom Cmdr. G. S. Fíuher, MSC, USN, NAMRU-2, Taipei, Taiwan. The

venom and antiserum dosages were the same as those used in previous tests.

Results are presented in Tahle IV. The rlata are similar lo the results obtained

with coral snake venom neutralizations. Antiserum must he given within 30 mi-

nutes for protection of mice and il failed lo prolong death limes.

IN VIVO NEUTRALIZATION OF CROTALUS D. TERRIFICUS VENOM IN MICE BY

HOMOLOGOUS ANTISERUM PRODUCED IN HORSES

To study the in vivo neutralization of a crotalid venom mice were given

Crotalus d. terrijicus venom followed by a commercially prepared antivenin * pro-

duced in horses with a mixture of four crotalid venoms. This product has wide

neutralizing activity for many species of lhe CROTALIDAE of lhe Américas.

In establishing the dose lhe antivenin potency was based on ils ability to neu¬

tralize Crotalus d. terrijicus venom. The antivenin dosage was adjusted to lhe

same range as lhose used in the previous studies. The results of this experi¬

ments are shown in the lower half of Tahle IV. Mice were protected only if

antivenin was given immediately after envenomation. There was only slighl

prolongation of death times when antivenin was given after 30 minutes. These

neutralizations were slightly lcss effective lhan those observed with cobra and

coral snake venom.

* Wyeth Laboratories, Philaclelphia, Pennsylvania, U.S.A.

SciELO

10 11 12 13

Mcm. Inst. Butantan

Simp. Internac.

33(3):881-886, 1966

PINYA COHEN

885

TABLE IV

IN VIVO NEUTRALIZATION OF NAJA ATRA VENOM IN MICE BY

HOMOLOGOUS HORSE ANTISERUM

Route venom

injected

Route antise¬

rum injected

Survivors as a function of

antiserum given

time

Prolongation

of death

time by

antiserum

(Hours)

Time

Alive

Decid

Intraperitoneal

Intravenous

0 min

Control

Antiserum *

30 min

Control

Antiserum

IN VIVO NEUTRALIZATION

OF CROTALUS D. TERRIFICUS

VENOM

IN MICE BY

CROTALID ANTIVENIN

Prolongation

Route venom

Route antise-

Survivors as a function of

time

of death

injected

rum injected

antiserum

given

time by

antiserum

Time

A live

Dead

(.Hours)

Intraperitoneal

Intravenous

0 min

Control

Antiserum **

30 min

Control

Antiserum

1 hr

Control

Antiserum

* Antiserum dose neutralized 13.4 mouse LD M in vitro.

** Antiserum dose neutralized 16.2 mouse LD M in vitro.

Keferences

1. COHEN, P„ and SELIGMANN, E. B. JR., Mem. Inst. Butantan, Simp. Internac.,

33 (1), 339-347, 1966.

2 3

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ) : 887 - 892 , 1966

AVELINO BARRIO y MARTA E. MIRANDA

887

28. LAS DIFERENTES POBLACIONES DE BOTHROPS ALTERNATA

DUMÉRIL Y BIBRON (OPHIDIA, CROT ALIDAE) DE LA AR¬

GENTINA, CONSIDERADAS DESDE EL PUNTO DE VISTA

MORFOLÓGICO Y ANTIGÉNICO

AVELINO BARRIO y MARTA E. MIRANDA

Instituto Nacional de Microbiología “Carlos G. Malbrán”,

Buenos Aires, Argentina

El crotálido Bothrops alternata es una espeeie considerada monotípica y de

amplia dispersión que abarca ciertas regiones dei sur y centro dei Brasil, sur

dei Paraguay, Uruguay y Argentina. En lo que respecta a su distribución’geo¬

gráfica en la Argentina ocupa una amplia área continua al este dei meridiano 64

que se extiende desde el limite septentrional dei país hasta el paralelo 34 apro¬

ximadamente. A partir de esa latitud aparecen en la província de Buenos Aires

tres conjuntos poblacionales totalmente desconectados entre sí, tal como uno de

nosotros lo hiciera notar en trabajo anterior (1) (Fig. 1).

Hasta el momento no se han senalado subespecies de esta entidad a pesar

de su vasta distribución. La comprobación de diferentes patrones de diseno

así como posibles diferencias inmunoquímicas de sus venenos nos llevaron a

estudiar comparativamente muestras poblacionales tanto desde el punto de vista

morfológico-estadístico como inmunológico con el fin de poder establecer la exis¬

tência de áreas de prevalência de determinados caracteres y en caso positivo

considerar el nivel de diferenciación alcanzado por estas poblaciones.

Material y métodos

Para nuestro estúdio se utilizaron ejemplares de Bothrops alternata llegados

al Instituto Nacional de Microbiología “Carlos G. Malbrán” desde diferentes

puntos dei país, los que posteriormente a su extracción de veneno fueron fijados

e ingresaron en nuestras colecciones (CHINM). Se investigaron las precipitinas

por el método de la doble difusión según Outcherlony (2), Grassei y col. (3)

y microdifusión empleado por Schenberg (4, 5). El veneno fue desecado al

vacío y guardado separadamente por ejemplar; en el momento de su empleo se

diluyó en una proporción de 10 mg/ml de solución fisiológica y se sembraron

0.05 ml en cada cúpula. Se utilizo suero antibotrópico dei Instituto Nacional de

Microbiología con un título tal que 1 ml de suero neutraliza 2,5 mg de veneno

de Bothrops alternata, siendo diluído al 50% en solución fisiológica.

Desde el punto de vista morfológico se analizaron estadísticamente los si-

guientes caracteres: número de escamas dorsales, ventrales, subcaudales y número

de disenos básicos de los ejemplares procedentes dei área de distribución conti¬

nua y de cada una de Ias poblaciones bonaerenses.

SciELO

10 11 12 13 14 15

888 LAS DIFERENTES POBLACIONES DE BOTHROPS ALTERNATA DUMÉRIL Y

BIBRON (OPHIDIA , CROTALIDAE) DE LA ARGENTINA, CONSIDERADAS

DESDE EL PUNTO DE VISTA MORFOLÓGICO Y ANTIGÉNICO

Fig. 1 — Mapa de Ia distribución geográfica de Bothrops alternata en la

Argentina. Poblaciones de distribución continua: 1, províncias de San Luis

y sudoeste de Córdoba. Poblaciones bonaerenses de distribución discontinua:

2, Gral. Lavalle; 3, Tandilia; 4, Ventania.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

■Simp. Internac.

33(3) .'887-892, 1966

AVELINO BARRIO y MARTA E. MIRANDA

889

Resultados

Anál is is inmunológico

El estúdio de las precipitinas demuestra que, mientras la difereneiaeión mor¬

fológica es apenas incipiente, como se verá, las variaciones geográficas registra¬

rdes en la composición anligénica de su veneno son mucho más notahles.

Se com[)rol)ó que existe homogeneidad dentro de las muestras procedentes

dei área de distribución continua a pesar de su extensión. No obstante, en el

enfrentamiento de muestra de ponzonas de localidades extremas se ponen de ma-

nifiesto ciertas líneas de precipitinas no comunes a la totalidad de ellas. Esta

divergência es mucho más notoria al comparar venenos procedentes de las pobla-

ciones de distribución discontinua de la província de Buenos Aires con las dei

área continua (Fig. 2, 3). También resulta interesante la verificación de la

inexistência de una total identidad inmunoquímica entre las tres poblaciones

bonaerenses, aunque debemos notar que la divergência es menor entre las po¬

blaciones de Tandilia y Ventania que entre cada una de éstas con la de Gral.

Lavalle (Fig. 4).

Fig. 2 -— Espectro de bandas de precipi¬

tinas. S.F: Santa Fe; C: Corrientes; V:

Ventania (Bs. As.), S: suero antibotró-

pico. 1: entrecruzamientos correspondien-

tes a fracciones antigénicas no comunes.

Fig. 3 — Espectro de bandas de precipi¬

tinas. S.L: San Luis; L: Gral. Lavalle.

(Bs. As.), S; suero antibotrópico.

Fig. 4 — Espectro de bandas de precipi¬

tinas de las poblaciones de la província

de Bs. As. V: Ventania; T; Tandilia, L:

Gral. Lavalle; S: suero antibotrópico.

1, 2 y 3 entrecruzamientos que demues-

tran ia no total identidad antigénica.

SciELO

890

LAS DIFERENTES POBLACIONES DE BOTHROPS ALTERNAVA DUMÉRIL Y

BIBRON (OPHIDIA , CROTALIDAE) DE LA ARGENTINA, CONSIDERADAS

DESDE EL PUNTO DE VISTA MORFOLÓGICO Y ANTIGISNICO

A nálisis morfológico

Kl análisis estadístico nos muestra que en lo referente al número de escamas

dorsales, ventrales y subcaudales no hay diferencias apreciables y significativas

entre los ejemplares dei área de distriliución continua y los de las poblaciones

de ia provincia de Buenos Aires, ni entre estas últimas entre sí (Fig. 7). En

cuanto al número de dibujos básicos, si bien cl análisis de variancia no permite

extraer conelusiones definitivas, parecería existir una lendencia a aumentar su

número en el sentido norte-sur. Despeeto de la eoníormación de dieho diseno

fundamental también éste varia de norte a sur entre dos palrones extremos, desde

la forma arrinonada más característica (Fig. 5) basta la forma de f] con sus dos

ramas interrumpidas cerca de su extremo inferior (Fig. 6). Paralelamente a

esta modificación se observa una tendencia de los disenos contralaterales a

fusionarse en la línea medio dorsal, configurando de este modo un dibujo en

forma de X. Por conseguiente, de acuerdo a este supuesto se aprecia una mayor

incidência en estos patrcnes en las poblaciones más meridionales tales como Ias

de las provindas de Buenos Aires. San Luis y sur de Córdoba (Fig. 6).

Fig. 5 — Bothrops alternata. Procedência: Sta. Clara de Buena Vista (Santa Fe).

CONCLUSIONES

Los espectros de las precipitinas consignados ponen eri evidencia que existe

entre los antígenos dei veneno una diferenciación inmunoquímica que es más

acentuada entre las poblaciones no conectadas en su distribución que entre las

colindantes. Diferencias inmunoquímicas interpoblacionales de venenos de lio-

throps neuwiedi y Bothrops jararaca ya fueron senaladas por Schenberg (4, 5).

En cuanto al aspecto morfológico no estamos en condiciones de afirmar cate¬

góricamente la existência de “clines” relativos al número y a las variaciones de

los patrones de diseno anotados, aunque los hechos hablarían a favor de tal su-

posición.

SciELO

10 11 12 13 14 15

892

LAS DIFERENTES POBLACIONES DE HOTHROPS ALTERNA TA DUMÉRIL Y

BIBRON ( OPHIDIA , CROTALIDAE) DE LA ARGENTINA, CONSIDERADAS

DESDE EL PUNTO DE VISTA MORFOLÓGICO Y ANTIGÉNICO

También cabe senalar que, dentro de la variabilidad ya comentada, la homo-

geneidad antigénica de los venenos esludiados es más constante en cada una de

las poblaciones que la de los rasgos morfológicos eorrespondientes. Esto guar¬

da relación con lo ya senalado por diferentes autores (6, 7, 8), en cl sentido

de que el estúdio dei veneno de diferentes procedências aporta dalos de indu-

dablc valor filogenético al permitir detectar nuevas formas en vias de segregación.

Si bicn en algunos casos estos câmbios se producen paralclamenle a la módifica-

eión de los caracteres morfológicos, en la mayor parte de las veces la evolución

de los componentes químicos de las ponzonas precede a la de los rasgos morfo¬

lógicos.

Por todo lo antedicho creemos que estamos ante un proceso evolutivo de

inieiacióm relativamente reeiente. Esto puede inferirse de la imprecisa diferen-

eiación morfológica y de la ausência de áreas netas de predominância de cicrtos

caracteres, que evidencian el aún escaso grado de divergência poblacional. No

obstante ello. todo parecia indicar que las poblaciones bonaerenses sou reliclos

fragmentários de una anterior ocupación subtotal de la província dc Buenos Aires.

Estas, tal vez, estuvieron vinculadas por más liempo con las dei extremo sudo¬

este dcl área de distribueión continua y, por el contrario, la inlerrupción dei

intercâmbio genético con las poblaciones dei litoral parano-platense fue más

precoz.

Como conclusión final y siguiendo entre oiros a Wilson y Brown (9) e

Inger(lO), opinamos que las comprobaciones efectuadas no nos autorizan a

separar a las diferentes poblaciones bonaerenses de esta especie en entidades de

uivei subespecífico.

SlJMMAKY

The different populations from Argentina of the monotypic crotalid Bothroy s

alternata have been studied from the morphological statistic and immunotogicat

point of view. Morphologically, a tendency to increase the number of basical

designs and to modify their characteristic confcrmation from North to South has

been observed. This is especially noticeable on the three populations from Buenos

Aires and in the southwestern extreme of the continuous distribution area. From

lhe point of view of the antigenic composition of the venoms, immunochemical

analysis evidences greater differences between distant populations than between

the neighbouring ones. There was not a complete identity among the spectrum

of precipitin bands of the three populations from Buenos Aires. The reported

faets do not allow us to make a separation of the diverse populations of this

species into subspecific entities.

Reeerences

1. BARRIO, A., Physis, 22 (63), 211, 1961.

2. OUTCHERLONY, O., Acta yath. microbiol. scand., 25, 187, 1948.

3. GRASSET, E., PONGRATZ, E., et BRECHBUHLER, T„ Ann. Inst. Pasteur,

91 (2), 162, 1956.

4. SCHENBERG, S., Ciência e Cultura, 13 (4), 225, 1961.

5. SCHENBERG, S., Toxicon, 1 , 67, 1963.

6. BARRIO, A., Rev. Inst. Malbrán, 16 (3), 204, 1954.

7. BARRIO, A„ Physis, 22 (63), 141, 1961.

8. VELLARD, J., Acta Zool. Lilloana, I, 55, 1943.

9. WILSON, E. O., and BROWN, W. L. Jr„ Syst. Zool., 2 (3), 97, 1953.

10, INGER, R. F., in W. F. BLAIR. (Editor), Vertebrate Speciation, Univ. of

Texas Press., Austin, 1961.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 893 - 898 , 1966

LATIFI, M. and MANHOURI, H.

893

29. ANTIVENIN PRODUCTIOX

M. LATIFI and H. MANHOURI

Department of Herpetology and Anlivenin of Razi Institute, Iran.

L\troductio\

The production of antpvenins in Iran was beset with many difficulties.

Since lhe date of preparation of this serum at Razi Institute, continued efforts

have been rnade lo improve the therapeutic properties of the produet. From

1960 till now a great nurnber of poisonous snakes from various localities of Iran

have been made to improve the therapeutic properties of the produet. From

month for venorn. The venoms are lyophilized and kept for hyperimmunization

and titration. Razi Institute produces the following antivenins:

a) Monovalent antivenins against the venorn of:

Naja naja, Vipera lebetina, Vipera xanthina, Vipera pérsica pérsica,

Echis carinatus, Agkistrodon Iialys.

b) Polyvalent anlivenin against lhe venorn of:

Naja naja, Vipera lebetina, Vipera xanthina, Vipera pérsica pérsica,

Echis carinatus, Vipera ammodytes, Agkistrodon halys.

c) Polyvalent antivenin against the venorn of:

Naja naja, Bungarus fasciatus (Krait), Echis carinatus, Vipera russelli.

A11 antivenins are produced as pepsin digested, ammonium sulphate concen-

trated solution containing 10-13 gr per cent protein.

Materials and Methods

We produce our antivenins according to methods used in Butantan institute,

São Paulo, Brazil. Production begun as early as 1960 and serum was issued

for the rapeutic use in 1961. The snakes are obtained by purchase from people

who calch them and bring them to the lahoratory. The snakes are milked once

a month for venoms. For obtaining the venorn from the poisonous glands in

Naja, the fangs are inserted on the rubber of the dishes and in VIPERIDAE

lhe fangs are pressed directly inside the dishes down to the glass. The venorn

is refrigerated at — 18°C. for 24 hours and dried in dessicator under vaccum

over P 2 0 5 . The dried venorn is kept away from light and humidity. For im-

munization as well as serum titration we always employ a pool of dried venorn

from several batches for preparing our stock. The dried venorn is wemhted

SciELO

10 11 12 13 14 15

894

ANTIVENIN PRODUCTION

veiy carefully in an analytical balance. A ten per cent solution is preparerl for

hyperimmunization using 50% sterile physiological sall and glycerin willi a piece

of camphor, as stock solution. To determine the CLD. LD 50 and serum polency

litration, one per cent solution is prepared using sterile physiological salt as the

initial dilution; determinations were made in white mice weighing 16-18 gr

through lhe intravenous route.

than five years

young and old

For the production of the antivenin, healthy horses more

old (usually 7-81 are seleeted. Observations indicate that very

horses do not respond very well to immunization procedures. The general scheme

followed in the immunization of horses is to gradually huihl up their antivenin

titre hy the regular subcutaneous injection of increasing doses of the unmodified

venoms employed for hyperimmunization which is prepared hy diluting the

stock solution as already mentioned. According lo our observations, it is hetter

lo start the injections of the horses with a small amount of venom as indicated

in Table I. lo avoid producing toxic symptoms and also to gel a higher titre in

polyvalent antivenins. The range of the mixture of venoms is shown in Table II;

lhe initial dose contained lhe equivalent of 0.1 mg in 10

injections were given twice a week up to the amount of

followed weekly in volume of 20 ml intervals, the highest

venom is 120 mg; Re-immunization hegan with 2 mg of

During lhe period of immunization three injections of one

adjuvant is sufficient. The oil is prepared for adjuvant using 75 gr arabic

white gum, 300 ml olive oil. hy distilled water up lo 600 ml total volume, issued

and autoclaved.

ml of volume, lhe

10 mg, then were

amount of injected

unmodified venom.

ml sterile oil. as

TABLE I — PROTOCOL OF IMMUNIZATION SCIIEDULE OF HORSES

Period of

injection

A n t i g e n

Administration

Venom

mg.

Total volume

with saline

0,1

10 ml

Subcutaneous

0,2

10 ml

Subcutaneous

0,4

10 ml

Subcutaneous

0,7

10 ml

Subcutaneous

10 ml

Subcutaneous

10 ml

Subcutaneous

10 ml

Subcutaneous

10 ml

Subcutaneous

+ 1 ml oil = 20

Subcutaneous

20 ml

Subcutaneous

+ 1 ml oil = 20

Subcutaneous

20 ml

Subcutaneous

+ 1 ml oil = 20

Subcutaneous

100

20 ml

Subcutaneous

bleeding

6 to 8 lit. for

each

horse

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internar.

33(3) :S93-S9S, 1966

LATIFI, M. and MANHOURI, H.

895

TABLE II — RANGE OF POLYVALENT MIXTURE OF

VENOMS EACH 1/12 EQUIVALENT TO 200 MG DRY

VENOM IN 2 ML SOLUTION

Venoms

P 2

Naja naja

3/12

4/12

Vipera lebetina

3/12

1/12

Echis carinaius

2/12

3/12

Vipera xanthina

1/12

—

Vipera pérsica pérsica

1/12

—

Vipera ammodytes

1/12

—

Agkistrodon liaiijs

1/12

—

Vipera russelli

—

2/12

Bungarus fasciatus (Krait)

—

2/12

PURIFICATION AND CONCENTRATION OF ANTIVENTN

Methods devised for the purification and concentration of antitoxins huvc

I)een applicable to antivenins also. 1 he technique adapted in this laboratorv is

tliat originated by Pope and developed by Grasset and Christensen (1947). and

llelsal and Mirchamsy (1953). The routine procedures follovved in Hazi Insli-

tute are outlined below.

1 he immune horses are bled inlo Sodinm citrate. Six to eight liters of

blood are removed from each horse on each of the tliree occasions in three

days. The plasma is separated from lhe red cells by siphon, and sufficient phe-

noíether mixtnre is added to give a phenol concentration of 0.5 per cent. 100

litres of plasma are mixed with 200 Iitres of Saline and the mixture is adjusted

l>y pH 3.2. pepsin (1/10000 titre) is added to 0.5 gr per cent of plasma and

lhe reaction is brought to pH 3.2. After 30 minutes, digestion al 30"C. 0.2 per

cent of trisodium phosphate. 0.1 per cent of toluine. 14 per cent of ammonium

sulphate are added to the mixture, while being stirred meehanically. The reaction

is brought to pH 5.2. the mixture is steam-heated rapidly to 55"C and main-

tained at this temperature for 60 minutes. At the end of the heating period

lhe mixture is water-cooled and the denatured protein is removed by filtration

through filter cloth under slight positive pressure. The filtrate is added 0.2 per

cent of trisodium phosphate and adjusted to pH 7.4, with 17 per cent of ammo¬

nium sulphate. Antibody-carrying protein precipitate is removed hy filtration

through filter cloth, pressed, weighted, dialized for 48 hours in running waler.

The volume of the dialized solution is determined and 40 per cent of aluminium

gel is added (Aluminium Gel prepared at Willstater technique). Then it is

heated al 50"C. for 60 minutes; the gel is removed hy filtration or centrifu-

gation and the requisite amounts of salt and phenol are added. pH Ó.8-7.2,

phenol concentration 0.35 per cent, protein 10-18 per cent. alhumine negative.

The solution of refined globulins is sterilized by filtration, checked for pH,

protein, electrophoreses, phenol, sterility, innocuity. pyrogenicity and potency

and ampouled for issue. The final results of polyvalent antivenin are a reco-

very 37-65 (52%), purification indices 1.1-2.39 (main 1.76), concentration Índi¬

ces 1.30-2.7 (main 1.85). The titration is made hy different methods, hui the

SciELO

10 11 12 13 14 15

896

ANTIVENIN PRODUCTION

routine procedure is outlined liclovv. Different amounts of pooled standard

venom are added lo a series of haemolytic tubes containing 0.1 ml of serum,

lotai volume 0.2 nd is injected intravenously lo white mice vveighling 16-18 gr.

Each dilution is injected to five mice and neutralization is calculated as mg weight

of dry venom neutralized one ml of serum. For example the poteney tesl of

polyvalent antivenin for tvvo hatches as indicated in Tables III and IV.

TABLE Iir — TITRE OF PLASMA AND SERUM AGAINST VARIOUS SNAKE VENOMS

OBTAINED IN IRAN. THE TITRE ARE GIVEN IN MG. VENOM NEUTRALIZED BY

1 ML OF PLASMA OR CONCENTRATED SERUM. THE M.L.D. OF VENOMS IN MG

Antivenin issued

for Iran

Naja

naja

Vivera

Jebe-

tina

V i pera

xan-

thina

Vipera

pérsica

Echis

cari¬

natus

Vipera

ammo-

dytes

Agkist.

halyè

Polyvalent P,

Plasma

0.2

0.8

0.2

0.4

0.5

0.3

Refined Serum

0.6

2.2

1.4

1.7

1.2

4.4

M.L.D.

0.009

0.010

0.007

0.028

0.004

0.010

0.014

Monovalent

Plasma

0.6

1.5

0.4

0.6

0.8

0.6

Refined Serum

0.8

1.6

2.4

1.2

0.8

TABLE IV —

- TITRE

OF PLASMA AND SERUM AGAINST THE VENOMS

OF FOREIGN ORIGIN

Antivenin issued for

neighboring countries

N a ja

naja

Bung. fase.

(Krait)

Echis

carinatus

Vipera

russeUi

Vipera

lebetina

Polyvalent P.,

Plasma

0.2

0.8

1.2

0.5

0.4

Refined Serum

0.6

1.6

1.2

1.6

M.L.D.

0.009

0.020

0.015

0.020

0.010

Acknowledyement — The authors are grateful to Prof. Dr. M. Kaveh, General

Director of Razi Institute for his encouragement and for providing us the materiais

needed for this work; Dr. H. Mirchamsy, Assistant Director, for his useful data

and comments. We wish lo extend our thanks to Prof. Dr. A. Rafyi, Dean of

the Veterinary College-Iran, for his encouragement aud valuable help and interest.

Also we are grateful to Prof. Dr. R. Furlanetto, Head of the Dept. of Immunology of

Butantan Institute — Brazil, for his continuous interest in our work and his kind

attention. We are thankful and indebted to the following colleagues: Dr. S. Ko-

rourian, Dr. F. Nazari, Dr. M. Eliazan, and Dr. R. Farzanpy for their help and

assistance.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3):893-898, 1966

LATIFI, M. ami MANHOURI, H.

897

References

1. CHRISTENSEN, P. A., South-African Snake Venoms and Antivenoms, 1955.

2. DELSAL, J. L., and MIRCHAMSY, H., Rev. Immunol., 17, 110, 1953.

3. GRASSET, E„ and CHRISTENSEN, P. A„ Royal Soc. trop. Med. Hyg., 41,

2, 1947.

4. GRASSET, E„ Trans. royal Soc. Med. Hyg., 26, 1932.

5. GRASSET, E„ Buli. Wld. Hlth. Org., 16 (7), 122, 1957.

6. GRASSET, E., Quart. Buli, Hlth. Org. of the League of Nations, 1936.

7. HAZRA. A. K„ Buli. Wld. Hlth, Org., 12, 15-46. 1945.

8. LEON, W„ and SALAFRANCA. E., Phillipine J. Sei., 85, 1956.

9. POPE, C. G„ Brit. J. exp. Path., 20, 1939.

SciELO

10 11 12 13 14 15

biochemistry

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(3):S99-902, 1966

MARCUS V. GOMEZ and CARLOS R. DINIZ

899

30. SEPARATION OF TOXIC COMPONENTS FROM THE BRAZILIAN

SCORPION — T1TYUS SERRULATUS — VENOM

MARCUS V. GOMEZ and CARLOS R. DINIZ

Dejxirtment of Biochemistry, Faculty of Medicine, U.F.M.G.,

Belo Horizonte, Brazil

Separation of protein components from T. serrulatus venom has already been

made using mainly electrophoresis techniques (1). In lhe present paper results

will be reported on lhe separation of toxic components from the venom of T.

serrulatus hy a combination of extraction and chromatographic techniques usiii"

dextran gels and lhe resin carhoxymethylcellulose. The procedure used allows

the separation of a toxic component in highly purified form. Columns of

29 X 2.5 cm were packed with the fine grade suspension of Sephadex G-25

in distilled vvaler, following lhe instructions of Gelotte (2). CM-cellulose was

used in 0.6 X 22 cm columns prepared according to Peterson and Sober (3).

Buffers were prepared from analytical grade reagents dissolved in glass distilled

water. Scorpion venom obtained hy electrical stimulation was supplied hy Insti¬

tuto Butantan and was used in all lhe experiments. Cellulose acetate paper

electrophoresis was run according to Kohn(4). Determinations of the LD : ,„ (5)

were made hy the intraperitoneal route in mice.

Results

Venom obtained hy electrical excitation contained insoluhle suhstances that

were separated hy successive cold water extractions. The water soluble extract

was chromatographed on Sephadex G-25 columns using water and 0.1 TRIS

Iuiffer or 0.1 M ammonium acetate as eluents. Two main protein peaks P, and

T, (Fig. 1) appeared in the water eluates. Component T, contained approxi-

mately 30% of the toxic aclivity evaluated hy injection in mice. TRIS Iuiffer

eluted two additional components P 2 and T 2 (Fig. 1), the bulk of loxicity heing

found in peak T 2 . Elution with ammonium acetate following water elution

(Fig. 2), showed a small inactive protein peak and a large peak where the toxic

activity was found. Electrophoresis on cellulose acetate paper revealed that peak

T 2 ohtained either with TRIS or acetate buffers still contained several compo¬

nents. For further purification elution with ammonium acetate was chosen and

Aided by Fundação de Amparo à Pesquisa do Estado de São Paulo.

SciELO

10 11 12 13 14 15

900

SEPARATION OF TOXIC COMPONENTS FROM THE BRAZILIAN

SCORPION — TITYUS SERRULÂTUS — VENOM

Fig. 1 — Chromatogram of 100 mg of total venom on a eolumn oi Sephadex G-25,

29 X 2.5 cm Flow rate 30 ml/h. Stepwise elution with vvater and Tris 0.1 M.

2 — Chromatogram of 80 mg of total venom on a eolumn of Sephadex G-25,

X 2.5 cm Flow rate 30 ml/h. Stepwise elution with water and ammonium

acetate 0.1 M.

lhe component was lyophylized and suspended in 0.01 M ammonium acetate

pH 7.7. An inactive precipitate formed and was removed hy centrifugation.

The soluble supernatant was transfered to tho CM-cellulose eolumn and eluted

with 0.01 M ammonium acetate pH 7.7. Several inactive protein peaks appeared

hut the toxic material was retained. Following several atlempls the elution of

the toxic fraction could he achieved hy 0.15 M ammonium acetate adjusted at

pH 9.0 (Fig. 3).

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internae.

33(3): 899-902, 1966

MARCUS V. GOMEZ and CARLOS R. DINIZ

901

Fig. 3 — CM-cellulose column 0.6 X 22 cm. Chromatogram of 5.5 mg T„ obtained

by gel filtratlon. Stepwlse elution. Starting buffer: Ammonium Acetate 0.01 M,

pH 7.7. Flow rate 1.5 ml/h.

The main loxic peak behaved as a single component when submitted to

paper electropboresis or rechromatography. Specific activities and yields of lhe

loxic component in lhe various steps are shovvn in Tahle I.

TABLE I — PURIFICATION OF A TOXIC COMPONENT FROM

T. SERRULATUS VENOM

Fractions

Protein

ld m

Toxic

unlts

Specific

activity

Yield

Total venom

205,0

3.150

15,3

100

Aqueous extract

136,0

43,4

3.140

23,0

99,5

Sephadex G-25

26,9

20,6

1.300

48,5

41,5

C.M.-cellulose

7,5

8,6

870

116,2

27,5

Discussion

The procedure now reported to separate the protein components from the

venom of T. serrulatus may he useful for preparative purposes. The toxic acti-

vity, which was assayed on mice hy LD,,, measurements and hy ohserving into-

xication symptoms, particularly salivation and lacrimation, was resolved in two

components on Sejihadex G-25. Further investigations are necessary to interpret

this finding as meaning the presence of at least two toxins in the venom. Chro-

matography on CM-cellnlose of the main toxic component from the Sephadex

columns, disclosed non toxic components and led to the preparation of a highly

purified toxin, which is homogeneous on paper electrophoresis at different pHs.

SciELO

10 11 12 13 14 15

902

SEPARATION OF TOXIC COMPONENTS FROM THE BRAZILIAN

SCORPION — TITYUS SERRULATUS — VENOM

Miranda, Rochat and Lissitzky (6), used dextrangels of lovv porosity as

a step in lhe purification of scorpioloxins. The usefulness of ihis procedure is

confirmed in our vvork and, as it is pointed out hy these authors, lhe combi-

nation of slight ion exchange and adsorption properties of lhe gel (2) are res-

ponsihle for the separation of lhe hasic proteins of the venom.

SüMMARY

A procedure is presented for the separation of a highly purified loxic com-

ponent from the venom of T. serrulatus.

Acknowledgment — One of us (C.R.D.) wish to thank the Faculdade de

Medicina de Ribeirão Prêto, U.S.P., the permission to work in this Department.

References

1. DINIZ, C. R., and GONÇALVES, J. M., Biochim. Biophys. Acta, 41, 470, 1960.

2. GELOTTE, B., J. Chromatog., 3, 330, 1960.

3. PETERSON, E. A., and SOBER, H. A., in S. P. COLOWICK, and N. O.

KAPLAN (Editors), Methods of Enzymology, Vol. 5, Academic Press, Inc., New

York, 1962, p. 3.

4. KOHN, J., in I. SMITH (Editor), Chromatographic and Electrophoretic Techni-

ques, Vol. 2, William Heinemann Books Ltd., London, 1960, p. 56.

5. BURN, J. H., Biological Standardization, Oxford University Press, London, 1937.

6. MIRANDA, F„ ROCHAT, H„ and LISSITZKY, S., Toxicon, 2, 51, 1964.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3): 903-906, 1966

H. BACHMAYER and H. MICHL

903

31. ON THE TOXIN OF TRITLRUS MARMORATUS LATR.

H. BACHMAYER and H. MICHL

Chemical Institute, University of Agriculture, Vienna, Áustria

Iytroduction

The range of the newt Trituras marmoratus includes Portugal, Spain and

lhe Southern parts of France. The amphibian is green with hlack speckles above

and grey to brown with hlack or white dots heneath. lt reaches a length of

16 cm and a weight of 10 g. Its toxin is supposed to be one of the most

active newt toxins with a DLM of 1.8 mg/g white mouse (s.c.) (1). Since no

Chemical datas were available we studied some chemical and biochemical pro¬

pordes and compared them to the propordes of olher toxins.

Material and methods

Toxin: The newts were rinsed with destilled water, placed in a heaker and

treated with 0.2 ml ether. The ether stimulated the excretion of the skin secrelion.

The product so obtained forms a characteristically smelling foam which was

dissolved in dest. water and freeze-dried. A well fed mature newt yields 20-50 mg

dry material. The first milking yielded enlirely soluble products. After re-

peated milking the solubility of the toxin decreased.

For better comparison with other amphibian toxins this method was pre-

fered to obtain the toxin by squeezing the paratoid glands.

Gel-chromatography: Sephadex G-200 was used in a column 80 X 2 cm.

The void volume of 85 ml was determinated with blue Dextrane. 5 ml of a

solution containing 1 per cent newt venom and 0.9 per cent sodium chloride was

placed on the column. The elution was performed with saline. 2 ml fractions

were taken.

Gel-electrophoresis : A disc electrophoresis system has heen used (2). The

small pore gel was prepared by dissolving 750 mg acrylamide (Fluka P 50135),

20 mg N,N’-methylene-bisacrylamide (Huka P 55626), 0.06 ml 10 per cent

solution of N,N,N’,N’-tetramethylenethylenediamine (TEMED. Fluka 57580) in

10 ml TRIS-HC1 buffer jiH 8.9. After deaeradon the polymerizadon was started

with 7 mg ammonium persulfate. Glass tubes (10X0,5 cm) were filled with

1.2 ml solution to a levei of 6 cm and allowed to polymerize under a layer of

destilled water.

A large pore gel solution was prepared by dissolving 250 mg acrylamide,

6 2 mg N,N’-methylenebisacrylamide, 0.17 ml 10 per cent TEMED in 10 ml

TKIS-HCl buffer pH 6.7, and starting with 3.5 mg ammonium persulfate. 0.2 ml of

SciELO

10 11 12 13 14 15

904

ON THE TOXIN OF TR1TVRUS MARMORATUS LATR.

tliis solulion were placed on top of the small pore gel and polymerized again

•mder a layer of dest. water. After the polymerization is complete the water is

replaced by 20-50 /j. 1 of a 1 per cent solulion of lhe toxin in TRIS-HC1 buffer

,,H 6.7. After 90 min a sufficient amount of lhe sample was soaked into lhe

large ]>ore gel. The rest was removed.

Electrophoresis, staining and destaining has been done as descri bed be-

fore (2, 3).

Hemolysis and enzyme assay. These determinations were done as describcd

before (4, 5, 6, 7). Surface lension was measured stalagmometrically.

Results AND DISCUSSION

The enzyme aetivilies of the Triturus marmoratus toxin are similar to those

of other amphibians (5, 6). E.g. the phosphatase (Fig. 1) is an unspecific

phosphomonoesterase similar to acid prostatic phosphomonoesterase. The maxi-

mum of activity is at pH 5.0 (p-nitrophenylphosphate). An amylase similar to

imk amylase could he detected(5). The arylamidase has an optimum of acli-

vity at pH 7.0-7.5 (L-leucine-/?-naphthyIamide). It hydrolyses (Table I) the

/3-naphthy lam ides of alanine, leucine, methionine and proline at a high rate. The

hydrolysis rates of the /3-naphthylamides of valine, glycine, lysine, arginine, iso-

leucine and cysteine decreases in the given seqnence down to some per cents of

TABLE I — HYDROLYSIS OF L-AMINO ACID /3-NAPHTHYLAMIDES

(THE VALUE FOR L-LEUCINE /5-NAPHTHYLAMIDE IS GIVEN AS 100)

Bombina

species

Triturus

cristatus

Triturus

marmoratus

ala

leu

100

met

100

met

100

pro

ala

leu

100

leu

100

giy

arg

vai

met

vai

hpro

pro

hpro

ser

try

lys

arg

his

giy

ileu

ser

his

thr

glu

thr

lys

asp

glu

arg

cys

his

asp

ser

tyr

cys

glu

asp

ileu

try

cys

tyr

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3):903-906, 1966

H. BACHMAYER and H. MICHL

905

the first group. The sequence of lhe hydrolysis rales of Trituras cristalus toxin

•liffers from T. marmoratus toxin mostly in respect lo lhe /Tnaphthylamides of

lysine and arginine. The firsl mentioned loxin hydrolyzes these substrates mueh

more readily than the latter. The natural substrates of ihis enzyme are of course

not /I-naphthylamides but peptides, il would be probably more correct to term

ihis enzyme as peptidase.

Triturus marmoratus toxin hemolyses human erythrocytes down to dilutions

of more than 1:10 millions. ín this respect it is markedly more active than

the toxins of T. cristalus or Bombina variegata. The hemolysing principie is

considered to be a direct hemolysing protein hy the same reasons (e.g. no phos-

pholipase activity, inactivation by proteolytic enzymes) as discussed in a previous

paper (6). Such proteins occur in snake venoms(8). In bee venom the direct

hemolysing factor is a polypeptide with 26 amino acids (9). In both cases the

factor was accompanied hy phospholipase. The factors themselves were strongly

basic.

In disc electrophoresis the hemolytic protein of Triturus marmoratus loxin

migrates al pH 8.9 with appr. 80 per cent of the mohility of bromphenolblue

(Fig. 2), it is therefore an acidic reacting substance. From Sephadex G-200 it

is eluted by 1.9 times lhe void volume (Fig. I). This corresponds an apparent

molecular size of appr. 40.000. Similar data have heen found for Triturus cris-

tatus and Bombina toxins (6). T riturus toxins lower lhe surface tension

markedly more than other proteins of similar mole size and electrophoretic be-

haviour(l). Hovvever. since the hemolytic activity of surface active substances

does not always parallel the influence on the surface tension this is not necessary

lhe only explanation for the strong hemolytic action of the T riturus toxin.

Fig. 1

Triturus marmoratus — Chromatography on Sephadex G-200.

SciELO

10 11 12 13 14 15

906

ON THE TOXIN OF TR1TVRVS MARMORATUS LATR.

Large pom qeL ——smalL por& geL

lilfO ■ II

BromphenoL-

bLue

Amimo otcid

amidcnse

NemoLt/sis

Fig. 2

Triturus marmoratus

Electrophoresis in polyacrylamide.

SlJMMARY

Amylase, acidic phosphomonoesterase, arylamidase and a direcl hemolysing

protein were demonstrated in the toxin of Triturus marmoratus Latr. The enzy-

ines and the hemolytic factor were characterized l»y chromatography on Sephadex.

gel electrophoresis and suhstrate specifioity.

References

1. GESSNER, O., Arch. Exy. Pathol. Pharmakol., 165, 350, 1932.

2. DAVIS, B. J., Ann. N. Y. Acad. Sei., 121, 404, 1964.

3. MICHL, H., and PASTUSZYN, A., Mikrochim. Acta, 880 1963.

4. KISS, G., and MICHL, H., Toxicon, 1, 33, 1962.

5. MICHL, H., and PASTUSZYN, A., Monatsh. Chem. 95, 978, 1964.

6. BACHMAYER, H„ and MICHL, H„ Monatsh. Chem., 96, 1166, 1965.

7. MICHL, H., and MOLZER, H„ Toxicon, 2, 281, 1965.

8. DOERY, H. M„ and PEARSON, J. E„ Biochem. J„ 78, 820, 1961.

9. KREIL, G., Monatsh. Chem., 96, 2061, 1965.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

•Simp. Internac

33 (3): 907-912, 1966

D.MEBS and H. W. RAUDONAT

907

32 - BIOCHEMICAL I.WESTIGATIOXS ON HELODERMA VENOM

D. MEBS and H. W. RAUDONAT

Institut fuer gerichtliche und soziale Medizin der Universitaet,

Frankfurt a.M., Germany

Introduction

Beside lhe snakes the reptiles contain only one venomous lizard family, lhe

(,lla m °nster, HELODERMATIDAE. They exist only in two species, the North

mencan Gila monster,, Heloderma suspectum and its Mexican eounterpart, H elo-

erma lorridum. In contrary to the snakes their venom glands are located in

the Jower jaw, the ducts leading to the exterior terminate between the langs and

I h owei lip. During the hite, the venom mixed with saliva is conducted into

I i(- wound hy caj)illai ity. Each fang possesses an indistinct groove along which

lhe venom moves.

The present Work deals with the investigation of the venom with modern

luochenncal and pharmacological methods.

Methods and Material

l oi the investigations lyophilized venom of Heloderma suspectum and Ilelo-

derma horridum from the MIAMI SERPENTARIUM, Miami, U.S.A.. was used.

lhe LD 50 was determined in mice by subcutaneous injection. The hyaluroni-

dase activity was estimated after the turbidimetric method of DORFMAN and

OTí with hyaluronic acid as substrate and compared with a standard prepa-

ration of hyaluronidase from ovine testes (500 IU/mg, SERVA, Heidelberg, Ger-

many), the phospholipase A activity was determined by the Marinetti method,

lhe proteolylic activity, according Kunitz, and the kinin-releasing activity from

plasma globulines was tested on the isolated guinea pig ileum.

Results and Discussion

The venom of Heloderma suspectum and Heloderma horridum consists, foi-

lhe most part, of macromolecular proteins, which can he precipitated with tri-

chloracelic acid and ammoninm sulfate.

After 48 hours dialysis against destilled water none of the venom had passed

the dialysis membrane. By spectroanalysis it was possible to find potassium,

sodium, calcium, magnesium as well as traces of aluminium, zinc, cooper, iron

and Silicon.

SciELO

10 11 12 13 14 15

908

BIOCHEMICAL INVESTIGATIONS ON HELODERMA VENOM

The LD 50 for Heloderma siispeclum was 0.82 frg/g mouse, for Heloderma Iwrri-

diini 1.4 gg/g mouse, s.c. injection. The loxic faelor is heat slahle; heating

lhe venom al pH 7.2, 100°C for 15 min no loss of toxicity was observed.

A comparativo estimation of cobra venom toxicity ( Naja naja) resulted in a

LD -,0 of 0.8 /tg/g mouse, thus in effect lhe toxicity of Heloderma venom

is comparable witb that of cobra venom.

An enzyme analysis of Heloderma venom was run for phosphomono-

and diesterase, acetylcholinesterase, nucleotidase, ATPase, DNAase and RNAase,

aminoacid oxidase, fibrinogenocoagulase, protease, phospholipase A and hyaluro-

nidase. Of all these enzymes we succeeded only in finding phospholipase A,

protease and hyaluronidase. A direct hemolytic faetor for example as one can

find in small amounts in cobra venom could not be proved, washed buman ery-

throcytes were not hemolysed by H elo d e r m a venom.

The hyaluronidase in Heloderma venom showed a very high activity

and was 20-40 fold higher than that of the ovine testes preparation, for 1 mg

Heloderma venom 10-20.000 IU could be calculated (Fig. 1).

600

0.7

0.4

0 3

0 2

0.1

0 5 1 0 2.0 • 5 0 12.5 25 50 100 200 500

log pg venom resp enzyme

Fig. 1 — Hyaluronidase activity of Heloderma venom and Hyaluronidase

from ovine testes, in dependence on the turbidity of unaffected hyaluronic acid

after incubatíontime for 1 h, 37°C.

The phospholipase A whirh is contained in many animal venoms could be

also detecled in II e lo d e r m a venom. Fig. 2 shows the enzyme activity of

H e l o d e r m a venom measured on the rate of clearing of an egg yolk emulsion

at |)H 7.2, 25“C under the influente of 200 /xg venom each, in comparison with

cobra venom ( Naja naja).

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3):907-912, 1966

D.MEBS and H. W. RAUDONAT

909

H e Iode r m a venom showed only a small proteolytic activity with a

pH-optimum from 8.0-8.5, tested on casein (Fig. 3). As well as casein, dena-

lured hemoglobin and gelatine were hydrolysed in smail rates, likewise benzoyl-

arginine-ethyl ester. The proteolytic activity was not affected by soybean trypsin

inhibitor.

Heloderm a venom incubated with bovine plasma globulin releases a

substance which causes an atropin resistant contraction on isolated guinea pig

ileum. This substance is heat stable and can be extracted from the incubate

with hot alcohol and further precipitated with ether from the extract dissolved

in acetic acid. Dialysing the alcohol extract against water, even after a relati-

vely short time (3 hours), a pari of the ileum contracting activity is found in

the dialysate. By the addition of chymotrypsin to the incubate, tbe ileum con¬

tracting factor is rapidly inactivated. A contraction caused by venom-globulin

incubate cannot be prevented by antihistaminica.

These facts indicate, that the substance which is released by venom from

plasma globulin seems to be bradykinin or a substance like bradykinin. Kinin-

release and casein hydrolysis showed the same pH-optimum (8.0-8.51. Paper

electrophoresis showed the similarity of tbe venoms. At pH 6.5. 0.06 M plios-

phate buffer, seven fractions were observed (lig. 4), at pH 8.0 only five. Beside

the strongly loxic fraction which contains tbe protease and a part of the hyalu-

ronidase, a further fraction, migrating to the catbode with a lesser toxicity, was

separated. The other fractions were nontoxic and without enzymatic activity.

H e l o d e r m a venom, as it is obtained, is a mixture of venom and saliva, so

these inactive proteins could be originated from tbe cavity of the mouth.

SciELO

10 11 12 13 14 15

910

BIOCHEMICAL INVESTIGATIONS ON HELODERMA VENOM

pH-optimum of protease activity

• H.horridum

'• H.suspectum

6.5 7.0

7 5

8 0 85

9.5 pH

Fig. 3 —• pH-optimum of the proteolytic activity of H e X o d e r m a venom,

tested on casein splitting, incubation of 1% casein solution for 5 h, 37°C, with

250 ng venom each.

Paper electrophoresis of Heloderma venom

0 06 M phosphate buffer pH 6 5 10 V/cm

start

- Toxicity

- Proteolytic activity

- Bradykinín releasing activity

- Hyaluronidase

- Phospholipase A

Fig. 4 —- Paper electrophoresis of Heloderma venom, 0.06 M phosphate

buffer pH 6.5, 10 V/cm.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3) :907-912, 1966

D.MEBS and H. W. RAUDONAT

911

SüMMARY

The venom of Heloderma suspectum and Heloderma horridum possesses a

toxicity eomparahle with that of cobra venom. It contains a very active hyahiro-

nidase, a phospholipase A and a kinin-releasing enzyme with small proteolytic

activity and a pH-optimum of 8.5. Electrophoretically l>oth venoms are similar.

References

1. DORFMAN, A., and OTT, M. L., J. bioh Chem., 172, 367, 1948.

2. MARINETTI, G. V., Biochim. Biophys. Acta, 98, 554, 1965.

3. KUNITZ, M., J. gen. Pliys., 3», 291, 1947.

SciELO

10 11 12 13 14 15

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Simi). Internac

33(3): 913-920, 1966

J. C. VIDAL ”, B. N. BADANO, A. O. M. STOPPANI

and A. BOVERIS

913

33. INHIBITION OF ELECTRON TRANSPORT CHAIN BY PURIFIED

PHOSPHOLIPASE A FROM BOTHROPS NEUWIEDI VENOM

J. C. VIDAL *, B. N. BADANO, A. O. M. STOPPANI and A. BOVERIS

Institute of Biochemistry, School of Medicine, University

of Buenos Aires, Argentine

íntkoihjction

lt is known that snake venoms inhibit mitochondrial electron transfer

Systems (T-5). Badano and Stoppani (6, 7) studied the aclion of crude and

heated venoms of several species of Bothrops on nonphosphorylating lieart

muscle extracts (Keilin and Hartree preparation) and found that the inhibitory

power was proportional lo the phospholipase A activity of those venoms. In this

Paper studies with purified phospholipase A (phosphatide acyl-hydrolase, E.C.

3.1.1.4) from B. neuwiedi venom are descrihed.

Materials and methods

Enzyme preparativas — Keilin & Hartree heart-muscle preparations were

ohtained from pig heart according to Slater (8). Protein eoncentration was

measured with the biuret method(9).

Measurement oj catalytic activity of heart-nmscle preparation — These were

earried oul spectrophotometrically at 30" (6, 7. 10). Reaction mixtures (final

vol. 3.0 ml) were made up as follows: a) NADH 2 -oxidase: 0.25 mM NADH,.

0.04 mM cytochrome c, 0.13 M phosphate buffer, pH 7.4; b) NADH 2 -cytochrome

c reductase: 1 mM KCN, 0.07 mM cytochrome c (oxidant) and other conditions

as in (a); c) NADH 2 -CoQo reductase: 0.13 mM Qo (oxidant); other con¬

ditions as in (b) ; d) Menadiol oxidase. Conditions as descrihed by Colpa-

Boonstra and Slater (11); e) NADH 2 -dehydrogenase. Conditions as descrihed

by Minakami et al. (12); f) Succinate oxidase: 28 mM succinate, 0.02 mM

cytochrome c and 0.13 M phosphate buffer (pH 7.4); g) Succinate dehydroge-

nase: conditions as descrihed by Arrigoni and Singer(13); h) Cytochrome oxi¬

dase: conditions as descrihed by Smith (14).

* Fellow of Conselho Nacional de Investigaciones Cientificas y Técnicas.

Abbreviations: NADH,, nicotinamide adenine dinucleotide, reduced form; Q„, 2, 3-di-

methoxy-3'-methyl-p-benzoquinone; EDTA, ethylenediaminetetra-acetate; DEAE-celluiose, die-

thylaminoethylcelluio.se,' A, absorbancy; cyt. c, cytochrome c; K ;I H 2 , menadiol; PMS, phe-

nazine methosulphate; Tris, tris (hydroxymethyl) aminomethane.

SciELO

10 11 12 13 14 15

914

INIIIBITION OF ELECTRON TRANSPORT CHAIN BY I URIFIED

PHOSPHOLIPASE A FROM BOTHROPH NEUWIEDI VENOM

Measurement oj phospholipase A activily — Tlie following methocls were

used: a) The turbidimetric method of Marinetti (15). In lhose conditions one

unit of enzymatic activily was defined as lhe amonnt of venom that, in 10 min

produced a decrease of 0.010 in ahsorbaney at 925 m/x. lí. neuuiiedi venom

presents a “lag” period before enzymatic activily is observed, and lherefore the

change in absorbancy at 925 m/x was considered after lhe end of lhe “lag”

period. b) The method of Habermann and Nenmannl 10). The inhibition of

(oagnlation of an eggyolk suspension incubated with venom is compared with a

control sample incubated in lhe same conditions but without venom. With this

method one unit of enzymatic activily was defined as the amount of venom that

inereases in one minute the coagulation time of the control. This method does

not detect the "lag” period. c) The method of Magee and Thompson (17) was

used with purified phospholipase fractions. Lecithin hydrolysis was followed hy

measuring lhe concentration of ester bonds liy hydroxamate formation, according

to Synder and Stephens (18). The hydrolysis required Ca ++ ions as it haa

been established for phospholipase A obtained from other sources(19).

Protein concentration was measured hy absorbancy at 280 m/x with lhe cor-

responding correetion for nucleotide absorbancy.

Results

Purification oj phospholipase A — The method deseribed hy Saito and Ha-

nahan (20) slightly modified (21) was applied to dried venom.

Table I shows the several steps of enzyme purification. In gel filtration

(step 2), all material with the phospholipase A activity was exeluded and an

another fraction labout 16%) without enzymatic activity remained included in

the column.

TABLE I

PURIFICATION OF PHOSPHOLIPASE A FROM VENOM

OF BOTHROPS NEUWIEDI

Experimental conditions as in Fig. 2. Enzyme activity measured by

the turbidimetric method (ref. 15).

Step.

Total

activity

Specilic activity

units

(Units)

Yield

mg of

protein

(a)

Crude venom

3234

100

( 1.0)

1. Heat-treatment at pH 3.0

3168

115

( 2.1)

2. Gel-filtration through Sephadex G-25

3798

117

168

( 3.1)

3. Chromatography on DEAE-cellulose:

F, (Tube No. 7-19)

380

( 1.7)

F 2 (Tube No. 53-65)

2887

1750

(32.0)

F 3 (Tube No. 79-84)

491

614

(11.4)

(a) Increase in specific activity obtained

at each

step.

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33(3):913-920, 1966

J. C. VIDAL ", B. N. BADANO, A. O. M. STOPPANI

and A. BOVERIS

915

The elution diagram of DEAE cellulose chromatography (Fig. 1) shows

ihree fractions (Fj, F 2 , and F :í ) having phospholipase A activity.

■ 0.10

■0.05

0005

■200

■100

Fig. 1 — Chromatography of Step 2 fraetion on DEAE-ceiiulose. 41 mg of protein

were applied on top of a 21 X 1.2 cm DEAE-cellulose. Elution with potassium

phosphate buffer (pH 7.4). The gradient was initiated at arrow G. Arrows (1)

and (2) indicate the points at which 0.1 M potassium phosphate buffer pH 7.0, and

1.0 M potassium phosphate buffer, pH 7.0, were used as eluants. c Absorbancy at

280 jrtm; O phospholipase specific activity (units/mg) measured by the turbidimetric

method (15).

The phospholipase A activity in the crude venom samples presented an

appreciahle “lag” period (Fig. 2) that was not affected by the concentration of

venom in the reaction mixture, by the addition of Ca + + ions or by increase of

the temperature at which activity was measured. On the other hand, the active

fraetion obtained after Steps 2 and 3 of the purification procedure did not pre-

sent any "lag'’ (Fig. 3). Similarly, autolysis of crude venom samples suspended

in saline at 15"C produced a progressive disappearance of the “lag” period with

simultancous increase in total phospholipase activity (Fig. 4). The effect of

autolysis could be observed only if venom proteases were active.

These facts suggest the presence in crude venoms of a low molecular weight

peptide inhibitor of phospholipase A. The inhibitor would form a termostable

complex with the enzyme at pH 4.5 but at pH 7.4 the complex would dissociate

and then lhe components could be separated on Sephadex G-25. This interpre-

tation is confirmed hy the direct demonstration of an inhibitor of phospholi¬

pase A in the included fraetion obtained from Sephadex G-25. In faet, in ano-

ther series of experiments (21). it was found that when the included fraetion was

added to the active one, it provoked a "lag" with the same characteristics as

that of lhe crude venom and simultaneously, the included fraetion produced a

strong inhibition of phospholipase activity. No inhihitory action was found in

the included fraetion obtained from samples of “autolyzed” venom (see abo ve),

nor in samples of the included fraetion preincubated with trypsin.

SciELO

10 11 12 13 14 15

SPECIFIC ACTIVITY

916

INIIIBITION OF ELECTRON TRANSPORT CHAIN BY PURIFIED

PHOSPIIOLIPASE A FROM BOTHROPS NEUWIEDI VENOM

MINUTES

Fig. 2 — Effect of venom concentration, temperature and Ca 2- * - addition on the

"lag" period. Enzyme activity measured turbidimetrically (15). The reaction mix-

tures were incubated at 25° with 88 ng (Ol or 42 ng (A. , j _ ] ) o£ crude venom,

or at 15° with 88 n g of crude venom (/^). VE: crude venom. The other curves

(•) were obtained at 25° with lhe stated amount of F,.

Fraction F 2 which appears homogeneous after electrophoresis on polyacryla-

micle gel, was tested as inhibitor of oxidizing heart muscle extracls.

According lo Table II (Expts. A and F), treatment with phospholipase A

produced a significant inhibition of lhe NADIT>-oxidase and succinate oxidase

syslems. The addition of cytochrome c (0.01 mM) lowered the inhibition of the

NADH 2 -oxidase system to 78%.

In order to establish the phospholipase-sensitive sites, the activity of the diffe-

rent components of electron transport chain was measured using adequate electron

acceptors (or donors) and/or blocking cytochrome oxidase activity with KCN.

Neither NADIF-dehydrogenase (Expt. E) nor succinate dehydrogenase (Expt. G)

were affected after phospholipase treatment. Tliis result is very interesting be-

cause it has Iteen demonstrated (9, 22) that digestion with phospholipase A pro¬

duced the solubilization of NADFL-dehydrogenase. However, it is possible that

in lhe experiments described in Talile II the concentration of enzyme used was

not sufficient lo |)roduce liberation of a significant amount of NADH 2 -dehydro-

genase. It must lie recalled that under similar experimental conditions Badano

and Stoppani (7) found complete inhibition of succinate dehydrogenase after

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3): 913-920, 1966

J. C. VIDALi *, B. N. BADANO, A. O. M STOPPANI

and A. BOVERIS

917

Fig. 3 — Effect of autolysis on the

“lag” period. Samples containing 250 jtíg

of crude venom vvere dlssolved in 1 ml

0.9% (w/v) NaCl and incubated at 15°

for the time (minutes) stated at the end

of each plot. Phospholipase activity vvas

measured by the turbidimetrie method (15).

The data in parentheses indicate the re-

lative phospholipasic activity of the au-

tolysed sample versus that of the origi¬

nal control sample.

Fig. 4 — Rate of hydrolysis of lecithin

by purified F, and F, measured the

turbidimetrie method (15). 10 mg soni-

cated lecithin in aqueous suspension was

incubated vvith enzyme as stated. Re-

action mixture: lecithin 5 mg/ml; 50 mM

Tris-HCl buffer (pH 7.4), 1 mM EDTA

and 5 mM CaCl 2 . Finai volume 2.0 ml.

treatment of Keilin-Hartree preparation with crude venom. Since succinate dehy-

drogenase is inhibited by treatment with proteolytic enzymes, tbey interpreted (7)

the enzyme inbibition as due to the venom proteases. The present results support

that interpretation.

Reduction of Qo (Exp. C ) by the phospholipase treated preparation

(NADH 2 -Qo reduetase activity) was partially inhibited hut the magnitude of

lhis inhibition was not sufficient to explain the inhibition of the whole system.

0n the other hand, menadiol oxidase (Expt. D) and NADEG-cytochrome c re¬

duetase (Expt. B) activities were strongly inhibited, in agreemenl with the inhi-

bition of the whole system. Einally, cytochrome oxidase activity (Exp. //) was

partially inhibited.

In order to ascertain whether the inhibitions presented in Tahle II were due

to alteration of the lipid constituents of the electron-transport particles, phospho-

lipase-treated and control preparations were extracted according to Fleischer

et ai (23) and analysed by thin-layer chromatography on Silicagel G (24). After

revelation, phosphorus was determined in each spot according to Doizaki and

Zi eve (25). Such studies showed a decrease in the concentration of lecithin,

phosphatidyl-ethanolamine and a third component (probably phosphatidic acids)

and a correlative increase in the respective lysoderivatives by an amount that

represenls about 40% of the total phospholipid content (26).

SciELO

10 11 12 13 14 15

918

INIIIBITION OF ELECTRON TRANSPORT CHAIN BY PURIFIED

PHOSPHOLIPASE A FROM BOTHROPS NEUWIEDI VENOM

TABLE II — EFFECT OF TREATMENT WITH PHOSPHOLIPASE A ON THE CATA-

LYTIC

PROPERTIES OF

THE KEILIN-HARTREE IIEART-MUSCLE

PREPARATION

Expt.

Reaction

Control

preparation

(Sp. activities)*

Inhibition of activity by

phospholipase A (%)

10 min

incubation

80 min

incubation

NADH, _

o 2

0.674

NADHj _ ►

3 +

cyt. c

0.175

nadh 2 _ ►

Q „

0.483

k..h 2 _►

o 2

10.0

NADH, _ >

Fe(CN);:

8.4

Suc. ->.

0 2

0.539

Suc. - ►

FMS

2.5

-2

3 +

Cyt. c — >

0 2

125.0

* Specific aclivitíes:

Expts. A, B. C, and E,

^mole of

NADHj oxidized/min/mg

protein;

F and G, /imole of succinate oxklized/min/mg proteln;

— 1 —i

D and H, fcVmin (mg protein/ml) (fc', constant of the first order equatlon).

Discussion

The role of phospholipids in electron transpor! has been studied in detail

by Brierley et o/. (27) and Fleischer et al. (23). It is well knovvn that lipids

represent 26% of mitochondrial weight; lhat phospholipids represenl 90% of

lotai lipids and that they are indispensable for electron transport. In fact, ex-

Iraction with adequate solvents or enzymatic hydrolysis by phospholipases inhi-

bits lhat transference.

The present study indicates that phospholipase A produces ils inhibitory

effect by acting on at least three points. 1) The segment Qo-cytochrome c is

the most sensitive to phospholipase A attaek. Eleetive inhibition of electron

transport between qninone and cytochrome c scems to be a rather common pro-

perty of phospholipases (28). 2) The fact that lhe quinone (Qo) reductase acti-

vity was affected, while NADH 2 -dehydrogenase aclivity did not show any de-

crease, indicated that another site lies between the flavoprotein (NADHj-dehy-

drogenase) and Qo. 3). The third ]>oint is near the cytochrome oxidase.

These conclusions which are in agreement with claims by other au-

ihors (2, 3, 5), show lhat lhe inactivation is in fact due to phospholipase A

action, but it remains undecided whether il is caused by the hydrolysis of phos-

pholipid constituents of lhe electron transport particle (“structural darnage”), by

the action of the products of hydrolysis on the electron carriers, or by both. When

extracted submitochondrial lipids were treated with phospholipase A and the hy-

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3):913-920, 1966

J. C. VIDAL ♦, B. N. BADANO, A. O. M. STOPPANI

and A. BOVERIS

919

drolysate was added to the Keilin and Hartree preparation there was some inhi-

bition of NADH 2 -CoQo reductase and menadiol oxidase activities but in a lesser

extent than after direct treatment of NA D H 2 -oxidase with phospholipase A. Succi-

nate oxidase and cytochrome oxidase activities were not inhibited by the hydro-

lysed lipid extract. The difference between lhe "direct” and “undirect” inhi-

bition of the electron transport system must be attributed to “structural damage”

by phospholipase A (261.

Summary

Phospholipase A from Bothrops neuiviedi venom was purified by a proce-

dure involving heat treatment at pH 3.0, gel-filtration through Sephadex C-25

and ehromatography on DEAE-cellulose. Three active fractions were isolated

(F,, F, and F g ) with specific activities 1.7, 32 and 11 times, respectively,

higher than that of the crude venom sample. F 2 and F ;s migrated as homoge-

neous proteins after electrophoresis on polyacrylamide gel. The crude venom

contains an inhibitor of phospholipase A activity which is responsible of the

initial “lag” period in the development of enzyme activity and can be separated

by filtration through Sephadex G-25 since it remains in the included fraction.

When this fraction was added to the active fractions it provoked a prolonged

“lag” and a strong inhibition of activity. The inhibitory effect was nullified

after treatment with trypsin or hy autolysis of entire venom at 15°. The in-

cubation of submitochondrial heart muscle particles (Keilin-Hartree preparation)

with F, intensely inhibited NADH 2 -oxidase, succinate and NADfT-cytochrome

c reductase activities. There was a less effective inhibition of menadiol oxidase.

NADH-,-Qo reductase and cytochrome oxidase, but NADfT-dehydrogenase and

succinate dehydrogenase activities were not affected after treatment with phos¬

pholipase A.

Acknowledgments _ This work was supported by grants of Consejo Nacional

de Investigaciones Científicas y Técnicas and The Jane Coffin Childs Fund for

Medicai Research. Dr. A. Barrio kindly supplied Bothrops newwiedi venom.

1 .

References

BRAGANÇA, B. M„ and QUASTEL, J. H„ Biochem. J., 53, 88, 1953.

2. NYGAARD, A. P., and SUMNER, J. B., J. Biol. Chem., 200, 723, 1953.

3. NYGAARD, A. P., J. Biol. Chem., 204, 655, 1953.

4. EDWARDS, S. W., and BALL, E. G., J. Biol. Chem., 209, 619, 1954.

5. M1NAKAMI, S., SCHINDLER. F. J.. and ESTABROOK, R. W., J. Biol. Chem.,

234, 2042, 1964.

6. BADANO, B. N., and STOPPANI, A. O. M., Rev. Soc. argent. Biol., 38, 342,

1962.

7. BADANO, B. N„ and STOPPANI, A. O. M„ Rev. Soc. argent. Biol., 39, 153,

1963.

8. SLATER. E. C., Biochem, J., 45, 1, 1949.

9. CLELAND, K. W., and SLATER, E. C.. Biochem. J., 53, 547, 1953.

SciELO

10 11 12 13 14 15

920

INHIBITION OF ELECTRON TRANSPORT CHAIN BY PURIFIED

PIIOSPHOLIPASE A FROM BOTHROPS NEUWIEDI VENOM

10. CHANCE, B„ J. Biol. Chem., 197, 557, 1952.

11. COLPA-BOONSTRA, J. P„ and SLATER, E. C„ Biochim. Biophys. Acta, 27,

122, 1958.

12. MINAKAMI, S., RINGLER, R. L., and SINGER, T. P., J. Biol. Chem., 237..

569, 1962,

13. ARRIGONI, O., and SINGER, T. P„ Nature, 193, 1256, 1962.

14. SMITH, L., Arch. Biochim. Biophys., 5», 285, 1954.

15. MARINETTI, G. V., Biochim. Biophys. Acta, 98, 554, 1965.

16. HABERMANN, E., and NEUMANN, W., Hoppe-Seylers Z. physiol. Chem., 297,

179, 1954.

17. MAGEE, W. L., and THOMPSON, R. H. S., Biochim. J., 77, 526, 1960.

18. SYNDER, F., and STEPHENS, N., Biochim. Biophys. Acta, 34, 244, 1959.

19. KATES, M., in K. BLOCK (Editor), Lipid Metabolism, John Wiley & Sons,

N. Y., 1960, Chapt. 5.

20. SAITO, K., and HANAHAN, D. H„ Biochemistry, 1, 519, 1962.

21. VIDAL, J. C., and STOPPANI, A. O. M., Rev. Soc. argent. Biol. (in press).

22. CREMONA, T„ and KEARNEY, E. B., J. Biol. Chem., 239, 2328, 1964.

23. FLEISCHER, S., BRIERLEY, G. P., KLOUWEN, H., and SLAUTTERBACK,

D. B., J. Biol. Chem., 237, 3264, 1962.

24. HORROCKS, L. A., J. Am. Oil Chemisfs Soc., 40, 235, 1963.

25. DOIZAKI, W., and ZIEVE, L., Proc. Soc. exp. Biol., N. Y., 113, 91, 1963.

26.

BADANO, B. N., STOPPANI, A. O. M„ VIDAL, J. C„ and BOVERIS, A.,

Rev. Soc. argent. Biol., 41, 224, 1965.

27. BRIERLEY, G. P., MEROLA, A. J„ and FLEISCHER, S. — Biochim. Biophys.

Acta, 64, 218, 1962.

28.

VIDAL, J. C., BADANO, B. N„ STOPPANI, A. O. M„ and BOVERIS, A.,

Rev. Soc. argent. Biol., 41, 181, 1965.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3):921-928, 1966

MAURÍCIO GOMES LOMBA, JULIO KIEFFER EZF-

QUIEL WAISBICH and OSWALDO VITAL BRAZIL

921

PREPARATION AND PROPERTIES OF 131 I-LABELLED CROTOXIN

MAURÍCIO GOMES LOMBA, JULIO KIEFFER, EZEQUIEL WAISBICH

and OSWALDO VITAL BRAZIL

Department of Pharmacology, University of Campinas, Campinas, São Paulo,

Brazil and Radiobiology Division, Atomic Energy Institute, São Paulo, Brazií

The in vivo distribution of snake venoms is still very little known. Gennaro

and Ramsey (1) applied the radioisotope trace technique to get some information

concerning tliis important aspect of venom pharmacology. They Iabelled the

eottonmouth moccasin (Agkistrodon piscivorus) venom with iodine-131 and de-

termined the radioactivity of various tissues from mice injected with this ma¬

terial. However, the utilization of Iabelled crude venom does not seem to be well

suitable for this purpose. Snake venoms are complex mixtures of proteins whose

molecular weight, composition in amino acids. toxicity and pharmacological

actions widely differ. Suppose, for instance, the specific activities of the various

components of the Iabelled venom under investigation are not the same. Then

lhe radioactivity of the tissues from the animais injected with it will not give a

real picture of its distribution. Moreover, the correct distribution of lhe more

toxic components, what is really the important thing to be known, can not be

clarified by using Iabelled crude venoms due to presence of inactive and little

toxic Iabelled proteins in them. Therefore, it seems better to labei venom com¬

ponents, instead of whole venoms.

A good knowledge of the pharmacology and role played in the physiopatho-

logy of envenomation of the component lo be Iabelled is highly desirable. The

Iabelled component may be used, in distribution studies, either alone or incor-

porated in the crude venom. Experiments with the last mentioned material seem

to be necessary as some venom components may alter the rate of ahsorption of

other venom components and the biological barriers of the organism, thus affecling

I heir distribution.

An investigation on distribution, points of fixation in tissues at cellular

and molecular leveis, rate and pathways of ahsorption and excretion of snake

venoms lias been planned according to the criteria outlined. The venom of lhe

Southamerican rattlesnake (Crotalus durissus terrijicus) was selected for this

study and the lahelling of crystalline crotoxin, its main toxin, with radioiodine,

tried. The method of iodination used to labei crotoxin as well as the physico-

chemical and biological characteristics of the radiocrotoxin obtained are reported

in this paper.

This research was supported by a grant from Fundação de Amparo à Pesquisa do

Estado de São Paulo.

SciELO

10 11 12 13 14 15

922

PREPARATION AND PROPERTIES OF ‘“'I-LABELLED CROTOXIN

Material and methods

Crotoxin — Crystalline crotoxin was prepared according lo llu> methods

developed hy Slolta and Fraenkel-Conrat (2). Solutions for iodination were made

at lhe time of the experiments hy dissolving 30 mg of crotoxin in 10 ml of

saline. The pH of the Solutions, originally hetween 4.5 and 5.0 was adjusted

lo 8.0.

Counting — A Nal (Tl) 3" X 3" well-type scintillation counter was

used with the pulse-height analyzer set al lhe l: 'l keV photopeak. Counts were

accumulated during a time sufficient to achieve a statistical error not greater

than 3%.

["'/] iodide — Sodium [ 131 I] iodide, carrier-free, was supplied hy the Ra

diochemistry Division of lhe Atomic Energy Institute, São Paulo, in a stronglv

alcaline solution (pH 12.0). The radioiodine used was free of reducing agents

iodination — McFarlanes (3) method was used. Pre-oxidation of the pro

tein as suggested hy McFarlane was employed in two preliminary experiments

lt was carried oul with a solution of iodine-iodide at pH 4.5 followed hy fil

tration of the mixture through a IR-4B Amherlite column in order to remove tlu

excess of iodine. The pH of the eluales was adjusted to 8.0 or 9.0. No pre-oxi¬

dation was used in the 12 suhsequent experiments.

After iodination, unhound radioiodine was usually remove from the mixture

hy an ion exchange resin (Amherlite IR-4B). In one of the experiments, part

of lhe mixture was suhmitted, for this purpose, to dialysis against running water

for 24 hours at room temperature.

The adopted proeedure to labei crotoxin with iodine-131 was as follows.

The |)H of a sodium [ liM I] iodide solution with total activity from 3 lo 8mCi

was adjusted to pH 8.0 or 9.0 with hydrochloric acid and huffered with alca¬

line glycin (pH 8.5). Then, 0.7 ml of a 127 I Cl solution containing 2.94 mg

of iodine were added in order lo obtain the 13, I Cl (Iodine-131 monochloride)

hy isotopic exchange. The crotoxin solution was jet sprayed over this “iodination

mixture”. and after strong agitation and 2 to 4 minutes of rest, passed through

the anionic resin Amherlite IR-4B. The total activity was then determined.

1‘hysicochemical assays — The radiochemical purity of the iodinated ma¬

terial was investigated hy thin layer ehromatography (silica gel G, 250 u tliick).

a mixture of acetone, n-hutanol, concentrated amónia and water (65:20:10:5)

heing used as solvent. The running time was 25 minutes. Crotoxin which re-

mained at the origin (Rf — 0) was developed hy nihidrin, and iodine, that

migrated with lhe solvent (Rf = 0.7), hy lead acetate. After drying, the plates

were divided in three parts and counted. Unhound iodine in the 131 I-labelled

crotoxin solution was also estimated hy paper electrophoresis (Whatman n.° 1).

It was performed with barbital-sodium harhital huffer pH = 8.6, ,/c = 0.05

and 2 mA per st ri p for 50 minutes. The labelled crotoxin did not migrate

(Rf t= 0).

Iodination of the crotoxin molecules was verified hy the trichloroacetie acid

test. The protein was precipitate hy adding the acid to the mixture, the pre-

cipitate ohtained heing washed several limes and its radioactivity finally deter¬

mined.

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(31:921-928, 1966

MAURÍCIO GOMES LOMBA, JULIO KIEFFER EZE-

QUIEL WAISBICH and OSWALDO VITAL BRAZIL

923

Labelled crotoxin was compared with lhe original crotoxin by rneans of

paper electrophoresis. The buffer, ionic strength and pH were as referred.

Running time of 4 and 18 hours were used. The eleclrophoretic strips containing

lhe radiocrotoxin were also analyzed in lhe “Vanguard radioscanner”.

Protein âetcrrninaãoii — Protein (crotoxin) concentration in the Amber-

lite eluated was estimaled hy determining its nitrogen content. The micro-

Kjeldahl and the ultra-violet (260 mg, and 280 mg.) differential spectrophoto-

metry methods were used. The nitrogen content of the original crotoxin was

also determined.

Biological assays — The toxieity of different batches of labelled crotoxin

was compared with thal of the original ones by determining their median lethal

doses for piedgeons and mice. Four dose leveis in geometrical progression

(common factor 1.5) and six or exceptionally ten animais per dose were used.

The animais were ohserved for a period of 24 hours. The median lethal doses

and their 95 of 100 confidence intervals were calculated hy Weil procedure (4).

The method of Cesari and Boquet (5) was employed for evaluating the

haemolytic (phospholipasic) activity of the labelled and unlabelled crotoxin pre-

parations. Inactivated sera and hlood cells from dogs were used in these assays.

Four conscious dogs were intravenously injected with 0.25 mg/Kg of the

radioiodinated crotoxin. Three of them were prepared as described in a previous

paper (6), for recording contractions of the tibialis anterior muscle and arterial

hlood pressure, after being anaesthetized with sodiutn pentobarbital (20 mg/Kg.

i.v.). Two dogs were prepared 24 and one 48 hours after the injection of the

labelled crotoxin. Stimulation of the peroneal nerve was carried out with supra-

maximal square wave pulses of 0.2 msec and 0.1 c/sec. When stimuli up to

100 v did not elicit muscle responses, it was assumed thal neuromuscular trans-

mission was completely hlocked. Direct muscle stimulation was done with shocks

of 50-100 v, 2 msec and 0.1 c/sec.

One of the four dogs was sacrificed nine days after the administration of

the radioiodinated crotoxin. The kidneys of this dog and those from the animal

which was used for studying neuromuscular transmission 48 hours after radio-

iodinated crotoxin administration were removed for histopathological examination.

The urines from these dogs were examined for albumin and haemoglobin.

The action of lhe labelled crotoxin was compared with that of the original

one on lhe isolated guinea-pig ileum. The intestine was suspended in an organ

hath with Tyrode solution at 37°C. A frontal inscription isotonic lever was em¬

ployed. The preparation was oxygenated with air.

Results

A good labelling effieieney did not occur when pre-oxidation was performed.

In the remaining experiments, a yield of 35 to 40 per cent of the initial radio-

activity was obtained. The speeific activity of the radioiodinated crotoxin was

in this case always greater than 120 /xCi/mg.

The evaluation of unbound iodine by chromatographic and eleclrophoretic

methods gave identical results. There was from 2 to 6 per cent of free 131 1 in

the different preparations. On the other hand. the trichloroacetic aeid test

SciELO

10 11 12 13 14 15

924

PREPARATION AND PROPERTIES OF "'I-LABELLED CROTOXIN

showed lhat frcm 94 to 98 por cent of the total radioaetivity were present in the

precipitates.

No differcnces were detected in the electrophoretic behaviour of crotoxin

and radiocrotoxin (Fig. 1). Analysis of the electrophoretic strips of labelled

crotoxin in the radioscanner revealed the presence of two peaks (Fig. 1)..

Fig. 1 —- Top — Crotoxin and radiocrotoxin eiectrophoresis (pH 8.6, ionio strenght

0.05, 2 mA, veronal-sodium veronal buffer) strips. Bottom — Radioscanner of the

labelled crotoxin.

The toxieily of radioiodinated crotoxin and lhat of crotoxin for pidgcons

and for mice did not differ significantly. The median lethal dose, for instance,

of a batch of crotoxin for pidgeons was 3.15 (1.62 to 4.18) mcg per Kg;

afler iodination, its LD.-,„ was found to be 3.44 (3.40 lo 3.45) meg per Kg.

Table I and figure 1 show the median lethal doses of different batches of

crotoxin and radioiodinated crotoxin for mice. The symptoms presented hy the

animais injected with crotoxin and radiocrotoxin were the same.

The haemolytic aetivity of the labelled crotoxin preparation which were

passed through lhe anionic resin resnlted somewhat lower than lhat of the ori¬

ginal crotoxin. However, when the free iodine was removed hy dialysis, the

haemolytic aetivity of both preparations was the same (Table II).

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(3) :921-928, 1966

MAURÍCIO GOMES LOMBA, JULIO KIEFFER EZE-

QUIEL WAISBICH and OSWALDO VITAL BRAZIL

925

TABLE I — TOXICITY FOR MICE (18 TO 21 g) OF SOME LABELLED AND

ORIGINAL CROTOXIN PREPARATIONS

Doses

Results (deaths in 24 hours)

micro-

grams

Experiment I

(07/08/65)

Experiment lí

(18/09/65)

Experiment III

(16/11/65)

Crotoxin

Radiocrotoxin

Crotoxin

Radiocrotoxin

0.66

—

0/6

1.00

0/6

1/6

2/6

1.50

0/6

4/6

5/6

2.25

5/6

6/6

3.37

6/6

—

tnog/Kg

150

Fig. 2 — Mean lethal doses (mcg/Kg)

and their 95 of 100 coníidence intervals

of the same preparations referred to in

Table I.

Vomits, defaecation, long lasting ílacid paralysis, albuminúria and haemo-

globinuria vvere lhe symptoms presented by lhe dogs injected with the radio-

iodinated crotoxin. They were identical to those showed hy dogs injected with

crotoxin (7).

Partial or complete neuromuscular hlockade (Fig. 3) was demonstrated in

lhe dogs injected with radiocrotoxin. Therefore, neuromuscular action was iden¬

tical to that of crotoxin (6).

The renal lesions found in the dogs injected with crotoxin (8) were also

observed in the kidneys of the dogs intoxicated by 131 I-crotoxin.

The lahelled crotoxin contracted the isolated guinea-pig ileus as crotoxin

does (9). There was cross dissensitization between crotoxin and radioiodinated

crotoxin.

SciELO

10 11 12 13 14 15

926

PREPARATION AND PROPERTIES OF 1 :I 1 I-LABELLED CROTOXIN

TABLE II — IN

VITRO HAEMOLYTIC

RADIOIODINATED

ACTIVITIES

CROTOXIN

OF CROTOXIN AND

Doses in

micrograms

Crotoxin

”'I Crotoxin

m I Crotoxin *

+ + +

+ +

Dialysed radiocrotoxin.

200

mm Hq

■

Fig. 3 ■— Records of the respiratory movements, blood

pressure, and of the tibialis anterior muscle con-

tractions of a dog (18 Kg, pentobarbital anaestesia),

injected 22 hours before with 250 mcg/Kg of radio¬

crotoxin. A — Stimulation of peroneal nerve with

square wave pulses of 0.2 msec., 0.1 c/sec and 100 v.

B — Direct mucle stimulation with pulse of 2 msec,

0.1 c/sec and 100 v.

Discussion

The results provided I>y the trichloroacetic acid tests and hy the analysis of

the electrophoretic slrips in the radioscanner showed that there vvas a real iodi-

nation of erotoxin, a protein or protein complex with a high aromatic amino

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

SirriB. Internac.

33(3):921-928, 1966

MAURÍCIO GOMES LOMBA, JÚLIO KIEFFER, EZE-

QUIEL WAISBICH and OSWALDO VITAL BRAZIL

927

acid content (9). On lhe other hand, lhe specific activity obtained by lhe method

of iodination used is sufíicient for crotoxin distribution studies.

lodination did not induced important physicochemical modifications in lhe

crotoxin molecule. This can be inferred from lhe results of lhe biochemical and

hiological assays. The smaller haemolytic activity of the radiocrotoxin was not

due lo iodination. It could be traced to the passage through the anionic resin:

A radioiodinated preparation submitted to dialysis instead of being passed through

the resin, presented the same haemolytic activity as the original crotoxin.

Crystalline crotoxin behaves as a homogeneous protein vvith a molecular

weight of the order of 30.000 in sedimentation and diffusion experiments carried

out with the ultracentrifuge (10). It behaves also as a uniform protein when

examined hy Cohu method (2). Signs of nonuniqueness of crotoxin have not

been observed by mean of electrophoresis either (11). However, in the present

investigation it was shown by the analysis of lhe electrophoretic strips of radio¬

iodinated crotoxin in the radioscanner the presence of two peaks of maxinal

radioactivity. This seems lo demonstrate that electrophoresis can start a sepa-

ration of crotoxin in two components, a fact not revealed by the common pro-

cedures. It is relevant lo remember that. according to Neuman am 1 Haber-

mann (12, 13) crotoxin is made up of two proteins: A phospholipase A of low

toxicity and a loxin of high toxicity (“crotactin") which are probably linked by

ionic bounds.

SuMMARY

McFarlane’s method of iodination was used for Iabelling crystalline crotoxin,

the main toxin of the South American raltlesnake venom. with radioiodine.

Yields of 35-40 per cent of the original radioactivity were obtained. Free iodine

was removed by means of filtration through an anionic resin (IR-4B Amberlile)

column. Radioiodinated crotoxin with specific activities grealer than 120 p,Ci

per gm was obtained.

Radioiodination of crotoxin was demonstrated by the trichloroacetic acid test

and by submitting the electrophoretic strips of the iodinated preparations to

analysis in the radioscanner.

Biochemical and hiological assays of 1,!1 I-labelled crotoxin revealed that the

iodination did not induced important physicochemical modifications in the cro-

toxin molecule.

In view of the results of the present investigation, the authors suggest that

the ,,!1 Mabelled crotoxin is a convenient tool for the investigation of the distri-

bution and rates of absorption and excretion of crotoxin as well as its points of

fixation in tissues at cellular and molecular leveis.

References

1. GENNARO, J. F. Jr., and RAMSEY. H. W., Nuture, 184 (Suppl. 16), 1244,

1959.

2 SLOTTA, C. H., and FRAENKEL-CONRAT, H. L., Mem. Inst. Butantan, 12,

505, 1938.

3. McFARLANE, A. S., Nature, 182, 53, 1958.

4. WEIL, C. S., Biometrics, 8, 249, 1952.

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5. CESARI, E., and BOQUET, P„ Ann. Inst. Pasteur, 55 (3), 307, 1935.

6. VITAL BRAZIL, O., Mem. Inst. Butantan, Sim]). Internac., 33 (3), 981, 1966.

7. VITAL BRAZIL, O., FRANCESCHI, J. P., and WAISBICH, E.; Mem. Inst.

Butantan, Simp. Internac., 33 (3), 973, 1966.

8. HADLER, W. A., and VITAL BRAZIL, O., Mem. Inst. Butantan, Simp. In-

ternac. 33 (3), 1001, 1966.

9. SLOTTA, K„ in ELEANOR E. BUCKLEY, and NANDOR PORGES (Editors),

Venoms, Vol. 44, A. A. A. S., Washington, D. C., 1956, p. 253.

10. GRALÉN, N., and SVERDBERG, T., Biochem. J., 32, 1375, 1938.

11. LI, C. H., and FRAENKEL-CONRAT, H. L., J. Am. Chem. Soc., 64, 1586,

1942.

12. NEUMANN, W. P., and HABERMANN, E., Biochem. Zeitschr., 327, 170, 1955.

13. HABERMANN, E., Biochem. Zeitschr., 329, 405, 1957.

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35. ACTION DU VENIN DE NAJA NIGRICOL/JS SUR LA

COAGULATION SANGUINE

J. MEAUME, M. JOUANNET, Y. IZARD et P. BOQUET

Institut Pasteur, Paris, France

Parmi les ELAPIDAE africains, ceux clt> 1’espèce Naja sécrètent un venin

donl ]'activité anticoagulante a été observée depuis fort longtemps (1).

Le mécanisme par lequel ces venins empêchent la coagulation du sang de

se produire demeure mal expliqué. Le venin de Naja nigricollis, par exemple,

ne présente aucun pouvoir anti-thrombique puisqu’il ne s’oppose ni à Ia coagu-

lalion du plasma, ni à celle du fibrinogène par la thrombine. L’effet inhibiteur

se manifeste par contre au cours de la coagulation du sang total, du plasma

recueilli sur oxalates et secondairement recalcifié, ou de la détermination du laux

de prothrombine par la méthode de Quick. LVxpérience montre également que

1 incubation du venin de Naja nigricollis avec la thromboplastine tissulaire amène

Ia destruction partielle de celle-ci. Malheureusement, dans des tests de coagu¬

lation plus complexes, la nature de 1’effet anticoagulant n’apparait pas claire-

ment (2) ; à peu près toutes les bypothèses ont été envisagées concernant la

destruction de tel ou tel facteur de coagulation et aucune certitude ne peut être

acquise.

Substituant au venin brut de Naja nigricollis une fraction três anticoagu¬

lante (fraction III), obtenue par filtration du venin sur Sephadex G 100 (3, 4),

uous avons cherché à determiner la nature du facteur responsable de 1’activité

anticoagulante.

Les expériences de diffusion-précipitation (3, 4) montrent en effet la pré-

sence dans Ia fraction UI de quatre antigènes.

L’un de ces antigènes est commun avec la fraction I. Un autre antigène

a pu être identifié à la phos[)holipase. Le fait que la fraction I, soumise à un

chauffage três bref à 96°, présente un pouvoir anticoagulant, suggère que le

facteur anticoagulant est cet antigène commun aux fractions UI et I. Mais sa

nature demeure inconnue. Nous ignorons s il s agit d une enzyme ou d’un inhi¬

biteur. Aucune activité protéolytique ou estérasique na pu lui être imputée:

la caséine, 1’hémoglobine, la sérum-albumine ne sont pas hydrolysées, les esters

synlhétiques (TAME, RAME) ne sont pas scindés.

Si nous admettons que le facteur anticoagulant principal du venin de Naja

nigricollis est cet antigène, devons-nous pour autant suivre Kruse et Dam (5) qui,

comme Fleckenstein et Fettig (6), dénient toute activité anticoagulante à la phos-

pholipase? Si l’on ajoute de très grandes quantités de phospholipides (Inosi-

thin) à un plasma soumis à 1’action de la fraction III du venin, on parvient à

réduire considérablement l’effet anticoagulant observé. II s’agit peut-être néan-

moins d’un effet non spécifique et cette expérience ne prouve pas que la phos-

pholipase dégrade les phospholipides nécessaires à la coagulation.

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COAGULATION SANGUINE

Oulre cette activité anticoagulante difficile à préciser, le venin des N a j a

africains est capable de eoaguler le sang de cheval. Boquet et Izard (7) obser-

vèrent eu effet qu’à fortes doses, ce venin était susceptible, non plus de retarder,

mais d’accélérer Ia coagulation.

L’addition de quantités croissantes de venin à du plasma humain exerce

dabord un effet anticoagulante qui augmente avec la dose de venin, puis un

effet coagulant três intense.

La séparation du venin en différentes fractions (3, 4) permet de retrouver

le facteur coagulant dans Pune d’elles (fraction I). La présence de calcium fa-

vorise Pactivité de ce facteur, mais n’est pas indispensable. L’hypothèse de

Pexistence d une enzyme de type thrombinique semble à exclure car Ia fraction I

est lotalement dépourvue (Paction sur le fibrinogène pur. La possibilité d’une

activation directe du facteur X par un mécanisme analogue à celui qui a été

décrit dans le cas du venin de Vipera russeli (9, 10) ne nous a pas semblé

devoir être retenue, car des plasmas de malades congénitalement prives de facteur

X sont coagules aussi vite que des plasmas normaux ou dépourvus des facteurs IX

ou VIL Par contre, des plasmas adsorbés sur sulfate de baryum ou sur plios-

phale tricalcique. dépourvus des facteurs IX. VII et X ainsi que de la prothrom-

bine vraie ne peuvent être coagulés. La présence de prothrombine apparait donc

comme indispensable à Paction du venin qui s’est révélé susceptible de eoaguler

seul une préparation contenant du fibrinogène pur et de la prothrombine purifiée. *

Certains auteurs actuels, parmi lesquels Magnusson (11), estiment que Paeti-

vation directe de Ia prothrombine en thrombine résulte (Pune j)rotéolyse. Or la

fraction I du venin de Naja nigricollis (fraction qui comporte plusieurs consti-

tuants) ne présente aucune activité protéolytique sur Pbémoglobine, la caséine,

la sérum-albumine et les acides aminés estérifiés. La nature de ce constituant

du venin pose de ce fait un problème particulièrement eomplexe. II s'agit d uii

constituant par ailleurs três sensible à la ehaleur: il disparait après quelques se-

condes de chauffage à 96". La mise en évidence d un facteur aecélérant la coagu-

lation du sang dans le venin de Naja nigricollis nous a incité à rechercher une

subslance analogue dans les venins des autres Naja africains.

I ne expérimentation récente a pu nous rnontrer que le venin de Naja haje

possède, comme celui de Naja nigricollis, un facteur coagulant.

II ressort de ce court exposé que Pétude de Pactivité des venins d’ELAPIDAE

africains sur la coagulation apporte des faits expérimentaux uliles à notre con-

naissance de la coagulation sanguine comme à celle du problème de la filiation

de ces serpenls au cours de Pévolution. Dans cet ordre de faits, il est inté-

ressant de noter qu’à la différence des N a j a vivant en Asie. les ELAPIDAE

australiens des espéces N o / e c h i s et /' s e u d e c h i s (12, 13, 14) présentent

un très net pouvoir coagulant. On peut se demander quelle relation existe entre

ce pouvoir coagulant el celui des ELAPIDAE africains que nous venons d exposer.

Heferences

1. NOC, F„ Ann. Inst. Pasteur, 18. 387. 1904.

2. MEAUME, J„ Toxicon, 4, 58. 1966.

3. BOQUET, P„ IZARD, Y„ JOUANNET, M„ et MEAUME, J., C. R. Acad. Sei,

262, 1134, 1966.

Préparée par le Doeteur J.-P. SOULIER.

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931

4. BOQUET, P., IZARD, Y., JOUANNET, M., et MEAUME, J„ Ann. Inst. Pasteur,

1966, à paraitre.

5. KRUSE, L, et DAM, H., Biochim. Biophys. Acta, 5, 268, 1950.

6. FLECKENSTEIN, A., et FETTIG, O., Arcli. exp. Path. Pharmak., 216, 415,,

1952.

7. BOQUET, P„ et IZARD, Y„ Expériences non publiées.

8. MEAUME, J., IZARD, Y., BOQUET, P., C. R. Acad. Sei., 262, 1650, 1966.

9. McFARLANE, R. G„ et ASH, B. J„ Br. J. Haematol., 10, 217, 1964.

10. ESNOUF, M. P„ et WILLIAMS, W. J., Thromb. Diath. Haemorr., 7, 198„

1962.

11. MAGNUSSON, S., Communication personnelle.

12. MARTIN, C. J„ J. Physiol., 15. 380, 1893.

13. HOUSSAY, B. A., et SORDELLI, A., C. R. Séanc. Soc. Biol., 81, 12, 1918.

14. EAGLE, H., J. exp. Med., 65, 613, 1937.

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L. GILLO

933

36. LES VENINS DE SERPENTS, SOURCE D’ENZYMES

ANTICANCÉREUX

I. ASPECTS BIOCHIMIQUES FONDAMENTAUX DU PROBLÈME

L. GILLO

Laboratoire d’Enzymologie Tumorale, Bruxelles, Belgique

Introduction

Nous nous proposons dYxposer ici les résultats d’une recherche de trois

années faite au Lalioratoire de Biochimie de 1’Institut Technique Supérieur de

Chimie Metirice de la Province de Brabant et au Laboratoire de Pharmacodyna-

mie de I Université Libre de Bruxelles.

Le but de cette recherche était de vérifier si les venins de serpents ont ou

uon une aetivité anticancéreuse et de tenter de situer cette action dans l’une ou

1’autre des fractious enzymatiques du veuin.

I u exposé exhaustif des bases théoriques et des hypothèses fondamentales

de cette recherche a été fait dans un article publié cette année par L. Gillo (1).

Ce problème n’était pas nouveau. La plupart des travaux expérimentaux sur les

tumeurs d’origine animale et en clinique humaine ont été publiés entre les années

1930 et 1940. Les résultats qui en découlèrent furent souvent positifs mais aussi

inconstants. Quatre groupes de savants dont Calmette, Saenz et Costil (2) attri-

buèrent au venin de Cobra une action antitumorale alors que cinq autres lui

déniaient toute action: Essex et Priestley (3), Morelli (4), Morelli et Focosi (5),

Julius (6), Vannfalt (7), Rosenbohm (8). La recherche d une aetivité antalgique

éventuelle donna environ 50% de résultats positifs (1).

Devant Finconstance des résultats, la thérapeutique du câncer par les venins

animaux fut ahandonnée vers 1950.

A la lumière des connaissances actuelles sur la composition enzymatique des

venins, nous pensons que cet échec doit êlre attribué à une erreur de conception

faite par les auteurs. En effet, leur hypothèse était que Faction anticancéreuse

etait due aux comjiosants nécrosants, aux agents cytolytiques, aux neurotoxines

du venin et donc intimement liée à leur toxicité. Ainsi Calmette (2), puis Chopra

et Chowhan (9) et Rousseau (10) ont attribué Factivité anticancéreuse aux lyso-

lécithinases. Yiengar (11) et d’autres y voyaiant Faction des protéases.

La plupart des chercheurs et cliniciens de Fécole française, Grasset et des

Ligneris (12), pensaient que cette action était liée aux toxines et titraient leurs

préparations en unités-souris, unités basées directement sur le degré de toxicité.

II est compréhensible que, partant de telles hipóthèses et devant la néccesité

d’utiliser des doses élevées pour atteindre le senil d’activité, on ait abouti à 1’into-

xication de Fanimal d’expérience ou du patient.

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La croissance et la multiplication cies cellules normales et eancéreuses ont

été éludiées par les voies cie la génétique biochimique et de l’enzymologie. L’étude

clu processus de croissance d’une cellule, de la synthèse des protéines cytoplasmi-

ques sous le controle des acides nucléiques, des enzymes intracellulaires et des

processus énergétiques rend très probable la possilcilité d’une intervention des

venins de serpents sur les processus cancéreux, par le moyen de leurs nucléases,

leurs phosphatases, leurs ATPases, leurs inhibiteurs de déshydrogénases et bien

dautres enzymes non toxiques.

Conlrairement à la conviction des premiers chercheurs, l’action antitumorale

ou antalgique éventuelle est due à des enzymes non toxiques. Les anciens cher¬

cheurs ont fait une erreur de conception en attribuant cette action aux toxines.

Par conséquent, dans les recherches expérimentales futures, les toxines et les lyso-

lécithinases devront être écartées du complexe venimeux pour laisser apparaitre

Taction des enzymes non toxiques responsahles de 1'effet antitumoral ou antal¬

gique.

Resultats et discussion

Dans nos expériences, nous avons choisi le même venin que Calmette, le

venin de Cobra, que a 1'avantage de n’être ni protéolytique ui nécrosant. Nous

avons étudié sur des milliers d’animaux 1’action antitumorale de doses de |4 à

1 gamraa de venin de Naja naja, Naja naja atra et Naja melanoleuca, sur 9 types

de tumeurs spontanées et greffées. Les mélhodes ntilisées à cet effet sont décrites

dans la communication du Docteur Wirtheimer.

Nous avons pu démontrer c|ue le venin total de Naja naja atra est tout aussi

actif sur le carcinome d Ehrlich que 1’Actinomycine D, médicament chimiothéra-

pique classique, inhibiteur clu RNA messager. Ce venin total est moins toxique

que 1’Actinomycine D. Pour savoir si cette activité antitumorale était due aux

toxines ou aux non toxines, nous avons approché ce problème par deux voies, la

première consistant à apporter la preuve que les toxines ne sont pas actives, la

seconde en démontrant que les enzymes, peu ou non toxiques, ont une activité.

Nous avons appliqué le test “in vitro” sur le carcinome d’Ehrlich à la cobra-

toxine par cinq fois recristallisée *. Cette cobratoxine est estrêmement toxique:

sa LD.-,n est de 60 y/Kg. Elle s’est révélée totalement inactive même aux doses

sublétales de 75 y/Kg qui entrainent rapidement la morte de plus de la moitié

des sou ris.

La même expérience fut faite avec la lysolécithinase extraite par nous de

notre venin de Cobra par chromatographie sur TEAE-coIlulose. En présence et

en absence de léeithine, cet enzyme réputé toxique et admis par beaucoup d’au-

leurs comme le principal agent anticancéreux n'a aucune action inhibitrice sur

le carcinome d’Ehrlich même aux doses sublétales. II est importanl d’ajouter

qu'à toutes doses 1’examen microscopique démontre que la lécithinase n’a aucune

action destructrice sur cette cellule conlrairement à ce qu’on a toujours pensé.

En conséquence nous pouvons conclure que ni la cobratoxine, ni la lyso-

lécithinase ne sont directement actives sur le câncer cLEhrlich.

Pour vérifier si cerlaines fractions, peu ou non toxicpies, du venin de N. n.

atra possédaienl une activité anticancéreuse, le venin fut soumis aux méthodes

* Cette cobratoxine nous a été aimablement envoyee par le Prof. Yang clu Kaohsuing

Medicai College — Kaohsuing — Formose.

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L. GILLO

935

classiques de fractionnement. Les pies obtenus furent caractérisés suivant leur

toxicité, leur composition enzymatique et de plus leur pouvoir d’arrêt de Ia crois-

sanee et de la propagation du carcinome d’Ehrlich.

Certaines fractions sont três actives et peu toxiques, quelquefois viugt cinq

íois moins hocives que Ie venin total dont elles étaient issues.

Le venin agirait à l’extérieur de la cellule en dégradant j)ar voie enzyma¬

tique le facteur de propagation (13).

Resume

II a été démontré que le venin de Naja naja atra a une action inhibitrice

sur le carcinome d’Ehr!ich.

Contrairement aux hypothèses des précédents chercheurs, cette action n"est

|>as liée aux fractions toxiques.

II a été démontré que les fractions le plus toxiques du venin, soit la eobra-

toxine et la lysolécithinase sont dépourvues d’activité anticancéreuse. D autre part.

des fractions qui sont jusque vingt cinq fois moins toxiques que le venin total se

sont révélées três actives.

R econnaissance

Nous tenons à exprimer ici toule notre reconnaissance à

la Province de Brabant, à la F. W. Breth Foundation de New York et à Monsieur

Mattens de Bruxelles dont la confiance et le soutien matériel nous ont permis

dYffectuer cette recherche.

SüMMARY

It was demonstrated that Naja naja atra venom has an inhibitory action on

lhe Ehrlich’s carcinoma.

Contrary to previously supported theories, this action is not due to the toxic

fractions: cobratoxin and lysolecithinase, the most toxic components of the venom.

show any anticancerous action. Fractions 25 times less toxic than initial venom

appearead to be very active.

Bibliographie

1. GILLO, L., Ann. Soc. Med. Nat. Bruxelles, 19, 121, 1966.

2 CALMETTE, A., SAENZ, A., et COSTIL, L., C. R. Acad. Sei., 197, 205, 1933.

3. ESSEX, H. E., et PRIESTLEY, J. T„ Proc. Soc. exp. Biol., 28. 550, 1931.

4. MORELLI, E., Sperimentale, 84, 443, 1930.

5. MORELLI, E„ et FOCOSI, M„ Z. Krebsforsch., 34, 473, 1931.

6. JULIUS, H. W„ Acta Brev. Neerland., 5, 49, 1935.

7. VANNFALT, K. A., Uppsala Lãkfôren Fôrh, 42, 315. 1936.

8. ROSENBOHM, A., Z. Krebsforsch., 53, 307, 1943.

9 CHOPRA, R. N„ et CHOWHAN, J. S„ Indian med. Gaz., 70. 445, 1935.

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C. WIRTHEIMER et L. GILLO

937

37. LES VENINS DE SERPENTS, SOURCE DTNZYMES

ANTICANCÉREUX

IJ. ÉTUDE EXPÉRIMENTALE

C. WIRTHEIMER et L. GILLO

Laboratoire dCEnzymologie Tumorale, Bruxelles, Belgique

Matériel et techniques

Nos recherches ont jiorté sur des souris porteuses de 10 espèces difíerenles

de tumeurs spontanées ou greffées.Notre choix fiit guidé ]>ar la facilite d’éle-

vage de ces animaux et la possibilite de disposer facilement de tumeurs variées:

TUMEURS SPONTANÉES (1)

Ces tumeurs de nature mammaire apparaissent spontanément entre le 5 e et

le 9 1 ' mois chez les souris de lignée C :i H ou R III dans des conditions parti-

eulières. Ces souris nous ont été fournies par le Centre Anticancéreux de Ville-

juif et par le Professeur Rudali de Elnstitut Curie de Paris.

Les tumeurs se développent en quelques mois d’une manière irrégulière tout

comme les tumeurs humaines et peuvent atteindre un volume enorme correspon-

dant au tiers du poids de 1’animal. Les métastases externes sont fréquentes et

on observe quelquefois un envahissement des organes internes. Nous n’avons

jamais observé de regression spontanée de ces tumeurs.

La nature histologique de cette tumeur est celle d’un adénocarcinome mam¬

maire. Elle est fortement vascularisée, de nature kystique tubulaire j)leine ou

mixte. Des zones de nécrose ne s’observent que dans les très grosses tumeurs.

TUMEURS GREFFÉES

Deux variétés de tumeurs greffées d origine mammaire nous ont été fournies

par les Professeurs Mühlbock d’Amsterdam et Neukomm de Lausanne (2, 3).

Elles soul greffées sur des souris C 3 H et Swiss et leur évolution est plus rapide

et plus régulière que celle des tumeurs spontanées.

Conlrairement aux tumeurs spontanées dont la Iocalisation se fait au hasard

au niveau des nombreuses glandes mammaires, on peut choisir une place d’im-

plantation favorable, notamment dans la paroi abdominale latérale.

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TUMEUR KOPROWSKA

Cette tumeur, greffée dans la région abdominale, nous a été fournie par

Madame Koprowska de Philadelphie. II s’agit d’une tumeur dorigine cervicale

utérine provoquée par des attouchemeuts au benzopyrène. La tumeur croít dline

manière rapide et possède un caractère bistologique stable.

CARCINOME D’EHRLICH (4, 5)

Ce carcinome a été obtenu à partir d’un adénocarcinome mammaire. Les

cellules lumorales isolées, iujectées par voie intrapéritonéale développent rapide-

ment un câncer ascitique à évolution aigüe; les mêmes cellules, iujectées par

voie sous-cutanée, donnent naissance à des tumeurs solides três malignes.

L’évolution des tumeurs malignes fnl contrôlée par la mesure régulière de

la surface tumorale ou par le poids de la tumeur. Les animaux furent pesés

régulièrement et une observation clinique journalière nous permit de juger de

létat de sanlé des animaux traités et des animaux témoins. Chaque expérience

fui contrôlée ]>ar 1’observation d’un nombre au molns égal d’animaux témoins

traités de façon identique par sérum physiologique. Nous avons noté la survie

en jours et 1’apparition éventuelle des métastases. Dans la plupart des cas, un

examen anatomo-pathologique fut pratiqué à la morl de 1’animal; il comprenait

un examen macroscopique de la tumeur et des principaux organes ainsi qu’un

examen histologique complet.

Nous avons utilisé, pour le traitement des animaux. un extrait stable de

venin de Cobra (Naja naja atra et Naja melanoleuca). La solution est pré-

parée à partir d’une poudre obtenue |>ar lyophylisation du venin frais, dans une

solution isotonique de chlorure de sodium. Ce venin est injecté en général à

des doses variant entre l/j, de gamma à 1 ganima. La voie utilisée pour le traite¬

ment dépend de la nature de la tumeur:

Les tumeurs solides sont trailées par des injections intratumorales de venin.

tous les deux jours, à la dose de 1 gamma en général. Dans certains cas parti-

culièrement résistants, la dose de 5 gamma a été utilisée. Enfin. dans une autre

variété de tumeur solide, le carcinome d’Erlich, la voir intrapéritonéales a été

essayée.

Les tumeurs ascitiques ont été traitées classiquement par des injections intra-

périlonéales à partir du 3* jour qui a suivi la greffe et pendant 6 jours.

Dans le cas du carcinome ascitique d’Ehrlich, nous avons utilisé une mé-

thode consistant à traiter par le venin les cellules cancéreuses in vitro avant leur

injection aux animaux, en concentrant 1’action de faibles doses dans un petit

volume. Ce test, sVffectue de la manière suivante: 011 prélève des cellules can¬

céreuses d Ehrlich sur un animal porteur d’ascile. On compte ces cellules et on

fait incuber, à 37° durant 3 heures, 1.000.000 de ces cellules en suspension dans

le sérum physiologique. On les injecte ensuite par voie intrapéritonéale à des

souris.

Le test d activité du venin est réalisé dans les mêmes conditions, les cellules

étant incubées au contact de 1 gamma de venin.

RÉSULTATS EXPÉRIMENTAUX ET DISCUSSION

Tumeurs mammaires spontanées

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Mem. Inst. Butantan

.Simp. Internac.

88 (3):937-942, 1960

C. WIRTHEIMER et L. GILLO

939

Pour la facilité de rexpérience, nous avons réparti ces tumeiirs en trois

classes suivant leur grandeur:

-— inférieures à 150 mm 2 ;

— comprises entre 150 et 350 mm 2 ;

-— supérieures à 350 mm 2 .

Les résultats obtenus sont repris dans le tableau ci-dessous.

Surfaee tumorale

Poids

Survie

Témoins

Traités

Témoins

Traités

Témoins

Traitt

(%>

(%)

(jours)

(jours

0,5 7

+ 177

+ 31

- 2

1 7

+ 177

- 63

- 2

- 6

NNA

1 7

+ 177

+ 35

- 2

+ 3

Classe

I sans

métastases

0,5 y

+ 177

- 13

- 2

+ 10

1 7

+ 177

- 63

- 2

- 6

NNA

1 7

+ 177

- 12

- 2

+ 3

Classe

Tumeurs 100 mm 2

Tumeurs entre 100

+ 151

- 51

et 150 mm 2

+ 223

+ 29

Résultats réunis

Sans métastases

+ 177

- 17

- 2

Avec métastases

+ 177

+ 6

- 2

Classe

0,5 et 1 y

+ 62

+ 5

- 6

NNA

1 7

+ 62

+ 15

- 6

Classe

III

1 7

+ 15

- 52

- 12

- 11

NNA

1 7

+ 15

- 13

- 12

- 13

Classe

ii + ui

+ 47

- 27

- 7

Classe

i + ii + ui

+ 84

- 20

- 4

SciELO

10 11 12 13 14 15

940

LES VENINS DE SERPENTS. SOURCE D'ENZYMES

ANTICANCÉREUX

Par im souci de rigueur, afin de ne pas fausser les stalistiques, tous les

animaux observés ont été considérés dans ce tableau, même ceux qui onl succom-

bé prématurément et subitement, par choc à la suite d’une injection de venin

ou de sérum physiologique.

Ce tableau analyse, en moyennes arithmétiques des valeurs relalives 1’évo-

lution des surfaces lumorales, des poids et de la survie.

II apparait clairement que les tumeurs des animaux témoins se sont déve-

loppées d’une manière impressionante, atteignant le triplo de la surface primitive.

Quant à la plupart des animaux trailés, ils moutrent un ralentissement net de la

croissance, un arrêt, une régression et parfois même une disparition de la tumeur.

La surface tumorale, dans la classe I, des témoins augmente uniformément

de près de 177%. Chez les animaux traités, cette surface tumorale subit une

régression allant jusque — 63% (1 gamma de venin de Naja melanoleuca).

L’augmentation de la surface, des tumeurs dans la classe II des animaux

témoins, est moins nette (+ 62%), les tumeurs étant déjà plus grosses à 1’origine.

Les animaux traités subissent un arrêt total de croissance.

Les tumeurs, dans la classe III, déjà considérables à 1’origine ne croissent

guère ( + 15%). Malgré cela, le traitement fait apparaitre une nette régression

allant jusque — 52%.

De 1’examen des trois classes réunies, on peut conelure que les venins de

Cobra ont une action freinatrice et régressive (—20%, témoins +84%).

On iTobserve pas d’action bien particulière du venin sur 1’évolution du poids

et la survie par rapport aux témoins. II faut remarquer à ce sujet que nous

avons injecté du venin total et qu il faut lenir compte de sa toxicité.

LMnhibition et la régression des tumeurs sous 1’influence du traitement par

le venin total de Cobra est confirmée par 1’examen anatomopathologique. Ces

examens des coupes des Iraitées montrent que les zones malignes sont progressi-

vement étouffées par des tissus fibronécrotiques. Les cellules cancéreuses sont

dispersées mais encore parfaitement reconnaissables. LIne telle image ne s’obser-

ve qu exceptionnellement dans les petites tumeurs témoins. Dans un certain nom-

bre de cas, le tissu cancéreux a comj)lètement dispam. II est remplacé par du

tissu fibronécrotique ou par du tissu inflammatoire. Les modifications des ca-

ractères histologiques que nous venons de décrire et qui correspondent à une

disparition progressive du tissu cancéreux se retrouvent dans 80% des tumeurs

Iraitées.

Tumeurs gre/fées solides

epi-

a I action du venin de Cobra. D’ailleurs

la même remarque au sujet des tumeurs

Nous avons étudié 3 tumeurs solides dont deux adénocarcinomes et un

thélioma.

Ce dernier s’est révélé insensible

tous les auteurs anciens avaient fait

provcquées par le goudron.

Dans l’adénocarcinome Gaspari, les animaux témoins et traités ont tous été

sacrifiés après trois semaines d observation ce qui correspond au temps d’évolu-

lion naturel de la tumeur témoin. Les tumeurs ont été disséquées et pesées. Le

tableau témoigne que les poids moyens des tumeurs des animaux traités ne sont

que la moitié du poids des tumers témoins. Cette action s’est révélée statistique-

ment significative après analyse de la variance.

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Mem. Inst. Butantan

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33(3):937-942, 1966

C. WIRTHEIMER et L. GILLO

941

Dans le cas de 1’adénocarcinome Mühlbock les tumeurs, ont été trailées par

des doses de 1 à gammas de venin total de Naja naja atra. A la dose de 1

gamma, aucun effect significatií n’apparait alors qu’à la dose de 5 gammas itne

activité se manifeste, statistiquement significative, aussi liien si l’on prend pour

critère 1’évolution de la snrface tumorale que si l’on se base sur le poids de

la tumeur.

Le traitement des tumeurs solides d’Ehrlich s’est fait par voie intratumo-

rale et intrapéritonéale à la dose de 1 gamma. Les rapports des moyennes des

surfaces tumorales en fin d’expérience sont de 512/316 par voie intratumorale

et 512/400 par voie intrapéritonéale. Si l’on prend eomme critère le poids des

tumeurs, le rapports sont respectivement de 12,1/6,7 et 12,1/7,6. L‘analyse de

la variance a confirmé 1'action freinatrice du venin sur ces tumeurs.

Les tumeurs ascitiques d’Ehrlich ont été provoquées par inoculation intrapé¬

ritonéale de 1.000.000 de cellules ascitiques d’Ehrlich à des souris Swiss. Trois

jours après 1’inoculation, on a injecté 1 à 5 gammas de venin total en répétant

cette opération pendanl 6 jours. A la dose de 1 gamma, aucune action statisti¬

quement significative n’est apparue et à la dose de 5 gammas, la moitié des ani-

maux a succombé. Dans ce cas également, 1’action sur 1’ensemble des animaux

est peu nette, mais nous avons été cependant frappés par le fait que certains

animaux ont survécu jusqu’à 37 jours alors que les témoins n’ont pas dépassé

21 jours.

Dans le test in vitro, après 1’injection des cellules ascitiques d’Ehrlich, lous

les animaux développent de I ascite à partir du 8 èrne jour et succombent au

20 eme jour. Conlrairement les animaux injectés avec les cellules ascitiques

d’Ehrlich incubées au contact avec 1 gamma de venin, ne font jamais d’ascile.

Nous avons vérifié la validité de ce nouveau test en 1’appliquant à iin chi-

miothérapique anticancéreux bien connu et bien étudié: l Actinomycine D. inlii-

biteur du RNA messager. A la dose de 0,5 gamma, l’Actinomycine D, tout eomme

0,5 gamma de Naja naja atra, supprime complètement in vitro la possibilité

d’apparition de l"ascite. II est intéressant de remarquer que le venin total de

Naja naja atra est moins toxique que 1’Actinomycine D alors qifil agit à la

même dose.

Nous avons appliqué notre test in vitro à 1’étude de l’action des deux prin-

cipales substances toxiques du venin de Cobra: la cobratoxine qui nous avait été

fournie par le Professeur Yang et la lysolécitbinase, isolée par nous.

A des doses croissantes allant jusqu aux doses sublétales, aucune action anti-

cancéreuse ne fut observée.

La queslion qui se pose ensuite est de savoir ce que deviennent les cellules

d’Ehrlich après 1’action du venin total. Nos premières expériences semblent

montrer quYlles ne sont pas simplement détruites eomme on pourrait le supposer

mais qidelles restent vivantes et Fexamen microscopique révèle quelles conservent

leurs caractères morphologiques normaux. Leur nomlire n’a pas diminué ainsi

qu en témoigne le comptage. Ce travail est actuellement en cours dans notre

laboratoire et fera 1’objet de la suite de nos recherches dans ce domaine.

SUMMARY

Ten different lypes of spontaneous or grafted tumors of the Mouse are

treated by Cobra venom.

SciELO

10 11 12 13 14 15

■942

LES VENINS DE SERPENTS, SOURCE D’ENZYMES

ANTICANCÈREUX

The product is injected hy inlratumoral and sometimes hy intraperitoneal

routes, particularly in the case of solid tumors.

Ascitic abdominal tumors are treated hy intraperitoneal route. The mam-

mary spontaneous tumors, treated in this way, regress about 20% whereas lhe

tumors of the conlrol group grow 177%.

The anatomopalhological examination confirms that there is a resolving

action on the cancerous cell due to a fibronecrosis process.

All lhe solid carcinomas that we studied showed a statistically significative

inhibition of their growlh.

Isolated Ehrlich’s cancerous cells, afler treatment with Naja naja atra venom

failled to produce ascitis when injected in mice.This action of the Cobra venom

can be compared to that of Actinomycin D at an equal dose.

The loxic components of the venom (Cobratoxin, lecithinase) showed no

activity on the Ehrlich’s cell.

Bibliographie

1. DOBROV OLSK AI A, Z„ C. R. Soc., Biol., 126, 287, 1937.

2. MÜHLBOCK, et BOOT, Câncer Res., 19, 402, 1959.

3. NEUKOMM, S., PEGUIRON, L., et HERVE, A., Action de la cystéamine sur

les tumeurs greffées irradies in loco — Problèmes de cancérologie contempo-

raine, Lausanne, 1961, p. 663.

4. EHRLICH, Z. Arztl. Forbild., 3, 205, 1906.

.5. SEGLER, K„ Câncer Res., 22, 1278, 1962.

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10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 943 - 950 , 1966

ANIMA DEVI, S. S. ASHGAR and N. K. SARKAR

943

38. 5’-NUCLE0TlDASE ACTIVITY IN SNAKE VENOMS

ANIMA DEVI* *, S. S. ASHGAR and N. K. SARKAR**

Department of Biochemistry, Post-Graduate Institute for Medicai Research

and Teaching, Chandigar, Punjab, índia.

5’-nueleotidases, the enzyme capaiile oi liyclrolyzing nucleoside 5’-monophos-

phates lo nucleosides and orthophosphate are widely distributed in nature. 5’-nu-

cleotidase activity has been found in animal tissues (1-3), buli semen(4), mi-

cro-organisms (5, 6) and in venoms of different species of snakes (7, 8). In

human tissues, lhe highest activity has been found in the posterior pituitary

gland (9, 10). Venoms generally exhibit high 5’-nucleotidase activity. This en¬

zyme is found with phosphodiesterase and non-specific phosphatases in venoms

which have been largely used in recent years to purify phosphodiesterase and

5'-nucleotidase as lhese enzymes are valuahle tools in the study of structure and

nucleotide sequence of nucleic acids (11-14).

After the findings of Gulland and Jackson (7) showed the presence of 5’-nu-

cleotidase in venoms, many attempts had been made to isolate it from venoms

in order to study lhe substrate specificity of the enzyme, its metal requirement,

and optimum pH (13-15). We have recently studied 5’-nucleotidase activity in

venoms of different species of snakes from widely separaled parts of the world

and the effects of various metallic ions, inhibitors, and activators on the enzyme

activity. The results are presented in this paper.

Methods

In the present study a 20 mg percent venom solution made in glass-distilled

water, the pH of which was previously adjusted to 7.4 with IN NaHCO :i was

used. The assay involved the measurement of orthophosphate liherated from

adenylic acid (5’-AMP) when a reaction mixture containing 0.1 ml of the venom

solution, 5 p.moles of 5’-AMP (Na salt), 1 /imole of Mg ++ and 20 /imoles

of glycine buffer, pH 8.5, in a total volume of 0.5 ml. was incubated at 37 U C

for 20 min. The reaction was terminated by adding an equal volume of 10%

cold trichloroacetic acid (TCA), and allowing it to stand at 0°C for 15 min

and then cenlrifuging it at 1200 X g at 0°C for 15 min. The clear liquid

(TCA extract) was used for the orthophosphate determination. The methods of

Eiske and Subba How (16), Cleland and Slator (17) were used to measure Pi

and protein.

•* Present acidress: Bioehemistry Section, Animal Research Institute, Research Brandi,

Canada Department of Agriculture, Ottawa, Canada.

* Present address: Department of Biology, Ottawa University, Ottawa, Canada.

SciELO

944 5’-NUCLEOTIDASE ACTIVITY IN SNAKE VENOMS

Results

Fig. 1 shows the 5'-nucleotidase activity of six venoms obtained írom lliree

different species of snakes. The rate of hydrolysis of 5’-AMP was linear lo the

enzyme concentration in each case, until 65 to 70% of the substrate was hydro-

lyzed. Of all lhe venoms tested for 5 -nucleotidase activity the venom of Crotalus

adamanteus exhibited highest activity and the venom of Bothrops jararaca the

least. The venom of Vipera russelli showed slighlly less activity than lhe venom

of C. adamanteus. The Km values calculated for the venoms of V. russelli and

water moccasin by Lineweaver and Burks equation (18) from the plots 1/V

against 1/S (Fig. 2) were found to be 2.2 X 10

id 1.66 X 10 -3 M.

Fig. 1

Ejjects of metallic wns, ortlio- and pyrophosphates and reducing agents

on venom 5 -nucleotidase activity.

Maximum activity was found in the presence of Mg" 1 "" 1 "; and in its absence

60-70% activity was noted. In the presence of EDTA, lhe activity dropped to

10-15%. However, when Mg ++ was added a twofold increase in activity was

found. Wilh dialyzed venom which showed only 20-22% of lhe activity of

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10 11 12 13 14 15

Mem. Inst. Butantan

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33(3) :943-950, 1966

ANIMA DEVI, S. S. ASIIGAR and N. K. SARKAU

945

Fig. 2

the undialyzed venom. a three- lo fourfold increase in activity was observed

when the activity was measured in the presence of 1 >otli EDTA and Mg ++ .

Zn ++ , Mn ++ , and I\i + + inhihited the enzyme activity strongly. Co + + at

low concenlrations stimulated the enzyme activity and at higli concentrations

inhihited its action (Table I).

Orthophosphate and pyrophosphate both inhihited 5’-nucleotidase activity.

Cystein and glutathione enhanced the enzyme activity, but methionine and cystine

inhihited its activity. NaF aeted as an inhihitor l)iit was less active than lhe

orthophosphate and pyrophosphate (Table I).

Effects of pH on venom 5' -nucleotida.se

From the pH-activity curve (now shown) il was concluded lhal venom

5’-nueleotidases have no well-defined pH optimum because lhe venom of V. russelli

exhibited maximum activity over the pH range 7.6 to 8.4, and C. adamanteus

venom was equally active over lhe pH range 8.0 to 10.0. At pH below 7.4

SciELO

10 11 12 13 14 15

946

5’-NUCLEOTIDASE ACTIVITY IN SNAKE VENOMS

TABLE I — EFFECTS OF METALLIC IONS, ACTIVATORS AND INHIBITORS ON

VENOM 5’-NUCLEOTIDASE ACTIVITY

Reagents

/imoles

added

Relative

Activity

Activation

(%)

Inhibition

(%)

Complete system

—

100

—

” - Mg

—

” + Mn + +

0.05

—

0.10

—

” + Ni + +

1.00

2.00

—

+ CO + +

0.10

1.00

—

„ ~\—h

” + Zn

5.00

0.05

—

0.10

—

” + cysteine

1.00

—

5.00

—

+ glutathione

1.00

—

5.00

—

1.00

+ Pi (Na-phosphate)

2.00

—

5.00

—

+ PP (Na-pyrophosphate)

1.00

—

2.00

—

5.00

” + NaF

1.00

2.00

—

5.00

—

The complete System contained 20 /imoles of glycine bufíer, pH 8.5, 1 /imole of Mg + +

5 /imoles of AMP (Na-salt) and 0.1 ml of a 0.02% venom solutlon. The reaction mixture

was incubated for 20 mlns. at 37°C. The amount of metallic ions, activators and inhi-

bitors added are shown in column 2. The degree of activation and inhibition of 5’-nucleo-

tidase is expressed as the percentage of the activity of the complete system, whieh has

been arbitrarily taken as 100 %.

and above 8.4, lhe 5’nncleotidase activity of V. russelli venom declined rapidly.

A sharp drop in 5’-nucleotidase activity of C. adamanteus venom was also noted

at pH below 8.0 and above 10.0.

Substrate specificity of venom 5’-nucleotidase

The ability of venom 5’-nucleotidase to hydrolyze other nucleotides is shown

in Tahle II. The results indicated that venom 5’-nucleotidase can hydrolyze almost

all nucleoside 5’-monophosphates and among them, 5’-AMP was hydrolyzed ma-

ximally (100%) and 5-CMP by 76%. Next lo it in order of effectiveness as

SciELO

10 11 12 13

Mem. Inst. Butantan

Simp. Internac.

33(3):943-950, 1966

ANIMA DEVI, S. S. ASHGAR and N. K. SARKAR

947

TABLE II — SUBSTRATE SPECIFICITY OF

VENOM 5’-NUCLEOTIDASE

Substrates

Relatlve Activity

5’-AMP

100

5’-UMP

5’-CMP

5’-GMP

5’-IMP

5'-dAMP

5’-dTMP

5’-dCMP

5’-dGMP

2’-AMP

3’-AMP

cyclic-2’, 3'-AMP

rlbose-5’-phosphate

a — glycerophosphate

P — glycerophosphate

The reaction mixture contained 20 amoles of gly-

cine buffer, 8.5, 1 /imole of Mg + +, 5 /imoles of

substrate (Na salt), and 0.1 ml of a 0.02% venom

solutlon in a total volume of 0.5 ml, ineubated

for 20 mlns. at 37°C.

substrates were 5’-UPM, 5’-GMP and 5’-IMP which were hydrolyzed by 36, 28

and 22%, respectively. Venom 5’-nucleotidase can also hydrolyze deoxynucleo-

side 5’-monophosphates viz 5’-dTMP could be hydrolyzed by 58%, 5’-dCMP,

5’-dAMP and 5’-dGMP by 54, 35 and 26%, respectively. Neither ribose-5-phos-

])hate nor cyclic-2’,3’-AMP could be hydrolyzed. Very limited hydrolysis vvas

noled with a- and /8-glycerophosphates.

Discussion

The twofold increase in lhe activily of cobra venom 5’-nucleotidase observed

by Kaye(19) after dialysis, was not noled by tis with lhe venom of V. russelli,

but a three- lo fotirfold increase in lhe activity of lhe dialyzed venom was noted

when measured in the presence of EDTA and Mg ++ . Probably the venom used by

tis did not contain any free Zn ions or other poisonous metais that can be removed

by diajysis, but contained traces of metais in bound form that can be removed

by EDTA. This perhaps explains the fourfold increase in activity noted by

Kaye (19) and us when the activity was measured in the presence of EDTA

and Mg ++ and not just in the presence of Mg + + alone. A 90% inhibition

SciELO

10 11 12 13 14 15

948

5'-NUCLEOTIDASE ACTIVITY IN SNAKE VENOMS

of enzyme aclivity was observed vvilli orthophosphate and pyrophosphate whereas

NaF inhibited only 65% of the activiíy. Activation by glutathione (reduced

form) and cysleine amounted lo 46 and 48% in eacli case (Table II).

Because venoras rich in 5’-nucleotidase activiíy did not hydrolyze a- and

/Tglycerophosphates al pH 5.0 and 9.0 and their aclivity was inhibited by JNi ++ ,

which did nol affect lhe hydrolysis of glycerophosphates by nonspecific plios-

phomonesterases, lhe hydrolysis of nucleoside 5’-monophosphates hy 5 -nucleoti-

dase cannot be related to the action of any nonspecific phosphomonesterase

present in the venoms. It can not either be linked to venom adenosine tri-

phosphatase since the hydrolysis of ATP by venoms was nol altered by Co ++ ,

which enhanced the hydrolysis of 5’-AMP by venoms at low concentrations and

inhibited al higher concentrations. Venom 5’-nucleotidase was nol identical lo

5’-nucleotidases obtained frorn other sources since the hydrolysis of 5-AMP by

them was not affected in lhe same way hy the same metallic ions and also because

the pH at which maximum hydrolysis of lhe suhstrate attained, was also diffe-

rent. Although venom 5’-nucleotidase hydrolyzed all deoxynucleoside 5’-mono-

phosphates such as 5'-dAMP, 5’-TMP, 5’-dCMP, and 5’-dGMP the affinity of lhe

enzyme for 5’-AMP was much greater than for 5"-dAMP. Other deoxy com-

pounds were also hydrolyzed but lo lesser degrees than the corresponding rihose

compounds. The hydrolysis of deoxy nucleotides by bactéria! 5’-nucleotidase

was firsl re])orted by Cárter (20). ll did not however hydrolyze 2’-AMP.

3-AMP or cyclic-2’,3’-AMP, nor catalyze lhe hydrolysis of ribose-5-phosphate.

5’-nucleotidases obtained from different sources appear to be different in many

ways, particularly in regard to their behaviour towards metallic ions and inhi-

bitors, and pH, their suhstrate specificity seems to be the same, and lherefore

should be considered as isoenzymes.

The existence of an enzyme capable of hydrolyzing a specific suhstrate

(adenylic acid), and as widely dislributed in nature as its own suhstrate possibly

im])lies the involvement of this enzyme in regulating the concentrations of orlho-

phosphate for glycogenolysis and adenylic acid (other monophosphates) for nucleic

acid synthesis in the cell. It is difficult to understand the reason for the higli

5 -nucleotidase activity in snake venoms since lhe amounl of enzyme present is

not sufficient lo inflict any toxic effect. It may be possible that the enzyme

5 -nucleotidase, like other enzymes present in the venoms, also contribute to a

limited extent towards lhe total toxicity of the venoms (21).

References

1 REIS, J. L„ Buli. Soc. Chim. Biol., 16, 385, 1934.

2. REIS, J. L., Enzymologia, 5, 251, 1938.

3. REIS, J. L„ Buli. Soc. Chim. Biol, 22, 36, 1940.

4. HEPPEL, L. A„ and HILMORE, R. J„ ./. B. C., 188, 665, 1951.

5. WANG, T. P., J. Bacteriol., 68, 128, 1954.

6. HERMAN, E. C„ JR„ and WRIGHT, B. E., J. B. C.. 234, 122, 1959.

7. GULLAND, J. M. ,and JACKSON, E. M„ Biochem. J., 32, 597, 1938.

8. ZELLER, E. A., The Enzymes, Vol. 1, lst Ed„ 1951, p. 986.

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Mem. Inst. Butantan

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33(3):943-950, 1966

ANIMA DEVI, S. S. ASHGAR and N. K. SARKAR

949

9. PEARSE, A. G. E., and REIS, J. L., Biochem. J., 50, 534, 1952.

10. AHMED, Z., and REIS, J. L„ Biochem., 69, 386, 1958.

11. RUZZELL, W. E„ and KHORANA, H. G., J. Biol. Chem., 234, 2114, 1959.

12. BJÕRK, W., J. Biol. Chem., 238, 2487, 1963.

13. SULKOWSKI, E„ BJÕRK, W„ and LASKOWSKI, M., SR„ J. Biol. Chem., 238,

2477, 1963.

14. BJÕRK, W., Biochem. Biophys. Acta, 89, 483, 1964.

15. NIKOUSKAYA, L. N„ KISHLINA, O. S., and TIKHONENKO, T. L„ Biokhi-

miya, 30, 107, 1965.

16. FISKE, C. H„ and SUBBA ROW, Y., J. Biol. Chem., 66, 375, 1925.

17. CLELAND, K. W.. and SLATOR, E. C„ Biochem., 53, 548, 1953.

18. LINEWEAVER, H., and BURK, D., J. Am. Chem. Soc., 56, 658, 1934.

19. KAYE, M. A. G„ Biochem. Biophys. Acta, 38, 341, 1960.

20. CÁRTER, C. E., J. Am. Chem. Soc., 73, 1537, 1951.

21. DEVI, A., and SARKAR, N. K„ in W. BÜCHERL, V. DEULOFEU, and E.

BUCKLEY (Editors), Venomous Animais and their Venoms, Vol. 3, Acad.

Press, 1968.

SciELO

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FARMACOLOGIA

PHARMACOLOGY

Mem. Inst. Butantan

Simp. Internac.

33 ( 3 ): 951 - 956 , 1966

I. S. DE LA LANDE and JANET C. LEVVIS

951

39. CONSTITUENTS OF THE VENOM OF THE AUSTRAL!AN BULL

ANT, MYRMECIA PYRIFORMIS

I. S. DE LA LANDE & JANET C. LEWIS

Department Human Physiology & Pharmacology , Univ. of Adelaide, Australia

Preliminary studies (1, 2), on lhe venom of an Australian buli ant, Myr-

mecia pyriformis (previously identified as M. jorjicata ) revealed the presence of

high concentrations of histamine (1-3% by weight of the dried venom) and

another suhstance possessing smooth muscle stimulating properties. 1 his latter

component, separated from the histamine by means of ascending paper chroma-

tography causes a slow, persistent contraction of the mepyramine-treated guinea

pig ileum and of lhe virgin ral uterus, and produces a prolonged hypotensive

response in the anaesthetized cat, distinct from the rapidly reversible depressor

response lo histamine. More recently Cavil! et al. (3) have reported in the

venom of a related species of ant, M. gulosa, lhe presence of hyaluronidase acti-

vity and a direct haemolytic component, in addition lo histamine and a smooth

muscle slimulant resembling that described for M. pyriformis. They achieved

partial separation of the components by means of low voltage paper electropho-

resis although this technique did not permit separation of the hyaluronidase

from lhe smooth muscle stimulant.

Subsequently, we have shown that lhe crude venom of M. pyriformis pos-

sesses hyaluronidase activity (determined turbidimetrically by the method of

Tolkesdorf et al.) (4) and the ability to lyse washed red blood cells using a

modification of the method described by Neumann, Habermann & Hansen(5).

I mg of the crude venom was found to be equivalent to 100 IU of hyaluronidase

(Rondase) and is therefore 10 times as polent as commercial testicular hyaluro¬

nidase on a weight hasis. Activity could be demonstrated in 10 /xg crude venom.

On account of lhe prolonged nature of the local responses in man to sting

by M. pyriformis, an investigation was made into lhe possibilily that lhe venom

contains components whieh might act indirectly ihrough the endogenous release

of pharmacologically aclive substances such as histamine and hradykinin. The

crude venom was lested for its ability to release histamine from isolated rat mast

cells (6) according to the technique described by Rolhschild (7), and its polency

in lhis respect was compared with 48/80. Saline extracts of venom or Solutions

of 48/80 were incubated with cells from the periloneal fluid of rats and after

incuhation the residual histamine content of the cells was assayed on the guinea

pig ileum. Activity was expressed in terms of % release. As little as 10 /xg

of venom was found capable of releasing appreciable amounts of histamine and

this dose was roughly equivalent to 2.5 /xg 48/80. In some experiments acti¬

vity was demonstrated by allowing live ants to release venom directly into the

incubating fluid containing lhe mast cells. Although the method was not suffi-

cientlv sensitive to show any response from 1 sting, lhe venom of 10 insertions

of a sting resulted in considerable histamine release.

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952

CONSTITUENTS OF THE VENOM OF THE AUSTRALIAN BULL

ANT, MYRMECIA PYRIFORMIS

The method used for lhe detection of hradykinin-rcleasing aclivily was based

on thal described hy Margolis et al. (8). Saline extraels of lhe venom were in-

eubated with bradykinin-free rabhil plasma in lhe presence of EDTA and lhe

bradykinin-released was assayed on the rat duodenum. In some experiments,

suspensions of human hradykininogen replaced lhe samples of rabhit plasma and

in all experiments eornparisons were made willi trypsin. Concentrations of venom

30 times lhose capable of produeing histamine release showed no evidence of

hradykinin-releasing activity.

Fnrther characterization of lhese pharmacologically active components has

heen attempted hy the application of fractionation procedures, boiling and di-

gestion with proteolytic enzymes. By these means it has heen possihle lo con-

clude thal the various aclivilies can be attributed lo separate substances, with the

exception of the haemolytie component which il has not heen possihle to separale

from the smoolh muscle stimulant.

Aseending paper chromatography was carried out in water saturated hutanol

acetic acid (5:4:1), these conditions having heen used lo separate the histamine

from the smoolh muscle stimulant in the preliminary studies. Chromatograms

were run for 24 hrs after which 1/2” stri])s were eluted in saline and eluates

submitted to tests for pharmacological and enzymic activity. Hyaluronidase acti¬

vity disappeared allogether under these conditions, histamine was clearly sepa-

rated from all other aclivilies, but lhere was considerable overlap of lhe hista¬

mine releasing, smooth muscle stimulant and haemolytie components. However,

a high percentage of the histamine releasing activity had a Hf value dose to

zero and it appears that a separate component is responsihle for al least pari of

lhe histamine releasing activity of the venom. The partial separation of this

faclor from the other active components hy means of low voltage paper electro-

phoresis at pH 6.24 again suggesls that there may be two fractions contributing

to histamine release, one of which is a distinet component and lhe other indistin-

guishable from the smooth muscle stimulant.

Electrophoresis was carried out at 4°C and a pH of 6.24 as described by

Cavill et al. ( 3). A eurrent of 0.3 mamps/cm was applied for 16 hours after

which the paper was divided into strips, eluted in saline and tested for lhe

various activities. Hyaluronidase and lhe smooth muscle stimulant could not be

separated and in our experiments the haemolytie activity moved towards the ca-

thode parallel lo the smoolh muscle stimulant. This is in contrast to the liaemo-

lytic component of M. gulosa which is reported lo move towards the anode.

Dialysis through Visking cellophane membranes was carried out foi' 24

hours at 4°C with the ratio of inner lo outer volume being 1:10. After dialysis,

the volume of the dialysale was reduced 10 fold hy freeze drying and redissolving

in the appropriate amount of distilled water.

With the exception of histamine which was always 100% dialysable, the

components were only slowly dialysable and their rates of dialysis were somewhat

variable. However histamine releasing activity and the smooth muscle stimulant

were always in detectable concentrations whereas hyaluronidase and haemolytie

activities were sometimes less lhan 10% dialysed, and could not be detected. The

behaviour of each component on chromatography, electrophoresis and dialysis is

summarized in lhe Table I. Table II shows lhe percentage activity of each

component remaining after digestion with trypsin, chymotrypsin and boiling for

10 minutes at pH 6.0.

6 SciELO 10

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Mem. Inst. Butantan

Simp. Internac.

33(3): 951-956, 1966

X. S. DE LA LANDE and JANET C. LEWIS

953

TABLE I :

COMPONENT

PAPER

CHROMATO-

GRAPHY

Rf value

ELECTRO-

PHORESIS

Rate of move-

ment to Catho-

de: ins/16 hours

DIALYSIS

% Dialysed in

24 hours

Histamine

0.22

(0.18 — 0.26)

5.25

(4.5 — 6.5)

90% (3)

Smooth muscle stimulant

0.14

(0.13 — 0.15)

1.3

(0.5 — 2.5)

13 — 30% (3)

Histamine release

0 — 0.15

2.25

(1.5 — 3.5)

36% (1)

Hyaluronidase

—

1.25

(0.5 — 2.5)

Undetectable

< 8% (2)

Haemolytic

0.14

(0.11 — 0.17)

2.0

(1.5 — 2.5)

Undetectable

< 12% (1)

* Figures in braekets refer

to number of

separate determinations.

TABLE II *

COMPONENT

BOILING

pH 6.0

% Activity

remaining

TRYPSIN

DIGESTION

% Activity

remaining

CHYMOTRYPSIN

DIGESTION

% Activity

remaining

Histamine

100% (3)

100 %, (1)

Smooth muscle stimulant

37% (3)

10% (4)

50% (2)

Histamine release

100 % (2)

10 — 20% (4)

30 — 40% (2)

Hyaluronidase

3% (3)

20 — 67% (3)

—

Haemolytic

31% (2)

10 % (1)

—

Figures in braekets refer to number of separate determinations.

The various procedures outlined above shed light on some characteristics of

lhe various components of the venom of Myrmecia sj). although detailed cha-

racterization of the constituents requires more satisfaclory methods of fractio-

nization. In many respects the venom resembles that of the bee and has

constituents belonging to each of the three classes described l)y Hahermann (9) for

HYMENOPTERA venoms -— namely biogenie amines, peptides and non-enzymic

proteins, and enzymes, — typified l>y histamine, the smooth muscle stimulant

and hyaluronidase respectively.

The smooth muscle stimulant fraction is slovvly dialysable through Visking

cellophane membranes and is only partially destroyed by boiling. However,

alrnost all the activity is lost by incubation with trypsin. It contracts the smooth

muscle of the guinea pig ileum, the virgin rat uterus and the blood vessels of lhe

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10 11 12 13 14 15

954

CONSTITUENTS OF THE VENOM OF THE AUSTRALIAN BULL

ANT, MYRMECIA PYRIFORMIS

isolated rabbit ear. Tachyphylaxis occurs, liut least markedly in the perfused

rald.it ear and consequently this preparation, treated willi mepyramine, has been

use d most frequently, doses lieing administered al inlervals of 20 minutes. An

example of this is shown in Fig. 1. Activily has been demonstrated vvilli 10 jug

of crude venom. There has been no clear-cut distinction between this factor and

the haemolytic component and it is possible that the smooth muscle stimulant

possesses haemolytic properties. It may also account for some of the histamine

releasing activity.

MEPYRAMINE 2 0 yg /1

Fig- 1 — Isolated Rabbit Ear perfused with Kreb's solutlon at 37°C. Perfusion rate

7 mls/min. Constriction reeorded by rise in perfusion pressure. Dialysate and residue

derived from a 25/ig equivalent of venom dialysed for 24 hours. Time scale — 1 minute.

Phospholipase A is widely distributed among itlsect and animal venoms and

il is conceivable lhat the venom of Myrmecia sp. also contains this enzyme, and

if this is so, it could contribute lo the histamine releasing activity. In this

conection attention is drawn lo two properties of Phospholipase A — namely

its heat stability and its ability to dialyse slowly, both of which apply to the

histamine releasing activily of the anl venom. Phospholipase A and lysolecithin

themselves possess various pharmacological activities including smooth muscle sti-

mulation, a delayed and prolonged hypotensive effect and increased capillary

permeability — all of which are properties possessed by lhe venom. Larger

scale separation and attempts at preparation of relatively pure fractions are in

progress, and it is hoped to embark on a more detailed analysis of the compo-

nents, particularly lhe smooth muscle stimulant and the histamine releasing factor

as soon as purer fractions become available.

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I. S. DE LA LANDE and JANET C. LEWIS

955

References

1. DE LA LANDE, I. S., THOMAS, D. W., and TYLER, M. J., Biochem. Pliar-

macol., 12, (suppl.), 187, 1963.

2. DE LA LANDE, I. S„ THOMAS, D. W„ and TYLER, M. J„ Proc. of the

Second International Pharmacological Meeting, Prague, 9, 71, 1965.

3. CAVILL, G. W. K„ ROBERTSON, P. L., and WHITFIELD, F. B., Science, 146,

79, 1964.

4. TOLKESDORF, S„ McCREADY, M. H„ McCULLAGH, D. R., and SCHWENK,

E., J. Lab. clin. Med., 34, 74, 1949.

5. NEUMANN, W., HABERMANN, E., and HANSEN, H., Arch. exp. Pathol.

Pharmakol., 217, 130, 1953.

6. THOMAS, D. W., and LEWIS, J. C., Aust. J. exp. Biol. med. Sei., 43, 275, 1965.

7. ROTHSCHILD, A. M., Biochem. Pharmacol., 11, 979, 1962.

8. MARGOLIS, J., BRUCE, S„ STARZECKI, B., HORNER, G. J„ and HAL-

MAGYI, D. F. J., Aust. J. exp. Biol. med. Sei., 43, 237, 1965.

9. HABERMANN, E., Proc. of the Second International Pharmacological Meeting,

Prague, 9, 53, 1965.

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Mem. Inst. Butantan

Simp. Internac.

33(3):957-960, 1966

A. P. CORRADO, A. ANTONIO and C. R. DINIZ

957

40. RECENT ADVANCES ON THE MECHANISM OF ACTION OE

BRAZILIAN SCORPION VENOM — T. SERRULATUS

A. P. CORRADO, A. ANTONIO and C. R. DINIZ

Departments of Pharmacology anã Biochemistry, Faculty of Medicine,

Ribeirão Prêto, São Paulo, Brasil

The mechanism of the pronounced syslemic hypertension produced by

scorpion venom is not yet completely understood. Most of the lheories relate

the hypertensive response to a sympathetic stimulation, which, aceording to the

different authors could be: peripheral ( 1, 2) (post ganglionic endings of the

sympathetic system), central (3) (spinal cord and sympathetic preganglionic neu-

rons) or both (4, 5). However. no attempt was made to suggest whether the

sympathetic stimulation produced by the venom is a direct one or mediated by

released caterholamines. On the other hand. it is known that the venom induces

a contraction of the guinea-pig ileum through the release of acetylcholine (6, 7).

Since the venom seems to interfere with hoth components of the autonomic

nervous system. we decided to investigate its actions on the isolated guinea-pig

lieart which gives opposite responses whether adrenergic or cholinergic agents

are tested.

Isolated hearts from eighteen guinea-pigs prepared aceording the Langendorff

method, were used.

The typical effect of lhe venom * is a bradycardia followed hy a conspicuous

increase in the force and frequency of the heart beatings; these effects are

achieved

doses

png from 5-10 meg which to correspond to a final con-

centration of the order of 2 X 10~ 6 to 1 X 10 -6 g/ml. In a few hearts

higher dose (20-40 meg) was necessary lo produce the bradycardia which usually

precedes the stimulating effect (Fig. 1). We have shown that the cardiosti-

mulating effect is indirect and due to the release of catecholamines since it was

blocked hy D.C.I. (9) and bretylium (2 X 10— 5 g/ml/min.) and was absent

in the reserpine treated animais** (Fig. 2).

In our experiments no potentiation of the cardiac effects of epinephrine by

lhe venom was seen indicating no interference hy the venom in the mechanisms

of inactivation of catecholamines.

The bradycardie response to the venom is better seen when the cardiosti-

mulating effect is blocked hy D.C.I. bretylium or previous reserpinization. 1 he

bradycardia is cholinergic in nature since it is blocked hy atropine (2 X 10

Instituto Butantan, São

* The whole venom, kindly supplied by Dr. W. Bücherl

Paulo, was obtained by electrical stimulation (8).

** Three guinea-pigs received 2 mg/kg of reserpine divided in two doses of 1 mg

given intraperitonealfy 48 and 24 hs. before the experiments.

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10 11 12 13 14 15

958

RECENT ADVANCES ON THE MECHANISM OF ACTION OF

BRAZILIAN SCORPION VENOM — T. ÜERRULA TUS

30 mcg

Fig. 1 — Normal guinea-pig heart —• Ef-

fect of 30 mcg of scorpion venom (VE —

final concentratlon 6 x 10 — ° g/ml) on the

Isotonic force of contractlon and frequency

of heart beatlng. Notice the bradycardia

preceding the cardiac stimulation.

NIC

15 mcg

30 mcg

NIC

30 mcg

-HEXA 50mcg/mm-

.05 mcg

Fig. 2 — Reserpinized guinea-pig heart — No cardiac stimulation is observed with

15 mcg of nicotine (NIC — final concentration 3 X 10 — “) or 30 mcg scorpion venom

(VE — final concentration 6 X 10 — “). Notice that hexamethonium (1 X IO- 5 g/ml/min)

blocks the bradycardic effect of nicotine but not that of the venom. At AI), epinephrine

(final concentration 1 X 10 — s g/ml).

g/nil) and potentiated I>y prostygmine (4 X 1() -6 g/ml). Moreover, the bra-

dycardic action of the venom seems lo he indirect since il is significantly reduced

hy hemicholinium (4 X 1() -5 g/inl/min.) in agreemenl with this view is the

observation that lhe contraction of lhe guinea-pig ileum elicited hy the venom

is markedly depressed hy morphine (6) and cocaine(lO). The site of action

of the venom is posl ganglionic since it was not affected hy hexamethonium

(1X10-= g/ml/min.) in doses which abolished the response lo nicotine (Fig. 2).

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Mem. Inst. Butantan

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A. P. CORRADO, A. ANTONIO and C. R. DINIZ

959

The site of actiou of the cardiostimulating effeet of the venom is not at the

sympathetic ganglion hecause il vvas not affected hy either hexamethonium or

atropine. On the other hand the effeet of lhe venom is higger and more resistant

to tachyphylaxis than that elicited hy tyramine; also the effeet of the venom but

not of tyramine is hlocked hy hemicholinium (4 X 10 -6 g/ml/min.).

It should he noted that the only drug ahle to hlock simultaneously the cho-

hnergic and lhe adrenergic effects of the venom is hemicholinium, which is a

compound knovvn lo interfere with the synthesis of acetylcholine 111). Sueli

result strongly suggests the participation of acetylcholine in both effects of the

venom, i.e., bradycardia followed hy cardiac stimulation. This hypothesis is

strengthened hy our ohservation that prostygmine 1 X 10 -7 g/ml is ahle to po-

tentiate the cardiac stimulation in the atropinized heart (Fig. 3). When we con-

IO meg

5 meg

10 meg

Prost 20 meg

Atrop 100 meg

Fig. 3 — Normal guinea-pig heart — Cardiac effects of 10 meg of scorpion venom

(VE — final concentration 2 X 10 — 6 g/ml) and acetylcholine (AC — final concentration

1 X 10 — ° g/ml) before and after the simultaneous administration of prostygmine (4 X 10 — c

g/ml) and atropine (2 X 10 — 3 g/ml). Notice the increase of the cardiostimulant effeet

of the venom after treatment of the atropinized heart by prostygmine.

sider all these facts they agree with and give further support lo the theory pro-

posed hy Burn and Hand (12) of the existence of a “cholinergic link" at the

sympathetic nerve endings. According to the theory of Burn and Hand. the

sympathetic post ganglionic stimulation produces a small discharge of acetylcho¬

line which in turn releases norepinephrine.

Therefore, the cardiac stimulating effeet of lhe scorpion venom resemhles

that of sympathetic nerve ending stimulation in lhe same way as it lias lieen

described and assumed for guanethidine (13).

References

1. HASSAN, A., and MOHAMMED, A. H., Lancet, 1, 1001, 1940.

2. MOHAMMED, A. H„ Lancet, 2, 364, 1942.

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11 12 13 14 15 16 17

960

RECENT ADVANCES ON THE MECHANISM OF ACTION OF

BRAZILIAN SCORPION VENOM — T. SERRULATUS

3. DEL POZO, E. C., ANGUIANO, L. G., and GONZALES, Q. J„ Rev. Inst. Salubr.

Enfer. troy., 5, 227, 1944.

4. RAMOS, A. O., and CORRADO, A. P., Ann. Fac. Med. Univ. 8. Paulo, 28, 81,

1954.

5. FREIRE-MAIA, L., and FERREIRA, M. C., Mem. Inst. Oswaldo Cruz, 59, 11,

1961.

6. DINIZ, C. R., and VALERI, V., Arch. int. Pharmacodyn., 71, 1, 1959.

7. TORRES, J. M., and DINIZ, C. R., Ciência e Cultura, 16. 197, 1964.

8. BÜCHERL, W., Mem. Inst. Butantan, 25, 53, 1953.

9. ANTONIO, A., DINIZ, C. R., and CORRADO, A. P., Ciência e Cultura, 15,

270, 1963.

10. DINIZ, C. R., and GONÇALVES, J. M., in E. E. BUCKLEY, and N. PORGES

(Editors), Venoms, Amer. Ass. Advanc. Sei., Washington, 1956, p. 131.

11. BURN, J. H., and RAND, M. J., Ann. Rev. Pliarmacol., 5, 163, 1965.

12. BURN, J. H„ and RAND, M. J„ Nature, 184, 163, 1959.

13. COSTA, E., and BRODIE, B. B„ in H. E. HIMWIDCH, and W. HIMWIDCH

(Editors), Biogenic Amines, Elsevier. Pub. Co., Amsterdam, 1964.

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Mem. Inst. Butantan

Simp. Internar.

33(3):961-964, 1966

E. C. DEL POZO, M. SALAS and P. PACHECO

961

41. EFFECTS OF SCORPION VENOM AT NEUROMUSCULAR JUNCTION

E. C. DEL POZO, M. SALAS and P. PACHECO

Instituto de Salubridad y Enfermedades Tropicales and Instituto

de Estúdios Médicos y Biológicos, México, D.F.

The squeletal musele activity provoked hy scorpion venom from mexican

Centruroides is due to effects on spinal motoneurons and neuromuscular

junctions 11).

The latter is a local action because the venom did not activate either de-

nervated musele or muscular nerves when directly applied.

Mechanograms of muscular responses to single shock stimulations in cats and

other mammals under scorpion venom intoxication render bigger and longer

contractions than those obtained in normal animais (Fig. 1).

Fig.l — Increase in amplitude and duration of muscular responses to maximal

stimuli under the action of scorpion venom (B). A, control before the venom; C and

D, decrease produced by the repetition of responses, (dei Pozo and Auguiano (1).

Simultaneous electrical recordings from the musele and its centrally cut

nerves show series of potentials in fast sequence after such single shocks are

applied lo the nerves. If the nerve is severed from the musele the repetitive

activity suddenly disappears and only the normal action potentials persist (2)

ÍFigs. 2 and 3).

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11 12 13 14 15 16 17

962

EFFECTS OF SCORPION VENOM AT NEUROMUSCULAR JUNCTION

5 mvj _

lOmseg

Fig. 2 — Repetitive activity as shovvn by

electrom'yogram. Several responses superim-

posed. Stimuli applied to the nerve. A, be-

fore the venom: B to D, aíter the injection

ot succesive doses ot scorpion venom.

I0m5«9

Fig. 3 — Simultaneous recording from

responses of sciatic nerve (above) and

gastrocnemius musele (below) to sti¬

muli applied to the nerve. A, before;

B, after the injection of a large dose

of scorpion venom; C, 5 minutes later;

D, after curare.

The present study was carried out wilh the purpose of analyzing the origin

and propagalion of the described repetitive electrical potentials.

Iii cats anesthetized with phenoharhital, electrical reeords were taken from

gastrocnemius muscles and from single fibers of the ventral spinal rools at L T .

The roots were centrally cut. Electrical stimuli were applied to the sciatic nerve.

The scorpion venom from Centruroides suffusus suffusus Pocock was injected

intravenously or directly into the recorded musele.

The repetitive potentials appeared first in the musele and after several se-

conds or in some cases minutes, in lhe anterior rools. When the sciatic nerve

was peripherally cut the repetitive discharges, disappeared. In several experi-

ments recordings eleetrodes were applied to the posterior spinal roots hut no

potentials were found (Fig. 4).

According to lhese resulls lhe antidromic propagation of the repetitive im¬

pulses by the efferent fibers may he affirmed. The single fiher reeords leaves

no doubt aboul the repetitive electrical activity in the functional unit.

In other series of experiments, the effecl of the venom on the end-plate po-

tential was studied. Silver macroelectrodes were applied to lhe region of lhe

end-plates of gracilis musele of cats previously curarized.

The recorded potential was local and propagation with a great decrement

was limited lo a very short area of lhe musele. E T nder scorpion venom the

electrogram showed an initial fast component probably corresponding to immediate

nerve terminais whieh was followed by a negative wave prolonged for ahout

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Mem. Inst. Butantan

Simp. Internac.

33(3):961-96-4, 1966

E. C. DEL POZO, M. SALAS and P. PACHECO

963

Fig. 4 — Spontaneous variation oí

the discharges to peripheral single

shocks applied to the sciatic nerve

after scorpion venom. Recordings írom

a thin ventral root filament at diffe-

rent intervals.

15 lo 20 mscc. With succesive doses of the venom lliis long potential gradually

changed in polarily and increased in duration lo ahout two fold the initial time

(Fig/5).

5 mseg

A B C GH

lOmseq.

D E F

Fig, 5 — Macroelectrodes recordings from end-plate region. Gracilis of cat

under the action of scorpion venom. Records from succesive stimuli at different

times after the injection of the venom.

Large doses of venom hlocked the neuromuscular transmission. This block

ocurred many times when the electrical discharges in the nerve were very marked

even increased at lhe time of the hlock.

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964

EFFECTS OF SCORPION VENOM AT NEUROMUSCULAR JUNCTION

Jii some recent experiments intracellular recordings of the end-plate polential

were done in frogs in order lo determine the place of action of scorpion venom.

Neuromuscular transmission was previously hlocked ljy curare. The venom did

nol produce significant changes in the end-plate potenlial either in amplitude or

duration. When the dose of venom was increased the end-plate polential suddenly

disappeared. Il was tested lhat these negative records were taken wilh the intra-

cellular electrode in ils ])lace as il was shown hy the resting potential appearing

when the electrode was taken out (Fig. 6).

Fig. 6 — Intracellular recordings írom frog sartorius muscle under the action

of scorpion venom. Successive responses to shocks applied to the nerve at 2

seconds intervals. The records are to be read from the lower to the upper

part, left column first.

Edwards working in this lahoratory hy measuring lhe quantal release of lhe

transmitter from lhe pre-synaptic nerve terminais in frogs under lhe action of

scorpion venom found evidence that this action takes place at the nerve endings.

The last two series of experiments described suggest that the action of

scorpion venom at the neuromuscular junction corresponds to aclivity at lhe

nerve terminais. This aclivity could explain the repelilive discharges in muscle

and nerve previously described.

The extracellular recording of end plate potentials are always diffieult lo

analize because of lhe complex spatial arrangement of lhe elements that contri-

hute lo the potential registered.

However, is should he kept in mind that the intracellular recordings were

done in frogs and the rest of the experiments correspond to cats and other

mammals. There is always possible differences due lo the species chosen.

References

1. DEL POZO, E. C., and ANGUIANO, G., Rev. Inst. Salubr. Enferm. trop.

(Mex.), 8, 231, 1947.

2. DEL POZO, E. C., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms,

Amer. Ass. Advanc. Sei., Washington, 1956.

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Mem. Inst. Butantan

Simp. Internac.

33(3): 965-972, 1966

TOSHIO SUGA

965

42. ON THE ACTION OF BUFOGENINS AND ALLIED COMPOUNDS ON

THE INTESTINE AND OTHER SMOOTH MUSCLE ORGANS

TOSHIO SUGA

Department of Pharmacology, Faculty of Medicine, Tokyo Medicai and Dental

University, No. 5-J/7, 1 — Chome. Yushima Bunkyo — ku, Tokyo, Japan.

Pharmacological studies ou lhe active principies isolated from Ch’an Sn. lhe

dried venom of the Chinese load, revealed some interesting activities. For exam-

ple, bufalin, a coprostáne-type steroid wilh a six membered unsaturated lactone,

has a very strong local anesthetic action and, as Dr. Oleada reporled in this

Symposium, its mode of action differs markedly from that of cocaine and resi-

bufogenin, a steroid with an expoxide in 14-15 position, which has a strong

slimulating action on respiration and blood pressure. This paper reports the re-

sult of an investigation on the pharmacological actions of hufogenins which were

mainly on guinea-pig smooth muscles (intestine, blood vessels and trachea), with

lhe expectation of ohtaining some usefiil data which could throw some more lighl

on their action mechanisms.

Their effects on intestinal tissues were principally studied hy using lhe ilenm

preparation according lo the Magnus perfusion method. The resibufogenin mi-

nimum effective doses for intestinal contractions were 5 X 10~ 8 to 10 7 , and

the dose-response curve was a sigmoid up to the concentration of 4 X 10~ G

(Fig. 1), in higher coneentrations, the responses were reduced. All hufogenins

studied act sirnilarly upon this smooth muscle preparation.

This contracting action on lhe ileum preparation is depressed lo some degree

hy atropine (10 8 ) and morphine (5 X 10~ 7 ), and enhanced by eserine (10 — fl )

(Fig. 2), suggesting the participalion of Ach release in the concentration me-

chanism. As it is nol influenced hy C6 the Ach releasing effect seems nol he

ganglionic, but post ganglionic (Fig. 2).

The guinea-pig ileum inhihiting effect induced hy atropine is nol reproduced

in lhe taenia-coli preparation. In lhe latter preparation, the contracting actions

of the so ealled Ach-releasers such as nicotine and picric acid were not ohserved

(Fig. 3). This seems to indicate, therefore, that its irresponsiveness seems to

result on account of its lack of nerve tissues, which release Ach.

The kymogram of Fig. 4 shows the intestinal contractions produced hy bu-

falin and the hloeking effects of atropine. The dose-response curves obtained

with the same preparation (Fig. 4) show that bufalin has a much more stronger

activily than resibufogenin, and that g-strophanthin is less active lhan resibu¬

fogenin. Though digitoxigenin is more active than resibufogenin the latter is

more active than digitoxin. Generally lhe actions of genins are stronger and

fasler in appearance than those of the glycosides.

SciELO

11 12 13 14 15 16 17

ON THE ACTION OF BUFOGENINS AND ALLIED COMPOUNDS

ON THE INTESTINE AND OTHER SMOOTH MUSCLE ORGANS

^ v^ v /v

Action oí Resibufogenin on the small Intestine of Guinea-pig.

Fig. 2 — Dose-Response Curve of Resibufogenin.

When a eomparatively high concentration of resibufogenin is applied to the

guinea-pig ileum, relaxation follows contraction considerably faster than with the

taenia-coli preparation. Supposing that a probable Ca ++ release would parti-

ci]>ate in this mechanism, we studied the effects of nethalide (jS-hlocker), reserpine

and dibenzyline on the mode of relaxation (Fig. 5). There was no remarkable

difference of the effects after lhe pretreatment by these suhstances.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3):965-972, 1966

TOSHIO SUGA

967

I leum

2x10

-Nic Mor Nic Rs Cg Rs

■-« SxIO^Sx.O" 7 ,0' 6 2.5 xI0' 5

Taenia col i

Pic Mor Pic Atr

5xlO’ 5 5x IO' 7 IO" 8

Rs 2x10

Nic W

2xlO’ 6

Pic Atr ACh Atr

5xlO' s IO’ 8 5xlO" 8 4xlO" 4

Fig. 3 — Comparison oi the Effeot of Resibufogenin on Ileum and Taenia coli.

Based on these results, we can assume that Ach release participates from the

contracting action of bufogenins, in addition to their direct actions on the exci-

tahle memhrane of the smoolh muscle, as reported by H. J. Schatzmann et ctl.

for the taenia-coli preparation. In the phase of relaxation there seems lo be no

participation of Ca + + release, this relaxation seems to depend upon the direct

inhibitory effect exerted on the excitable memhrane.

It is known that bufogenins and allied compounds, provoke a rise in blood

pressure, as a consequence of an augmentation of peripheral vascular resistance

resulting from vasoconstriction effects. In the spirally cut aortic strip preparation

SciELO

11 12 13 14 15 16

968

ON THE ACTION OF BUFOGENINS AND ALLIED COMPOUNDS

ON THE INTESTINE AND OTHER SMOOTII MUSCLE ORGANS

Bufalin

Af ter Atropine (IO - ®)

arranged according to G. Paterson, bufogenin did not cause sucli a remarkalde

contraction as in the ileum preparations, but only a slight rise of muscular tone.

However, they potentiated the contraction induced hy epinephrine and KC1. These

phenomena resemhles the effect of Ca ++ on the K contracture (Fig. 6). The

peak of maximum effect is in the 1.5 limes concentrated Ca ++ solution. This

1, | SciELO

Mem. Inst. Butantan

Simp. Internac.

33(3):965-972, 1966

TOSHIO SUGA

969

lleum, Guinea pig

After Reserpine

Fig. 5 ■ —■ Effect of Nethalide and Dibenzyline on the intestinal contraction

produced by Bufogenin.

Fig. 6 ■— Action of Bufogenin of the

Tracheal Strip immersed in Na-free

Krebs Solution.

concentration of Ca + + augmented the contraction produced hy K. Other diffe-

rences, between the actions of bufogenins upon the blood vessels and heart tissue

were observed and will be referred to later.

The effects of bufogenins on the tracheal smooth muscle were studied on the

spirally cut tracheal strip preparation described by J. W. Constantine. In con-

trast to their effects on the aorlic preparation, where we could not observe any

1, | SciELO

970

ON tiie: action of bufogenins and allied compounds

ON THE INTESTINE AND OTHER SMOOTH MUSCLE ORGANS

relaxation, even after the application of concentrated hufogenin Solutions, in the

tracheal tissue we observed relaxation following an increase in muscular tone.

Occasionally we observed a few cases presenting only relaxation. fn the stage of

relaxation, hufogenin reduced the contractions produced by Ach and histarnine

The effects of bufogenins and g-strophanthin ou lhe guinea-pig tracheal

strip preparations are shown in Fig. 8. Though relaxation following the increase

Fig. 8 — Effects of Bufogenins and

Allied Compound on Guinea-pig’s Tra¬

cheal Preparatton.

Fig. 7 — Effects of Bufogenins on the

Tracheal contractions of the Guinea-pig.

in muscular tone are the responses induced by resibufogenin and hufalin, g-stro-

phanthin did not cause any relaxation even after using high doses. Comparing

the actions of resibufogenin and hufalin. we observed lhal the inhibitory effect

of resibufogenin is faster in appearance in spite of its weaker contracting effect.

Studying the effects of ions upon the relaxing action, I found that the in¬

crease in muscular tone disappeared in the Na + free perfusion, and only the

relaxing effect persisted (Fig. 9). H. J. Schatzmann supposed. based on the re-

port of Caldwell and Keynes, that the first contracting phase of the taenia-coli

preparation is dependent on the inhibition of Na + efflux (Na + pump) result-

ing from the action of ouhain on the outer side of the muscle cells memhrane,

and the second relaxing |>hase is caused by the inhibition of Na + influx re-

sulting from its action on the inner side of the muscle cells memhrane after the

permeation of the cells.

This hypothesis permits an explanation lor the absence of lhe contracting

phase after the perfusion of Solutions Na-deprived, and I suppose that the diffe-

rence of action among resibufogenin, hufalin and g-strophanthin shown in Fig. 9,

depeneis on the difference of the permeative properties, such as shape and size

of Iheir molecules.

A comparision of the effects of bufogenins on the various smooth muscle

tissues and rectus abdominis muscle of frogs, and their action on the complelely

depolarized memhrane in isotonic K 2 S0 4 Krebs solution is shown in Fig. 10.

In the guinea-pig ileum and trachea we could recognize the relaxation after

the application of a high concentration of hufogenin. On the other hand. in

the aortie strip, heart tissue and rectus abdominis muscle of frogs we observed

oídy the contraction without the relaxation phase. All other tissues thau the car-

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3) : 965-972, 1966

TOSHIO SUGA

971

Fig. 9 — Eífects of Bufogenins and Ca on the K- and Adrenaline-Contractions

of Guinea-pig’s and Rabbifs Arterial Strip Preparations.

diac one lost both the contracting and relaxing responses to bufogenins after K

induced depolarization. Therefore, the potentiating action of cardic steroid on

the smooth contraction depends chiefly on the direct action on the cell membrane,

while on the cardiac tissue another mechanism participates llian the direct mem¬

brane action.

The above mentioned data, regarding my investigations on the aclions of

bufogenins upon the various smooth muscle tissues, seem to indicate that the com-

paratively rapid contracting response of the ileum is due to a Ach releasing me¬

chanism. In addition to this direct stimulating action, in the other muscle tissues

examined (taenia-coli, blood vessels, trachea and frogs rectus abdominis muscle),

the cell membrane seems to be chiefly involved, and there are some differences

in the forms the cell membrane is directly actuated by the bufogenins.

SciELO

10 11 12 13 14 15

972

ON TIIK ACTION OF BUFOGENINS AND ALLIED COMPOUNDS

ON THE INTESTINE AND OTIIER SMOOTH MUSCLE ORGANS

Bufalin

Depolarization mduced After K-Deporahzation

by isotonic K Solution

5mm 4xl0' 6 8x I,6 xIO' 5 3.2x

5min 2x1o' 7- 8x 2xl0' 6 8x 32x!0' 5

5min 4xl0' 6 8x I.6xI0' s 3.2x 6.4x

0 2x10 8x 32x10

nf7 t

-S

K 2 S0 4

Fig. 10 — Actions of Bufogenin on the Smooth Muscle Tissues and

K induced Depolarization.

2 3 4 5 6 SClELO 10 2.1 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3):973-980, 1966

OSWALDO VITAL BRAZIL, JÚLIA PRADO

FRANCESCHI and EZEQUIEL WAISBICH

973

43. PHARMACOLOGY OF CRYSTALLINE CROTOXIN. I. TOXIC1TY

OSWALDO VITAL BRAZIL, JÜLIA PRADO FRANCESCHI

and EZEQUIEL WAISBICH

Department of Pharmacology, University of Campinas, Campinas, S.P., Brasil

Croloxin is a crystallline loxin isolated for llie firsl lime in 1938 from lhe

venom of lhe Soulli American rattle-snake (Crotalus durissus terrijicus ) by Slotta

and Fraenkel-Conrat (1) working at the lalioratories of Butantan Institute. It

behaved as a single pure prolein when it was examined by E. J. Cohn’s me-

thod(l), electrophoresis (2) or sedimentation and diffusion experiments in the

ultracentrifuge (3). Its molecular weighl was estimated to be 30.000 (3). Ho-

wever, as Neumann and Habermann (4. 5) bave more recently shown, croloxin

can be separated into two components by column chromatography. One of these

exhibits the same toxicity as croloxin but is devoid of known enzymatic acti-

vities. It was called “crotactin” by Neumann and Habermann. The other is

a phospholipase A. wbicb according to the German authors, presents only a

little loxicity. Tbus crotoxin is now believed to be made up from two proteins

probably United by ionic bonds.

Pharmacological data concerning crotoxin are very scarce. In fact, little

more than its high toxicity for mice (6) and its stimnlatory effect on the iso¬

lated guinea-pig ileum (1), was known about ils pharmacological properties. The-

refore, a pharmacological study of crotoxin was undertaken and will be presented

in tbis and in the following Communications.

Material and methods

The venom used in tbis research carne from a serpentarium at Catalão.

Goiás. It was a dried pool of C. d. terrijicus venom devoid of crotamine.

Both methods devised by Slotta and Fraenkel-Conrat (1) for separating cro¬

toxin were utilized. Nearly all batches of it were obtained in crystalline form.

Crotoxin, as well as the venom was dissolved in saline immediatly hefore use.

However, in some experiments a solution kept in the refrigerator at 4°C for

no more than 7 days was also employed.

I he median lethal doses of crotoxin were determined in mice, rats, pigeons,

guinea-pigs and rabbits and lhose of the venom, in mice and rats. Four dose

leveis spaced in a geometric progression (common ratio 1.5) were employed.

Six animais per dose were used, except in the determination of the LD 50 for

pigeons. In this case, three animais per dose were employed.

This study was supported by a grant írom the “Fundação de Amparo à Pesquisa

do Estado de São Paulo”.

SciELO

10 11 12 13 14 15

974

PlIARMACOLOGY OF CRYSTALLINE CROTOXIN. I. TOXICITY

The intravenous route was used for lhe determination of lhe median lethal

doses of crotoxin and lhe venom for mice (17-21 g). In addition. crotoxin

median lethal dose hy subcutaneous route for miee was also determined. An

Agia micrometer syringe oulfil was used in this case to inject lhe crotoxin

solution. In other determinations, eroloxin or lhe venom was administered by

intravenous, inlraperitoneal or intramuscular route. Pigeons, guinea-pigs, mice

and rats were observed for 48 hours. The rabbits were discarded after lhe fifth

day. The median lethal doses and their 95 of -100 confidence intervals were

calculated hy lhe procedure described hy Weil(7).

The signs evoked by crotoxin in dogs were investigated in 36 conscious

animais. The inlravenous route was employed. The animais were observed for

5 lo 9 days. Ten dogs, however, were anaesthetized 24 or 48 hours after ero¬

loxin administration in order to prepare them for lhe study of neuromuscular

transmission. To some of these animais artificial respiration was givcn after

their spontaneous respiration had ceased. An automatic Takaoka respirator in

connection with an air or oxygen cylinder was used. The urine of nearly all

dogs was examined for alhumin and liaemoglohin. Diuresis was studied in 13

female animais whieh were kept for this purpose in metaholic cages for 5 or

more days afler crotoxin injection. In most instances, Controls were performed by

collecting lhe urines during the four days thal preceded crotoxin administration.

Sixteen of the dogs were used for the determination of the crotoxin median

paralysing dose. Four dose leveis in a geometric progression with a common

faetor of 1.25 were employed. The ED 50 was also calculated according to Weil

procedure. Three unanaesthetized dogs were also intravenously injected with the

venom.

The motor effects evoked hy crotoxin and gallamine (Flaxedil) were com-

pared in monkeys by using two Cebus sp.. lloth substances were intravenously

administered.

Conscious cats were injected with crotoxin by intravenous and inlra (cere-

bro) ventricular routes. Three animais were used in both instances. Crotoxin

was injected in one of the brain lateral ventricules througb a Feldberg cannula

inserted some days before.

Hesults

Crotoxin showed itself to be more toxic than the venom for both mice and

significantly (Table 1).

Great speeies variahility was found in lhe sensitiveness to crotoxin. Pigeons

proved to be extremely sensilive while rats were very resistent to its lethal action

(Table I). Crotoxin mean lethal dose by subcutaneous route for mice was 2.16

(1.15 to 4.02) times grealer than lhat determined by intravenous administration.

The crotoxin median paralysing dose for dogs was estimated to be 138 (106 to

179) rncg/Kg. Its mean lethal dose for this animal speeies was probably grealer

than 200 mcg/Kg (Table II).

Crotoxin given by the usual parenteral routes of administration elicited pa-

ralysis in all animal speeies in which it was injected. In nearly all (rats, gui¬

nea-pigs, rabbits, cats, dogs and monkeys) of them, the motor effects which

began by muscular hypotonia and muscular weakness and evolved to flaccid pa-

ralysis, were identical with those observed in animais injected whith curare. The

order of involvement of the various muscular grou])s was also the same in both

2 3

5 6

11 12 13 14 15

Mcm. Inst. Butanlan

Simp. Internac.

33(3): 973-980, 1966

OSWALDO VITAL BRAZIL, JÚLIA PRADO

FRANCESCHX and EZEQUIEL WAISBICH

975

TABLE I — CROTOXIN AND CROTALUS DURISSUS TERRIFICUS VENOM MEDIAN

LETHAL DOSES AND THEIR 95 OF 100 INTERVALS FOR VARIOUS ANIMAL SPECIES

Animal

Route of

administration

Crotoxin LD 50

(mcg/Kg)

Venom LD 50

(mcg/Kg)

Venom LD n0 /

Crotoxin LD. 0

Pigeon

Intravenous

2.17

(1.35 to 3.48)

—

Guinea-pig

Intramuscular

(29 to 50)

—

Mouse

Intravenous

(66.9 to 101.4)

168.5

(135.7 to 209.85)

2.05

(1.34 to 3.14)

Subcutaneous

177.5

(117 to 269)

—

Rabbit

Intravenous

110.23

(85.64 to 141.9)

—

Rat

Intraperitoneal

756

(564 to 1010)

1950

(1950 to 2120)

2.58

(1.77 to 3.75)

cases. The similarity of signs evoked by croloxin and curare vvas very convin-

cingly shown in monkeys (Fig. 1). Pigeons and mice were exceptions. In these

animais, oídy large doses produced lhe curare-like paralysis. Small doses elicited

an ascendenl lype of paralysis. Mice, for instance, injected wilh a dose of

croloxin near ils LD 30 frequently showed after a long delay, paralysis of their

hind limbs while lhe neck and fore limh muscles were not affected.

Fig. 1 — From left to right: (1) a monkey 15 hours after receiving 100 meg/Kg

of crotoxin i.v., (2) lhe same monkey 30 hours later after neariy complete recovery

and (3) another Cebus sp 3 to 5 min. after being intravenously injected with 300

mcg/Kg of gallamine (Flaxedil). Ptosis of the eyelids and of the jaw, paralysis

of the muscles of the neck as well as a generalized loss of muscle strength occurred

in both monkeys. They also became aphonic, could not swallow and presented dia-

phragmatic respiration. Consciousness in both animais seemed to be preserved.

SciELO

976

PHARMACOLOGY OF CRYSTALLINE CROTOXIN. I. TOXICITY

The diaphragm was one of lhe last muscles lo become paralysed in croloxin

intoxication. When it ceased contracting, lhe animais could lie maintained alive

nnder artificial res])iralion. This was demonstrated in cais and tlogs. Croloxin

paralysis proved to be reVersible in all animais except rabbits. These animais

always died before recovering from lhe paralysis even when it was not complete.

The first signs of lhe molor effects caused hy not too large a dose of cro-

toxin appeared afler a long delay and the paralysis lasted for long periods of

time. In dogs, for instance , the onset of loss ol muscle strength generally

occurred from 2 to 4 hours after the intravenons injection of 160 to 250 mcg/Kg

of croloxin and the animais became paralysed for 48, sometimes 72 hours, or

even more. However, the lag between crotoxin administration and the onset

of paralysis was usually a little shorler in lhe small laboratory animais. In

addition, the duration of paralysis in the animais did not last so long.

During crotoxin paralysis consciousness and sensitivity seemed to be pre-

served. Paralytic dogs, for instance, shook their tails when the observer whislled

or tiekled lhem, or made efforts to move when their skin was pinched. Pre-

servation of consciousness was also testified by the reaction that a paralytic dog

presented at the incidental approach of a cal. It became furious making des-

perate efforts to fight with the cat.

TABLE II — SOME EFFECTS OF CROTOXIN IN DOGS

Effects

Doses

of crotoxin

(mcg/Kg)

200

160

128

102.4

Defaecation

2/4

3/4

2/4

1/4

Vomiting

3/4

2/4

0/4

1/4

Salivation

2/4

1/4

0/4

Sllght muscular hypotonia (1)

4/4

Advanced muscular hypotonia and con-

siderable loss of muscle strength (2)

4/4

2/4

3/4

2/4

Flaccid paralysls(3)

4/4

2/4

1/4

Death

1/4

0/4

1/4

0/4

(1) Some incoordination and weakness

in muscular i

iction were

perceivable

when the

dogs were forced into locomotion.

(2) The dogs could not support themselves on their limbs because of the loss of mus¬

cular strength.

(3) The dogs lay dow helpless on one of their flanks.

Besides paralysis, other effects were observed in unanaesthetized dogs. Prior

to lhe appearance of paralysis, one or more of lhe following effects frequently

occurred: defaecation, vomiting, salivation (Table II). During the paralysis and

after its partial or complete regression, or even in its ahsence, albuminúria, haemo-

globinuria and oligúria or anuria (Table III) were verified. Opacity and exfo-

liation of the córnea as well as vomiting occurred in a few dogs after the third

day of crotoxin administration. Vomiting and salivation were also observed in

cats before lhe onset of paralysis.

I, | SciELO

Mem. Inst. Butantan

Simp. Internar.

33(3):973-980, 1966

OSWALDO VITAL BRAZIL, JOLIA PRADO

FRANCESCIII and EZEQUIEL WAISBICH

977

TABLE III

EFFECT OF CROTOXIN ON DIURESIS

Dog (Ç)

Weight

(Kg)

Crotoxin

Dose

(mcg/Kg)

D i u r e s i s

Before crotoxin

administration

Mean of 4 days

(mi/day)

After crotoxin

administration

Mean of 5 days

(ml/day)

Per cent

reduction

11.0

250

—

61.0

5.7

250

—

36.0 *

—

5.7

250

—

49.0

—

8.1

250

—

53.0

—

6.2

250

183.0

62.0

66.1

6.7

160

—

94.0

—

7.9

160

149.0

200.0

Inereased

6.4

160

212.5

109.0

48.7

7.9

160

312 5

168.0

46.2

6.1

128

2C0.0

137.0

16.5

5.9

128

337.5

52.0 **

84.5

4.9

128

140.0

105.0

25.0

6.5

102.4

185.0

84.0

54.6

Mean of 2 days.

Mean of 4 days.

Deaths of the dogs generally look place within the first 48 hours of cro-

toxin injection. They were due to respiratory arrest. However. a few animais

died afterwards when the paralysis vvas in regression. There vvas post-mortem

evidences that in these cases sliock was the cause of death. Renal lesions were

demonstratcd in the kidney of nearly all dogs injected with crotoxin. They vvill

he reported in a separate eommunication. Hemorragic foci were never seen in

the meninges or hrain suhstance of the dogs killed by crotoxin action.

When crotoxin (50 mcg) was injected hy intra (cerebro) ventricular route

in cats (1 to 5.5 Kg) seizures of clonic eonvulsions ending in death were pro-

duced 2 to 3 hours after its administration (Fig. 2). Taehypnoea, slight clonic

movements in the paws, salivation and vomiting were observed prior to the con-

vulsive seizures.

The venom evoked signs in mice which were similar lo lhose produced hy

crotoxin. In rats and dogs, however, the symptomalology differed frorn that

elicited hy crotoxin, chiefly in the latter species.

Rats injected with venom hecame very flaccid hui never showed complete

paralysis as the rats under action of crotoxin. Respiratory arrest seemed lo take

place before the motor effects attained that point.

The venom (250 mcg/Kg. i.v.) elicited the following effects in dogs:

SciELO

10 11 12 13 14 15

978 PHARMACOLOGY OF CRYSTALLINE CROTOXIN. I. TOXICITY

Fig. 2 — This cat (1.3 Kg) vvas injected with 50 mcg of erotoxin by intra (cere-

bro) ventricular route. The seizures of clonic convulsions (fotograph) appeared 2

hours and 20 minutes after erotoxin administration. Tachypnoea, salivation and

vomiting oecurred prior to the convulsive fits.

1. Immecliately after its injection, tachypnoea, defaecalion, micturition as

well as a very short period of equilibrium loss, and nystagmus were observed.

A short lime later défaecation, vomiting and salivation oecurred.

2. After a delay of 1 or more hours, seizures of tonic-donic convulsions

ending in dealli or clonic convulsive movements, usually of the limbs associated

with some degree of muscular hypolonia, and loss of muscular strenglh happened.

All three dogs died: one of them 2 hours after lhe injection of lhe venom,

another between lhe first and the second day and a third dog in from the third

to the fourth day. Post-mortem, this last animal showed small haemorragic foci

in lhe pia mater and zones of congestion and oedema in lhe lungs, besides inten-

sily congested abdominal viscera. The mucosa of ils intestines was congested

and haemorragic.

Discussion

The results obtained in this research point out the following order of sus-

ceptibility to lhe lethal action of erotoxin: pigeons > guinea-pigs > mice > rab-

bits > rats (see Table 1). lt seems lo be important, lherefore, to inquire if this

same order of sensiliveness occurs in relation lo the venom. Our results showed

Mem. Inst. Butantan

Stmp. Internac.

33(3) :973-9S0, 1966

OSWALDO VITAL BRAZIL, JÚLIA PRADO

FRANCESCHI and EZEQUIEL WAISBICH

979

that lhe venom was also mueh more toxie for mice llian for rats. Rrazil atui

Pestana (8), on lhe other hand, encountered lhe following valties for lhe mini-

mal lethal doses of lhe venom: I mcg (3.3 mcg/Kg, i.v.) for pigeons, 60 mcg/Kg

(i.m.) for guinea-pigs and 250 mcg/Kg (i.v.) for rahhits. Il can he inferred»

lherefore. frotn these restills that the order of susceplihility lo lhe venom among

pigeons, guinea-pigs, mice, rahhits and rats, is the same as that shovvn lo cro-

toxin. The venom was found lo he less loxic than crotoxin for mice and rats

(see Table I). The figures given hy Brazil and Pestana (8) refer lo the mini-

mal lethal doses of the venom and lherefore are not strictly comparahle with the

valties of the median lethal doses we determined for crotoxin. Nevertheless the

analysis of their data suggests that the venom is also less loxic than crotoxin

for pigeons, guinea-pigs and rahhits. As, in addition, the signs evoked hy small

doses of lhe venom and crotoxin in these animais are very close or even in-

distinguishable, il can he concluded that crotoxin is the component which pro-

duces the signs of envenomation and causes dealh in pigeons, guinea-pigs, mice

and rahhits when small doses of the venom are injected. In rats which are

very resistanl to crotoxin (and also to the venom), other venom componenls

participate in lhe envenomation. altering lhe symptomatology elicited hy crotoxin.

For dogs, the venom seems to he as loxic as crotoxin and, in addition, evokes

effects. when given hy intravenous route, which are very different fronr those

produced hy that suhstance. lt gives rise, for instance, to convulsions and, so-

metimes, lo small haemorragic foci in the pia. as first pointed out hy Brazil (8, 9)

and eonfirmed in this study. The convulsive action of the venom could he duo:

(a) to the penetration of crotoxin in the hrain interstitial spaces after lhe

breakdown of the hlood-hrain harrier produced hy anolher component of the

venom, or (h) to a toxin of the venom different from crotoxin. The first hypo-

thesis is suggested hy the convulsive action of crotoxin when introduced hy intra

(cerehro) ventricular route. The second one is based on lhe separation of a

fraction from the venom which caused convulsions not only in dogs hut in mice

as well (unpublished data).

Several signs of systemic envenomation produced in man hy the bite of lhe

South American ratlle-snake were ohserved in lhe animais injected with cro¬

toxin, specially monkeys and dogs. Such were ptosis of the eye-lids and of the

jaw, weakness of the neck and other skeletal muscles as well as general muscle

paralysis. Difficulty in swallowing occurred in dogs and monkeys and is a

complaint of some patients. Plosis of the eye-lids is one of the first signs of

envenomation and is probably displayed hy nearly all patients bilten hy C. d.

terríficas. Vomiting wilhin lhe first hours of lhe bile also occurs and was fre-

quently ohserved in dogs injected with crotoxin (Table 11). Albuminúria, hae-

moglohinuria, oligúria and even anuria, which were effects produced hy croto¬

xin in dogs. are frequently ohserved in patients (10). However, participation of

other venom components in the genesis of renal disturhances and vomiting is not

excluded.

SüMMARY

1. The toxicity of crotoxin was compared with that of the venom of Crotalus

durissus tervificus by determining their median lethal doses for mice (i.v.) and

rats (i.p.). Crotoxin showed itself to be more toxie than the venom for both

animal species.

2, The median lethal doses of crotoxin for pigeons (i.v.), guinea-pigs (i.m.)

and rabbits (i.v.) were also determined. The following order of susceptibility to

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PHARMACOLOGY OF CRYSTALLINE CROTOXIN. I. TOXICITY

its lethal action was encountered: pigeons guinea-pigs mice rabbits rats.

lt seems that the order of sensitiveness to the venom is the same.

3. Crotoxin evoked paralysis in all animal species used. In nearly all of

thern the paralysis was similar to that elicited by curare in the degree of flacci-

dity shown by the skeletal muscles and in the order of involvement of the various

muscular groups. Artificial respiration maintained the animais alive after spon-

taneous respiration had ceased. Consciousness and sensitivity seemed to be pre-

served in animais presenting crotoxin paralysis.

4. Besides paralysis, defaecation, vomiting, salivation, albuminúria, haemoglo-

binuria, oligúria and in some instances opacity and exfoliation of the córnea,

were observed in dogs.

5. When administered by intra (cerebro) ventricular route in cats, crotoxin

elicited convulsions instead of paralysis.

6. There are evidences indicating that the lethal effect of small doses of

the venom in pigeons, guinea-pigs, mice, rabbits and rats is due to crotoxin. In

dogs, however, it seems that death is produced by a component of the venom

different from crotoxin.

7. In human beings several signs observed after the bite of C. d. terrificus.

can be assigned to crotoxin action.

References

1. SLOTTA, K„ and FRAENKEL-CONRAT, H., Mem. Inst. Butantan, 12, 505„

1938-39.

2. LI, C. H., and FRAENKEL-CONRAT, H., ./. amer. chem. Soc., 64, 1586, 1942.

3. GRALÉN, N., and SVEDBERG, TH., Biochem. J ., 32, 1375, 1938.

4. HABERMANN, E., Biochem. Z., 329, 405, 1957.

5. NEUMANN, W. P„ and HABERMANN, E„ Biochem. Z„ 327, 170, 1955.

6. SLOTTA, K., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms. Amer.

Ass. Advanc. Sei., Washington, 1956, pp. 253-258.

7. WEIL, C. S„ Biometrics, 8, 249, 1952.

8. BRAZIL, V., e PESTANA, B. R., Rev. méd. S. Paulo, 12, 415, 1901.

9. BRAZIL, V., Rev. méd. S. Paulo, 4, 296, 1901.

10. WAJCHENBERG, B. L., SESSO, J„ and IN AGUE, T„ Rev. Ass. méd. brasil.

1, 179, 1954.

2 3

5 6

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Simp. Internac.

33(31:981-992, 1966

OSWALDO VITAL BRAZIL

981

44. PHARMACOLOGY OF CRYSTALLINE CROTOXIN. II. NEURO-

MUSCULAR BLOCKING ACTION

OSWALDO VITAL BRAZIL

Department of Pharmacology, University of Campinas, Campinas, S.P., Brazil

In the precedent communication (11. it was shown that the motor signs

observed in animais injected witli crotoxin are very close to lliose evoked liy

curare. This snggested a peri|)heral origin for the paralysis caused by crotoxin

and incited us to invesligate neuromuscular transmission in animais to which

ihis substance was administered. In the present paper, evidence is given indi-

cating that block of neuromuscular transmission is really lhe cause of the para¬

lysis. Some characteristics of the neuromuscular blockade elicited by crotoxin

was also investigated as well as ganglionic transmission in animais injected with

this venon component. In addition the effect il produces on the sensitiveness

of slriated muscle to acetylcholine or potassium was studied on the isolated and

cronically denervated rat hemi-diaphragm.

Material and methods

In order to decide if neuromuscular transmission is hlocked or not in lhe

animais showing the paralysis evoked by crotoxin, 10 conscious dogs were inlra-

venously injected with 0.2 to 0.5 mg/Kg of this substance. Twenty-four or

forty-eight hours thereafter, they were anaesthetized with sodium pentobarbital

(20 mg/Kg, i.v.) and prepared as follows. A drill was inserted in the superior

third of the tibia of one of tbe legs and attached through a special adjustable

block to a upright fitted on a iron base plate in order to immobilize the limb of the

animal. The peroneal nerve was exposed in the upper antero-lateral aspect of

the leg and sectioned. To its distai end platinum electrodes were attached. The

tendon of the tibialis anterior muscle was then exposed. sectioned. and connected

by means of a strong thread to a flat spring Steel myograph of a Brown-Schuster

myograph apparatus. Two needle electrodes were inserted in the muscle, one

in its belly. the other near the tendon. The trachea was exposed and cannulated.

A tracheal cannula provided with two upper lateral outlets was used. In most

experiments one of these was put in connection with a Marey tambour in order

to record the respiratory movements of the animal. A François Franck cannula

ivas then inserted in one of the carotids for recording the arterial blood pressure

(hrough a Ludwig’s manomeler. A polyethylene cannula was inserted in lhe

femoral vein of the opposite side to the prepared limb for intravenous adminis-

tration of drugs.

This investigation was supportec! by a grant from the “Fundação de Amparo à Pes¬

quisa do Estado de São Paulo.

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PHARMACOLOGY OF CRYSTALLINE CROTOXIN.

II. NEUROMUSCULAR BLOCKING ACTION

Nerve stimulation was carried out wilh supramaximal shocks of 0.2 msec

duration delivered at a rale of 30 per minute. When lhe muscle did not respond

lo stimuli of up to 100 v. applied lo the nervo, it was assumed that transmission

was eompletely blocked. Stimuli of a higher (50 c/s) frequency were also em-

ployed for short I 10 to 40 seconds) periods of stimulation. Direct muscle sti-

mulalion was made willi shocks of 2 msec. duration and 50 to 100 v. The stimuli

used were square wave pulses from a Grass S 4 Stimulator.

Neostigmine methylsulfate (0.1 mg/Kg) and calcium chloride (0.5 ml/Kg

of a 10% solution, as well as a 10% solution of sodium hiearhonate, were intra-

venously injected in some dogs.

The neuromuscular blocking action of crotoxin was also studied in cats.

Sixteen animais were anaesthetized with sodium pentobarhital (30 mg/Kg, i.v.)

and prepared as descrihed for dogs. Nervo stimulation was made with supra¬

maximal pulses of 0.2 msec duration delivered at a rale of 6 per minute. Shocks

of 2 msec duration and 50 to 100 v. were used for direclly stimulating the

muscle. Large doses (0.5 mg/Kg and 1 mg/Kg, i.v.) of crotoxin were used in

order to obtain neuromuscular blockade within the first three hours of its admi-

nistration. The effect produced by neostigmine methylsulfate (0.1 mg/Kg, i.v.),

edrophonium chloride (0.5 mg/Kg, i.v.) and succinylcholine chloride (0.02 or

0.05 mg/Kg, i.v.) on lhe neuromuscular blockade was investigaled.

Ganglionic transmission was studied in four of the cats which were utilized

in lhe investigation of lhe neuromuscular action of crotoxin. The vagus-sympa-

ihetic trunk was exposed and sectioncd. In two cats, the sympathctic chain was,

■ n addition, separated from lhe vagus nerve. Platinum electrodes were then

attached to the upper end of either the sympathetic chain or lhe whole vagus-

sympathctic trunk. The head of the animal was firmly fixed by a cal holder

and the nictitating membrane connected with a Starling heart lever. Pulses of

0.2 msec duration and 2 to 5 v. delivered at a rate of 5 per second were used.

Arterial blood pressure was also recorded.

Artificial respiration was employed whenever necessary.

Crotoxin-acetylcholine and crotoxin-potassium antagonism were investigaled

on the isolated and cronically denervated rat hemidiaphragm preparation. The

lefl phrenic nerve of rats was sectioned under ether anaesthesia as descrihed

in a previous paper (2). Afler 25 to 35 days, the rats were anaesthetized with

ehloral hydrate (300 mg/Kg, i.p.) and bled. The denervated hemi-diaphragm

was then removed and suspended in a organ bath containing 8 ml of Tyrode

solution with 0.2 per cent glucose at 37"G. The preparation was aerated with

a mixture of 95% CL and 5% CCL. The contractures were recorded wilh an

isotonic lever. Logarilhmically spaced doses (eommon ratio 2) of acetyleholine

or potassium chloride were added to the bath and allowed to act for 30 lo 40

seconds. Afler each addition of acetyleholine or potassium chloride the bath

fluid was changed four limes and the preparation left for 10 minutes. The

following doses of crotoxin were used in the experiments with acetyleholine:

0.5 X 10 _íi , 10—, 2 X 10 — 4 X 10—, In the experiments with potassium,

only one dose (2 X 10— 8 ) of crotoxin was employed. AI1 contractures were ex-

pressed as percentages of the contracture obtained by immersing the preparations

in a 0.1M K 2 S0 4 solution.

The crotoxin used in this investigation was prepared as indicated in lhe

precedent paper (1).

2 3

5 6

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33(3) : 981-992, 1966

OSWALDO VITAL BRAZIL

983

Results

Block of neuromuscular transmission was demonstrated in all of the 10 dogs

which had been injected 24 or 48 hours before the experiment with crotoxin

(Tahle I). The hlockade was complete in 4' of them and partial (Fig. 1) in

the remaining. Neostigmine, calcium chloride and sodinm bicarbonate did not

remove the neuromuscular hlockade nor ameliorate the transmission. A partial

recovery of muscle responses or a great increase of them always appeared after

TABLE I — NEUROMUSCULAR TRANSMISSION * AND ARTERIAL BLOOD

PRESSURE OF DOGS 24 OR 48 HOURS AFTER INTRAVENOUS

INJECTION OF CROTOXIN

Crotoxin dose

(mcg/Kg)

Hours after

injection

Neuromuscular

blockade

Arterial blood

pressure

(mm Hg)

500

complete

170

500

partial

120

500

partial

116

250

complete

104

230

partial

220

250

complete

250

partial

—

250

complete

230

200

partial

186

200

partial

160

* Sciatic nerve-tibialis anterior muscle preparation.

tetanic stimuli were applied lo the nerve (post-tetanic facilitation, Fig. 1). Ces-

sation of lhe spontaneous respiration occurred in three dogs in early stages of

the experiments; artificial respiration secured their survive. All dogs showed

flaccid paralysis and their respiration was diaphragmatic before being anaes-

thetized.

In the cal experiments, crotoxin produeed complete or partial neuromus¬

cular hlockade within the first 3 hours of ils administration. Inhibition was

complete at that time in 3 out of the 9 animais injected with 0.5 mg/Kg and

in 5 out of the 7 to which 1 mg/Kg of crotoxin was administered. Neostigmine

was without effect on the neuromuscular block. Edrophonium increased the

partially inhihited responses of the tibialis anterior muscle. Flowever, the effect

was shorl-lived (Fig. 2). Succinylcholine also increased the twitches in 6 out of

8 experiments (Fig. 3). This effect was produeed by doses of the depolarizing

drug which caused partial hlockade before crotoxin administration (Fig. 3).

The stimulation of the cervical sympathetic trunk elicited contractions of

the nietitating membrane in all four cats. In two animais, the contractions

attained a very good tension (Fig. 4). These cats had been injected with 0.5

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PIIARMACOLOGY OF CRYSTALEINE CROTOXIN.

II. NEUROMUSCULAR BLOCKING ACTION

iUülüLlMlüllllilHimii

Fig. 1 — Dog, 7 Kg, sodium pentobarbital, Records of arterial blood pressure and

ccntractions of tibialls anterior muscle. The peroneal nerve vvas stimulated at A

and B with supramaximal shocks of 0,2 msec duration delivered at a rate of 30 per

minute; at signals, shocks of a high (50 cycles/second) frequency vvere applied to

the nerve for 10 seconds. At C, direet stimulation (pulses of 100 v. and 2 msec

duration delivered at a rate of 30 per minute) of the tibialis anterior muscle.

This animal had been intravenously injected 48 hours before the experiment with

0.25 mg/Kg of crotoxin. It was paralytie vvhen anesthetized with 20 mg/Kg of

sodium pentobarbital to be prepared for recording arterial blood pressure and the

contractinns of the tibialis anterior muscle.

mg/Kg of crotoxin. In the other Iwo, the contractions were somewhat weakcr

hut had no tendency to decrease in tension during lhe experiments. Therefore.

il seems lliat there was also no ganglionic hlockade in these animais vvhicli had

heen injected willi ] mg/Kg of crotoxin. In all four cats, lhe res|)onses of the

tihialis anterior muscle were already hlocked and in three of them, the spon-

taneous respiration, in addition, had ceased hefore the beginning of the expe¬

riments.

Crotoxin produced a shift of the dose-response curve for acetylcholine to

the right. A slight decline of the slopes of the curves occurred (Fig. 5). The

inhihitory effeel produced hy crotoxin on the acetylcholine contractures could

not he removed hy washing repeatedly the preparation. However, some decrease

of it was obtained. The dose-response curve for potassium (Fig. 6) was slightlv

shifted to the left hy 2 X 10—“ of crotoxin. The contractures elicited hy ace-

tylcholine or potassium after Ireatment of the muscle with crotoxin were not so

sustained as hefore.

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33(3) :981-992, 196B

OSWALDO VITAL BRAZIL

985

Fig. 2 — Cat, 4 Kg, sodium pentobarbital. Records of arterial blood pressure and con-

tractions of the tibialis anterior muscle. The peroneal nerve was continuously stimu-

lated with supramaximal shocks of 0.2 msec duration delivered at a rate of 6 per

minute. At E, direct stimulation (shocks of 100 v., 2 msec timed 10 seconds apart)

of the muscle was done. At Cr., 1 mg/Kg of crotoxin and at ED, 0.5 mg/Kg of edro-

phonium vvere intravenously injected. The records at B, C, D, and E were taken 70,

105, 135 and 150 minutes after A.

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PHARMACOLOGY OF CRYSTALLINE CROTOXIN.

II. NEUROMUSCULAR BI.OCKING ACTION

Discussion

1 liis study showed thal thc‘ neuromuseular hlockadc produced hy crotoxin

can per se explain llie paralysis oliserved in lhe animais injected with il. This

does not mean, of course, lhal an action of crotoxin on lhe central nervous

systcm does nol contrihute lo lhe paralytic effect. However, lhe following residis

do not seen to support this hypothesis. First, llie experiments concerning lhe

effects evoked hy lhe inlra (cerehro) ventricular administration of crotoxin (1)

pointed out lhat lliis suhstance exerts a stimulating action, not a depressing one.

if il gains access to lhe hrain inlerslitial fluid. Secondly, consciousness and

sensihility seemed to he preserved in lhe animais showing lhe paralysis evoked

hy crotoxin. Thirdly, lhe order of involvement of lhe various muscular groups

during crotoxin intoxication was similar lo lhat elicited hy drugs vvhich act

peripherally, and differed, lherefore, from lhat produced hy compounds Iike

mephenesin vvhose loci of action are in lhe central nervous System. Therefore,

mm Hg

Fig. 3 — Cat, 2.4 Kg, sodium pentobarbital. Records of respiration, arterial blood

pressure and the contractlons of the tibialis anterior muscle. The peroneal nerve

was continuously stimulated with supramaximal shocks of 0.2 msec duration de-

livered at a rate of 6 per minute. Direct stimulation of the muscle (at D) was

done with pulses of 100 v.„ 2 msec timed 10 seconds apart, after complete neuro-

muscular block had occurred. At A, 0.05 mg/Kg of succinylcholine was intravenously

injected before crotoxin administration; at B and C, the same dose of the depola-

rizing drug was injected 105 and 122 minutes after the intravenous administration

of 1 mg/Kg of crotoxin. Artificial respiration (at C and D) was given after the

spontaneous respiration had ceased.

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10 11 12 13 14 15

v\rAw.v.'.^

Fig. 4 — Cat, 2.5 Kg, sodium pentobarbital. 1. Records of arterial blood pressure and

ccntractions of the tibialis anterior muscle. The peroneal nerve was stimulated with

supramaximal shocks delivered at a rate of 6 per minute. B, C, D, E and F are

records taken 30, 60, 90, 120 and 150 minutes after the intravenous administration

of 0.5 mg/Kg of crotoxin. 2. Records of arterial blood pressure and contractions of

the nictitating membrane. The cervical sympathetic Chain was stimulated with shocks

of 2v, 0.2 msec, delivered at a rate of 5 per second; the stimulations were done for

15 seconds periods followed by periods of 1 second rest. The records of the contractions of

the nictitating membrane were taken nearly 4 hours after the administration of crotoxin.

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988

PHARMACOLOGY OF CRYSTALLINE CROTOXIN.

II. NEUROMUSCULAR BLOCKING ACTION

it can be concluded thal in all probability the paralysis caused l>y crotoxin is

exclusively due lo an interruption of neuromuscular transmission.

The blockade produced by crotoxin is of the non-depolarization type as

can be inferred from a comparison of the results ohtained in ihis research with

lhose given by lhe study of the depolarizing drugs (3, 4). Edrophoninm and

succinylcholine, for instance, did not deepen lhe blockade as they would do if

it was caused by a persistent depolarization of lhe end-plate region of lhe

rnuscle fibres. Instead, both drugs increased although only to a small degree the

partially blocked response of the cat tihialis anterior muscle. Furthermore, during

crotoxin neuromuscular block, tetanization of the motor nerve was follovved by

a post-tetanic partial relief of it, a result which also would not have occurred if

transmission was interrupted by a depolarization blockade. The same conclu-

sion can be drawn from the absence of muscle fasciculations in cats and other

as from the lack of any stimulating action

rat hemi-diaphragm. Depolarizing drugs

skeletal muscles, not to flaccid paralysis.

animais injected with crotoxin as well

of this substance on the denervated

in birds give rise to contracture of

Small doses of crotoxin did not elicited

j>aralysis of the curare type in pigeons.

as previously reported(l), a flaccut

However, the symptoms presented by

Fig. 5 —- Dose-response curves for acetylcholine ln the denervated rat hemi-diaphragm

preparation in the absence of crotoxin and in the presence of various concentrations

(0.5 X 10 — “, 10 — “, 2 X 10 — “ and 4 X IO—») of it. Each dose-response curve was obtained

in a diferent denervated diaphragm as the effect evoked by crotoxin was, for praticai

purposes, irreversible.

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Mem. Inst. Butantan

Simp. Internac.

33(3):981-992, 1966

OSWALDO VITAL BRAZIL

989

Fig. 6 — Dose-response curves for potasslum chloride obtained in the denervated rat

hemi-diaphragm in the absence of crotoxin and in the presence of 2 x 10 — 0 of that

substance. Crotoxin potentiated the action of the potassium ions on the denervated muscle.

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990

PHARMACOLOGY OF CRYSTALLINE CROTOXIN.

II. NEUROMUSCULAR BLOCKING ACTION

lliese animais under crotoxin action were far from resembling those evoked hy

the depolarizing flrugs. On the other hand, crotoxin in large doses produced

a flaccid paralysis similar lo that evoked hy curare vvhile large doses of lhe

depolarizing drugs still give rise to contraclure of the skeletal muscles. it seenis.

lherefore, that it can confidently be concluded that crotoxin interrupts neuro-

muscular transmission hy producing a non-depolarization type of hlockade.

A non-depolarization block, in its largest acceptation, can have one or more

of the following causes: ía) a reduction in the amount of the transmitter li-

herated hy the motor nerve terminais, (b) a decrease of the sensitiveness of the

end-plate to the depolarizing action of acetylcholine and (c) a depression of

the excitahility of the musele fihre memhrane. Botulinum toxin (5), small doses

of hemicholinium (6, 7. 8) and the recently studied snake venom components,

/?- and y-bungarotoxin (9), for instance, block transmission hy the mechanism

mentioned in la) while the competitive neuromuscular hlocking drugs and pro-

hahly a-bungarotoxin (9) hy that referred lo in (b). A hlockade exclusively

produced hy a depression of the excitahility of the musele fihre memhrane is

very infrequently observed. However, it seems that an excess of calcium can

give rise to a block hy such a mechanism,, as the end-plate potencial is not

depressed during lhe hlockade elicited hy this ion(10). Large doses of pro-

caine and the local anestheties in general provoke a neuromuscular block which

is caused hy a comhination of the first Iwo or even of all three factors men¬

tioned (11, 12, 13, 14). The same can be said in relation lo an excess of

magnesium ions (15) and large doses of the antibioties of the streptomycin

group(16, 17. 18, 19), although in lhese cases the inain cause of the hlockade

certainly is a decrease in the amount of acetylcholine liberated hy the nerve

terminais. The experiments here reported do not permit to decide if crotoxin

diminishes the amount of lhe transmitter liberated at the end-plate hy lhe nerve

impulses. However, it can he inferred from the crotoxin-acetyleholine antagonism

ievealed in the denervated hemi-diaphragm preparation of the ral that the main

cause of the hlockade produced hy crotoxin is very probably due to a decrease

in lhe sensitiveness of the end-plate to the depolarizing action of acetylcholine.

This mechanism of hlockade seems also to he demonstrated hy the reversion of

lhe succinylcholine effect in cats showing a parlial neuromuscular block evoked

hy crotoxin.

If crotoxin blocks the action of acetylcholine in the denervated musele and

at the end-plates hy reacling wilh the acetylcholine receptors or al some other

point of the musele fihre memhrane can not yel he decided. The type of shift

produced in the dose-response curve for acetylcholine suggests that both mecha-

nisms of action operate. An action on the acetylcholine receptors is also hinted

hy the similarity of lhe order of involvement of the various muscular groups in

the animais injected with crotoxin or curare. In any case lhe fixation of cro-

loxin on lhe specific and/or non-specific receptors must not he a loose one as

lhe comhination of the majority of biologically active suhstances wilh receptors.

This seems to he demonstrated hy the extreme slowness of the reversibility of the

paralysis and henee of the neuromuscular block produced hy crotoxin.

Crotoxin exerts little or no effect on transmission at ganglionic synapses as

well as at adrenergic neuroeffector junctions. This can be inferred from the

results ohtained in the experiments here reported concerning the contraction of the

nictitating memhrane in response to electrical stimulation of the sympathetic

cervical chain in cats injected with large doses of crotoxin. As already stated,

contractions of lhe nictitating memhrane were always elicited in spite of the fact

2 3

5 6

11 12 13 14 15

Mem. Tnst. Butantan

OSWALDO VITAL BRAZIL

991

Simp. Internac.

33(3): 981-992, 1966

tliat transmission at lhe myoneural junction was blocked one or more hours

heíore lhe experiment. It is interesting lo note that such selectivity of aetion

on neuromuscular transmission seems to he only displayed hy some curarizing

drugs or some depolarizing agents. Callamine and toxiferine, for instance, among

the competitive neuromuscular hlocking agents oídy paralyze ganglionic synapses

when very large doses of them are administered lo cats (20, 21). On the

olher hand, lhe agents which interrupts neuromuscular transmission hy a pre-

junctional mechanism such as hotulinum toxin, hemicholinium, magnesium or

slreptomycin are usually quite as active in producing ganglionic bloekade as

they are in paralysing the myoneural junction.

SlJMMARV

1. Similarity of the motor signs evoked by crotoxin and curare suggested

the investigation of neuromuscular transmission in animais showing the paralysis

elicited by crotoxin. The experiments were earried out on the sciatic-tibialis

anterior muscle preparation of dogs to which crotoxin was injected with an ante-

cipation of 24 or 48 hours. A partial or complete block of transmission was

revealed in all experiments. Neostigmine, calcium, or bicarbonate were without

effect on the bloekade. However, the stimulation of the motor nerve with shocks

of a high (50 c/s) frequency alvvays caused a transitory post-tetanic improvement

on transmission.

2. The neuromuscular blocking aetion of crotoxin was also studied in cats

(sciatic-tibialis anterior muscle preparation). Large doses (0.5 mg/Kg and 1 mg/Kg)

were employed. A complete or partial neuromuscular bloekade appeared within

the first three hours of crotoxin administration. Edrophonium and succinylcholine

increased, although only to a small degree, the nearly completely blocked responses

of the tibialis anterior muscle. Their effects were transitory.

3. Ganglionic transmission in crotoxin injected cats was investigated by stimu-

lating the cervical sympathetic chain and recording the contractions of the nicti-

tating membrane. The experiments were always done after transmission at the

myoneural junction was blocked. The stimulation of the cervical sympathetic

chain invariably elicited contractions of the nictitating membrane whose tensions

did not decrease during the experiments. It seems, therefore, that no ganglionic

bloekade was caused by crotoxin.

4. The effect produced by crotoxin on the contractures evoked by acetyl-

choline or potassium in the isolated and cronically denervated rat hemi-diaphragm

was investigated. Crotoxin shifted to the right the dose-response curve for ace-

tylcholine. Only a slight decline of the slopes of the curves obtained in the

presence of increasing concentrations of crotoxin ocurred. On the other hand,

crotoxin did not antagonize, but potentiated, the aetion of potassium on the de¬

nervated muscle.

5. The following conclusions seem to emerge from the analysis of the results

obtained on this investigation: I. Block of neuromuscular transmission is probably

the sole cause of the paralysis evoked by crotoxin. II. Crotoxin interrupts neuro¬

muscular transmission by producing a non-depolarization type of bloekade. III. A

decrease in the sensitiveness of the end-plate to the depolarizing aetion of acetyl-

choline is the unique or the main cause of crotoxin neuromuscular bloekade.

iv. Crotoxin probably reacts with the acetylcholine receptors and also at other

sites of the muscle fibre membrane. Its fixation on the receptors can not be a

loose one as the combination of the majority of biologically active substances

with receptors.

Rekerexces

1. VITAL BRAZIL, O., FRANCESCHI. J. P., and WAISBICH, E„ Mem. Inst.

Butantan, Simp. Internac., 33 (3), 973, 1966.

2. VITAL BRAZIL, O., O Hospital. 68, 909, 1965.

2 3

5 6

11 12 13 14 15 16

992

PHARMACOLOGY OF CRYSTALLINE CROTOXIN.

II. NEUROMUSCULAR BLOCKING ACTION

6 .

7 .

8 .

10 .

11 .

12 .

13.

14.

15.

16.

17.

18.

19.

20 .

21 .

PATON, W. D. M., and ZAIMIS, E. J., Pharmacol. Rev., 4, 219, 1952.

ZAIMIS, E. J., in BOVET, BOVET-NITTI, and MARINI-BETTOLO (Editors).

Curare and curare-like agents, Elsevier Pub. Co., Amsterdam, 1959, p. 191.

BURGEN, A. S. V., DICKENS, F„ and ZATMAN, L. J., J. Physiol., 109, 10,

1949.

ELMQVIST, D., QUASTEL, D. M. J., and THESLEFF, S., J. Physiol.. 167,

47 P, 1963.

MacINTOSH, F. C., BIRKS, R. I., and SASTRY, P. B„ Nature, 178, 1181,

1956.

REITZEL, N. L„ and LONG, J. P., Arch. int. Pharmacodyn., 119, 20, 1959.

CHANG, C. C., and LEE, C. Y., Arch. int. Pharmacodyn., 114, 241, 1963.

KUFFLER, S. W., J. Neurophysiol., 7, 17, 1944.

CASTILLO, J., and KATZ, B., Proc. Royal Soc., B„ 146, 339, 1957.

HARVEY, A. M„ Buli. Johns Hopkins Hosp., 65, 223, 1939.

MacGREGOR, D. F., J. Pharmacol.. 92, 414. 1948.

STRAUGHAN, D. W., J. Pharm. Pharmacol., 13, 49, 1961.

CASTILLO, J., and ENGBACK, L„ J. Physiol., 124, 370, 1954.

ELMQVIST, D., and JOSEFSSON, J. O., Acta physiol. scand., 54, 105, 1962.

VITAL BRAZIL, O., Foi. clin. biol., 29, 20, 1960.

VITAL BRAZIL, O., Arch. int. Pharmacodyn., 130, 136, 1961.

VITAL BRAZIL, O., and CORRADO, A. P., J. Pharmacol., 120, 452, 1957.

DEPIERRE, F., C. R. Acad. Sei., 225, 956, 1947.

WASSER, P. G„ in BOVET, BOVET-NITTI, and MARINI-BETTOLO (Edi¬

tors), Curare and curare-like agents, Elsevier Pub. Co., Amsterdam, 1959,

p. 244.

6 .

8 .

10 .

11 .

12 .

13.

14.

15.

16.

17.

18.

19.

20 .

21 .

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3): 993-1000, 1966

OSWALDO VITAL BRAZIL, REINALDO FARInA,,

LUIZ YOSHIDA and VILSON ANTONIO DE OLIVEIRA

993

45. PHARMACOLOGY OF CKYSTALLINE CR0T0XIN. III. CARDIO¬

VASCULAR AND RESPIRATORY EFFECTS OF CROTOXIN AND

CROTALUS DU RI SSL! S TERRIFICUS VENOM.

OSWALDO VITAL BRAZIL, REINALDO FARINA, LUIZ YOSHIDA

and VILSON ANTONIO DE OLIVEIRA

Department of Pharmacology, University of Campinas, Campinas, S.P.. Brazil

The venom of C. d. terrijicus besides producing skeletal muscle paralysis

and renal insufficiency, gives rise to acute disturbances of blood pressure and

respiration as it was demonstrated hy Arlhus (1) and others (2, 3). The main

purpose of the present stndy was to investigate the actions of crotoxin on blood

])ressure and respiratory function so that lhe role it represents in the genesis of

the disturbances evoked by lhe venom could he evaluated. Experiments wilh the

venom were also done for adequately evidencing the differences of action and

potency between crotoxin and venom.

The mechanism through whieh acute respiratory disturbances are caused hy

the venom did not seem to he clear. Houssay (4) concluded from experiments

carried oul on the perfused and isolated head preparation of dogs that the venom

of C. d. terrijicus exerts direct actions on the central nervous system. He veri-

fied that signs of respiratory stimulation and depression appeared in the isolated

head following the injection of lhe venom in the perfuser dog. However, the

experiments of Houssay do not eliminate lhe possibility that the respiratory dis-

turbances could he also reflexly generated through stimulation of chemoreceptors

at the carotid and aortic bodies, or of receptors in the lungs. Therefore some

experiments were planned lo investigate this point. The results suggest that the

direct action on the medulla is perhaps less important in causing the acute res¬

piratory effects than the reflex ones.

Material and methods

lhe venom of C. d. terrijicus used in this research was extracted as pre-

viously reported (5) from snakes captured in Goiás; it helonged to the variety

devoid of crotamine. The hatches of crotoxin utilized in the experiments were

crystalline preparalions obtained according to procedures already referred to in

the previous paper (5).

This study was supported by a grant from the “Fundação de Amparo à Pesquisa do

Estado de São Paulo”.

'SciELO

D 11 12 13 14 15 16

994

PHARMACOLOGY OF CRYSTALLINE CROTOXIN. III. CARDIO¬

VASCULAR AND RESPIRATORY EFFECTS OF CROTOXIN AND

CBOTALUS DURISSUS TBRRIFICUS VENOM

The acute effecls evoked hy erotoxin and lhe venom on blood pressure and

respiration were compared in dogs anaeslhelized vvith sodium pentoharhital (30

nig/Kg, i.v.). Tlie effeel they produced on lhe haematocrit value was also

investigated in mosl oí lhe experiments. The dogs were prepared for recording

arterial lilood pressure and respiralion as it was descrihed in a previous work (6).

Samples of blood were withdrawn for lhe determination of lhe haematocrit

values 30 minutes and 1, 2 and 3 hours after lhe injection of the venom or

erotoxin. Twenty-four dogs were employed in lhe experiments: sixteen animais

were injected wilh erotoxin and eight with lhe venom. Crotoxin from two hatches

(erotoxin n.° 5 and n.° 14) was nsed. No haematocrit value determinations were

made in the experiments, in numbers of eight, in whieh crotoxin n.° 14 was

employed. The dose of 0.25 mg/Kg of crotoxin or of the venom was used in

all experiments. The arterial blood pressure and respiration were usually re-

eorded for 3 hours after administralion of the venom or crotoxin.

The effects produced hy erotoxin and the venom were also compared hy

injecting them in lhe same dog. Two or three doses of 0.25 mg/Kg, conve-

nientely spaced, of crotoxin were administered in order that the animal become

irresponsive to its hypotensive action. Then 0.25 mg/Kg of the venom were

injected.

The experiments planned lo invesligate the mechanisms involved in the

genesis of the respiratory effects caused hy lhe venom were carried oul in four

dogs with both vagi cut, and in ten whose earotid sinus nerves, in addition, were

seclioned. Occlusion for 30 or 45 seconds of the common earotid arleries was

made before the injection of lhe venom lo lest the denervation of the earotid

sinus regions. The dogs were anaeslhelized with sodium pentoharhital. The dose

of 0.25 mg/Kg of lhe venom was also employed in these experiments.

Results

Table I shows the percentages of lhe maximums of blood pressure falis

observed in the experiments in whieh erotoxin n.° 5 and crotoxin n.° 14 were

used, and their means 32 ± 0.5% and 25 ± 6.44%. The difference belween

these means is not statistically significanl (P > .05). Both of them, however,

are significantly smaller (P < .01) than the mean 58.3 ± 5.6%, obtained

from lhe experiments with the venom C. d. terríficas (Table II). The venom

was also mucli more polent in increasing lhe haematocrit value. Table I and

II sbow the percentages of the increases whieh were demonstrated 30 minutes

after the injection of crotoxin n.° 5 and the venom, as well as their means

20.2 ± 4.4% and 43.5 ± 5.9%. The difference belween these means are

highly significant (P << .01).

The hypotensive effect elieited hy crotoxin was always reversible; the arte¬

rial blood pressure usually attained its primitive levei in less than two hours

after its administralion. This did not always happen in the experiments with

the venom: In two out of eight dogs, the effect on blood pressure was irrever-

sible and the animais died from the hypotension in less than one hour. However,

the lilood pressure of lhe other dogs recovered within 2 or 3 hours. The effects

evoked hy lhe venom and crotoxin on lilood pressure were also qualitalively

different from each other (Figs. 1, 2).

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3): 993-1000, 1966

OSWALDO VITAL BRAZIL, REINALDO FARINA,,

LUIZ YOSHIDA and VILSON ANTONIO DE OLIVEIRA

995

TABLE I — MAXIMUMS OF BLOOD PRESSURE DEPRESSIONS AND INCREASES IN

THE IIEMATOCRIT VALUES CAUSED BY THE INTRAVENOUS ADMINISTRATION OF

0.25 MG/KG OF CROTOXIN

Crotoxin no.

5 *

Crotoxin

no. 14 *»

Experiment Maximum fali

(no.) of (%)

Increase in the

hematocrit

value (%)

Experiment

(no.)

Maximum fali

of blood

pressure (%)

1 40.9

7.5

2 30.6

17.0

3 56.2

40.0

4 37.7

17.0

5 19.3

12.0

6 14.2

17.8

7 55.5

39.4

8 6.5

10.6

Mean ± S.E.

32.6 ± 6.5%

20.2 ± 4.4%

25.6 ± 6.44%

* LD,„ for mice (i.v.) 82 (66.9 to 101.4) mcg/Kg.

** LD f(0 for mice (i.v.) 80.3 (60.8 to 108.9) mcg/Kg.

TABLE II — MAXIMUMS OF BLOOD PRESSURE DEPRESSIONS

AND INCREASES IN THE IIEMATOCRIT VALUES CAUSED BY

THE INTRAVENOUS ADMINISTRATION OF 0.25 MG/KG OF THE

VENOM OF C. D. TERRIFICUS

Experiment Maximum fali of

(no.) blood pressure

(%)

Increase in the

hematocrit value

(%)

37.7

44.7

71.7

50.0

70.0

44.2

73.3

21.5

50.7

35.1

60.2

56.4

69.7

71.7

33.3

24.3

Mean ± S.E.

58.3 ± 5.6%

43.5 ± 5.9%

'SciELO

D 11 12 13 14 15 16

996

PIIARMACOLOGY OF CRYSTALLINE CROTOXIN. III. CARDIO¬

VASCULAR AND RESPIRATORY EFFECTS OF CROTOXIN AND

CROTALUS DURISSUS TERRIFICUS VENOM

IHB

Fig. 1 — Dog, 10.5 Kg, sodium pentobarbital. Records of respiration and blood pressure.

At Cr., 0.25 mg/Kg ot crotoxin were injected. At B, C and D, records taken 30, 60

and 90 minutes atter A. Time 6/6 seconds.

Crotoxin produced a rather gradual blcod pressure fali which began to appear

írom 2 to 3 minutes after its administration. After the venom, on the other

hand, an abrupt and transient fali of blood pressure appeared within the firsl

30 seconds and was followed by a pressor effect of one or two minutes duration.

Thereafter a less abrupt and prolonged fali occurred in all experiments. lt was

iisually more intense than the first fali.

Respiration did not seetn to be acutely modified by crotoxin (Fig. 1). The

\enom, on the other hand, caused an intense and transient increase in the fre-

quency and amplitude of the respiratory movements a few seconds after its admi¬

nistration. This was followed by apnoea of brief duration and then by tachypnoea

which persisted for more than one hour (Fig. 2).

Haemolysis was demonstrated in all experiments in lhe blood extracted two

hours after the injection of crotoxin or venom.

SciELO

Mem. Inst. Butantan

Simp. Internac.

38(3) :993-1000, 1966

OSWALDO VITAL BRAZIL, REINALDO FARIÍIA,,

LUIZ YOSHIDA and VILSON ANTONIO DE OLIVEIRA

997

The striking differences in thc effects elicited hy crotoxin and venom were

also revealed hy injecting them in the samè dog. The venom cansed the usual

disturbances on hlood pressure (and also on respiration) alter lhe dog was niade

irresponsive (tachyphylaxis) to the hypotensive action of crotoxin. Fig. 3 shows

lhe records of an experiment in which the hlood pressure was not altered hy

crotoxin. Nevertheless, the venom produced, as usually, the characteristic effects

on hlood pressure and respiration.

The apnoea usually elicited hy the venom did not occur after its adminis-

tration in the dogs whose vagi had heen cut. In its place, a hrief phase of de-

creased respiratory excursions was observed (Fig. 4). The other acute respi-

ratory disturbances evoked hy the venom were definitely attenuated in all expe-

riments. In eight out of the ten dogs whose vagi and carotid sinus nerves had

heen sectioned, the venom gave rise to oídy a slight increase in the respiratory

frequency and to a very hrief phase in which a small diminution in lhe respi¬

ratory amplitude occurred. In two of these animais the respiratory effects were

alrnosl suppressed (Fig. 5). On the other hand, in two other experiments the

respiratory disturbances were nearly as intense as those observed in dogs with

the vagi and carotid sinus nerves intact. However, the apnoea did not occur.

Fig. 2 — Dog, 7 Kg, sodium pentobarbital. Records oí respiration and blood pressure.

At V., 0.25 mg/Kg of C. d. terrificus venom were injected. At B, C and D, records

taken 30, 60 and 150 minutes after A. Times 6/6 seconds.

SciELO

■ v^V****"* * vN**

LU..tU.U.lJLU

tf ,j

Fig. 3 — Dog, 10 Kg, sodium pentobarbital. Records of respiration and blood pressure.

At Cr. (panei A and B), 0.25 mg/Kg ot crotoxin were injected; at V. (panei C),

0.25 mg/Kg of the venom of C. d. terrificus were administered. At B, C and D, records

taken 1, 2 and 3 hours after A. Time 6/6 seconds.

DlSCUSSlON

The results of lhe present investigation show that lhe remarkahle acute dis-

turbances provokecl by the venom of the South American rattlesnake on cir-

culation and respiration are not due to crotoxin. Neither can they he atlrihiited

lo crotamine hecause the venom employed in the experiments did not contain

that loxin. Therefore, it can he inferred that, besides these, there are other

components of the venom of C. d. terrificus whose pharmacological actions must

jilay an important role in clinicai envenomation, especially in the genesis of

shock, a condition frequently exhibited by lhe patients bitten by that snake(7).

The separation as well as the pharmacological and immunological study of sueh

components is highly desirahle.

It is sur])rising that, being endowed with phospholipase A activity, crotoxin

exhibits so small hypotensive and shocking potencies. Lysolecithin, in effect,

besides being hemolytic, produces in dogs and cats an abrupt fali of blood pres¬

sure and releases histamine (8). Feldberg, llolden and Kellaway (8) attribuled

998

PIIARMACOLOGY OF CRYSTALLINE CROTOXIN. III. CARDIO¬

VASCULAR AND RESPIRATORY EFFECTS OF CROTOXIN AND

CROTALUS VURISSUS TERRIFICUS VENOM

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(3) :993-1000, 1966

OSWAI.DO VITAL BRAZIL, REINALDO FARINA,,

LUIZ YOSHIDA and VILSON ANTONIO DE OLIVEIRA

999

Fig. 4 — Dog, 6.0 Kg, sodium pento-

barbital, both vagi sectioned. Records ol

respiration and blood pressure. At ar¬

rows, C.25 mg/Kg o£ the venom of C. d.

terrifictis were injected. At B, record

taken 7 minutes after A. Time 10/10

seconds.

Fig. 5 — Dog, 6.5 Kg, sodium pento-

barbital, both vagi and carotid sinus

nerves sectioned. Records of respiration

and blood pressure. First arrows, com-

pression of the common carotid arteries

was made, second arrows, 0.25 mg/Kg of

the venom of C. d. terrificus were in¬

jected. Time 10/10 seconds.

much of the cardiovascular effects produced l>y the Indian cohra ( Naja naja)

venom to lysolecithin formation, an interpretation which can perhaps be questioned

in view of the present residis.

The triple effect, here reported, produced liy lhe venom of C. d. terrificus

on blood pressure, that is, the transient and abrupt fali of blood pressure which

is followed by an hypertensive period of short duration and a secondary de-

pression, was first described hy Arthus in rabbits (1). He reported that he did

not observe such a sequence of effects after administration of the other venoras

he had studied. Vellard and Huidobro, on the other hand, showed that the

venom of C. d. terrificus from some regions produces only depression of blood

pressure, a finding that was confirmed by one of us (3) with the venom from

rattlesnakes of Santiago dei Esteio, Argentina. The triple effect, as previously

shown (3), is not supressed in dogs (i) injected willi atropine, (ii) with lheir

central nervous system destroyed by lhe procedure of Galvão and Pereira,

liii) with their autonomic ganglia blocked by hexamethonium and finally (iv)

with their a adrenergic receptors blocked by dibenamine or chlorpromazine.

The brief period of apnoea can not be imputed lo the respiratory stimu-

iation caused by the venom as it was also observed in experiments in which only

a small stimulation of the dog respiration was elicited. Moreover, in the expé-

riments in which both vagi had been cut, the apnoea did not occur even in those

SciELO

11 12 13 14

1000 PHARMACOLOGY OF CRYSTALLINE CROTOXIN. III. CARDIO-

Aw VASCULAR AND RESPIRATORY EFFECTS OF CROTOXIN AND

CROTALVS nURIKSUS TERRIFICUS VENOM

ilogs whose respiration was inlensely stimulaled (two experiments in animais wilh

the vagi and carotid sinus nerves cut). lt seems, therefore, that this apnoea

originates from the stimulalion, in the Iungs, of vagus nerve filtres which conduct

inhihitory impulses to the respiratory eenlers. The hrief period of apnoea evoked

hy nieoline also arises, according to Aviado (9), from stimulalion of reeeptors in

lhe Iungs.

The respiratory stimulalion caused hy lhe venom was, in mosl cases, greatly

reduced in lhose dogs whose vagi and carotid sinus nerves had heen sectioned.

This suggests that is must be, in pari al least, reflexly generated, probahly hy

an action of the venom on lhe chemoreceptors of the carotid and aortic bodies.

Slimmary

1. A comparative study of the acute effects produced by crotoxin and the

venom of C. d. terrificus on blood pressure, haematocrit value and respiration was

carried out in sodium pentobarbital anaesthetized dogs. The venom used in the

investigation beionged to the variety devoid of crotamine.

2. Crotoxin was much less active than the venom in producing hypotension

and in increasing the haematocrit value. The effects elicited by them on blood

pressure were also qualitatively different from each other.

3. Respiration was not acutely modified by crotoxin. The venom, on the

other hand, caused an intense and transient increase in the frequency and ampli¬

tude of the respiratory movements within a few seconds after its administration,

a brief period of apnoea and one of tachypnoea which usually was very long

lasting.

4. The results of the present investigation show, therefore, that the acute

disturbances elicited by the venom on circulation and respiration can not be due

to crotoxin or to crotamine. There are other venom components whose pharma-

cological actions must play an important role in C. d. terrificus envenomation,

especially in the genesis of shock.

5. Some experiments were also made to clear up the mechanisms involved

in the genesis of the respiratory disturbances that follow the administration of the

venom. It was inferred from the results of these experiments that the brief

period of apnoea originates from stimulation of reeeptors in the iungs while the

respiratory stimulation is due to an activation of the chemoreceptors in the

aortic and carotid sinus bodies, and to a direct action on the respiratory centers.

RefERENCES

1. ARTHUS, M„ C. R. Acad. Sei., 156, 1256, 1913.

2. VELLARD, J„ and HUIDOBRO, F., Rev. Soc. argent. Biol, 17, 72, 1941.

3. VITAL BRAZIL, O., Ann. Fac. Med. Univ. S. Paulo, 28, 159, 1954.

4. HOUSSAY, B„ and HUG, E„ C. R. Soc. Biol.. 3!) (32), 1509, 1928.

5. VITAL BRAZIL, O., FRANCESCHI, J. P., and WAISBICH, E., Mem. Inst.

Butantan, Simp. Internac., 33 (3), 973, 1966.

6. VITAL BRAZIL, O., Mem. Inst. Butantan, Simp. Internac., 33 (3), 981, 1966.

7. WAJCHENBERG, B. L., SESSO, J„ and INAGUE, T„ Rev. Ass. méd. brasil.,

1, 179, 1954.

8. FELDBERG, W., HOLDEN, H. F„ and KELLAWAY, C. H„ J. Physiol. (Lond.),

91, 232, 1938.

9. AVIADO, D. M., in DrilVs Pharmacoloyy in Medicine, third edition, McGraw-Hill

Book Co., N. Y., 1965, p. 524.

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11 12 13 14 15 16 17

Mem. Inst. Butantan

Simp. Internac.

33(3): 1001-1008, 1906

WALTER AUGUST HADLER and

OSWALDO VITAL BRAZIL

1001

46. PHARMACOLOGY OF CRYSTALLINE CROTOXIN.

IV. NEPH ROTOXICITY

WALTER AUGUST HADLER and OSWALDO VITAL BRAZIL

Department of Histology and Embriology and Department of Pharmaeology

University of Campinas, Campinas, São Paulo, Brazil

Renal lesions identical lo those originally found in crush syndrome (haemo-

globinuric neplirosis, lower nephron nephrosis or tubulorhexis) have been des-

cribed in autopsy and biopsy tissue (1, 2, 5) from human cases of snakebite

caused by Crotalus durissus terrificus (South American rattlesnake). Similar

lesions, according lo Amorim and coworkers (3) ean be experimentally produced

by injecting dogs with the crude venom of this snake. Identification of the

componcnt or eomponents of the venom responsible for such histopathologic picture

is highly desirahle. A study was, therefore, undertaken of lhe renal lesions

produced in dogs by crotoxin, one of lhe main toxins of the venom. Resides

motor paralysis, this venom component elicits albuminúria, haemoglobinuria and

olyguria in dogs as it was shown in a previous paper (7).

Material and Methods

The histopathological study was made on the kidneys of twenty dogs intra-

venously injected with doses of 0.102, 0.128, 0.160, 0.200 and 0.250 mg/Kg

of crotoxin. The kidneys were removed one to nine days after crotoxin adminis-

tration from animais under sodium penlobarhital anaesthesia or from dogs re-

cently died of crotoxin intoxication.

Pieces of the kidneys were fixed in Bouin’s fluid, or in 10% formalin in

phosphate buffer t.pH 7.0), included in paraffin, cut and stained by haemoto-

xylin and eosine or Masson’s trichrome method. PAS, and benzidine reaction

for haemoglobin detection (5), were also made.

Results

Bolh glomeruli and renal tubules showed pathological alterations. There was

some correlation between intensity of lesions and the dose of crotoxin injected,

lhe lesions being more marked in the kidneys of those animais which received

higher doses. There were, however, some variations not correlated to the doses.

This study was supported by a grant from the "Fundação de Amparo à Pesquisa do

Estado de São Paulo”.

SciELO

10 11 12 13 14 15 16

1C02

PIIARMACOLOGY OF CRYSTAI.LINE CROTOXIN.

IV. NEPHROTOXICITY

The renal lesions showecl focal distribution : injuried areas were alvvays

found to be separated by apparently normal ones.

lt was possible lo recognize some differences between early lesions, wliich

occurred in the first four days follovving crotoxin ínjection, and late lesions,

observed afler lhe fourth day. Such differences concern the component of the

nepnron most attained by the lesions. In early lesions, the renal tubules were

less attained than the glomeruli; in late ones, tubular lesions predominated.

Early i.lsions

Glomerulus. The lesions most eonstantly observed were capillary congestion,

thickness of the basement membrane, deposil of PAS positive material between

the capillary loops (Fig. 1 ) and nuclear pycnosis of some glomerular cells.

Fig. 1 — Section of ktdney of dog Injccted with crotoxin. PAS reaction. Early

lesions. Cross section of glomeruli shovvs thickness of the basement membrane and

some amorphous PAS positive material among the glomerular loops.

Proximal tubule. Degeneralive lesions were present in some epithelial cells

of the proximal convolutions. The brush horder of the damaged cells desap-

peared at the same time lhat an alteration of the normal distribution as well as

a numerical reduction of the mitochondria occurred (Fig. 2). The cytopiasm

of the damaged cells appeared vacuolated and the nucleus was somctimes pycnotic.

lt must be emphasized lhat the degenerative lesions reached only some tu¬

bular cells, in very restricted areas. These areas were situated chiefly in the

internai zone of the renal córtex, eorresponding lo the terminal portion of the

proximal eonvoluted tubule.

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(3) :1001-1008, 1966

WALTER AUGUST HADLER and

OSWALDO VITAL BRAZIL

1003

Fig. 2 — Section of kidney of dog injected with erotoxin. Masson’s trichrome stain.

Late lesions. The degenerative lesions predominate in proximal tubules vvhose eells

lost thelr brush border and present little amount of irregularly distributed mitoehondria.

[nside the tubules, it was frequently found PAS positive and haematic casts.

Distai tubule. Degenerative lesions similar to those described for the pro¬

ximal tubular cells were found in distai tubules. However, the injuried areas

were smaller and the damaged cells less numerous lhan those in lhe proximal

convolutions.

Collecting ducts. The collecting duct cells were apparently normal. In the

collecting duct lumen, some PAS positive haematic casts could be seen.

Interstitial tissue. Some areas of oedema near the injuried tubules were

ohserved.

Late lesions

Glomerulus. The glomeruli showed in most cases a normal aspect. Howe-

ver, thickness of the basement memhrane coidd be ohserved in a few glomeruli.

Proximal tubule. The proximal tubules were intensely injuried. Although

focal in character, the lesions had a tendency to become diffuse through con-

fluence of neighboring injured areas.

The lesions predominated in terminal segments of the proximal tubules.

However, cells in the initial ones were also sometimes damaged.

SciELO

staJn. 3 Late ^ “«*»*'« trichrom,

brush bortler. The distai tubule eells as well as "hosf oT^m SWOl ' en Cells wllhoul

present o„ ly minür iesions. in thi instanee!, the theír^h “

mltüchondria pattern.

Mem. Inst. Butantan

SirriD. Internac.

33(3):1001-1008, 1966

WALTER AUGUST HADLER and

OSWALDO VITAL BRAZIL

1005

The fcllowing alterations could he seen in lhe epithelial cells of lhe damaged

tuhules: disappearence of lheir brush horder, a great decrease in the amount as

well as an irregular pattern of the mitochondria, swelling and microvacuolar

degeneration of the eytoplasm. When intense this degenerativo process gave a

foamy aspect lo the eells (Figs. 3, 4, 5 and 6). The intensely vacuolated cells

showed nuclear pycnosis and necrosis. Some PAS positive granules could he

ohserved in the eytoplasm of lhe vacuolized cells.

PAS positive casts were seen in the lumen of the proximal tuhules.

Distai tubule. The lesions were qualitatively identical to those seen in

the proximal tuhules. However, they were much less intense, cellular necrosis

being rarely seen. Disorganizalion of the mitochondria pattern and slight mi-

crovacuolization of the eytoplasm of the epithelial cells (Figs. 4 and 5) were the

predominant alterations ohserved.

The initial portion of the distai tuhules appeared more intensely damaged

than the distai one.

CollectiiiL' clucts. Collecting duets did not show lesions. In their lumen

some PAS positive casts

Interstitial tissue. Around the injured tuhules there was some chronic ii

rnatory infiltration and sometimes tissue neoformation.

Fig. 5 — Sectlon of kiclncy of dog injected with crotoxin. Masson’s trichrome stain.

Late lesions. Cells of proximal convoluttons show intense microvacuolar degene-

ration. In distai tubules the mitochondria pattern is altered and discret microva¬

cuolar degeneration of their cells can be seen.

SciELO

1006

PHARMACOLOGY OF CRYSTALLINE CROTOXIN.

IV. NEPHROTOXICITY

Fig. 6 — Section of kidney ot dog ínjected with crotoxin. Haematoxylin and eosine.

Late iesions. Intense mierovacuolar degeneration and eeltular necrosis of próxima!

convoluted tubules can be seen.

Discussion

The histopalhological picture shown hy the kidneys of dogs ínjected witli

crotoxin was not exactly the same as that which Amorim and Mello (1, 2)

described iii human cases of C. d. terrijicus snakebite. These authors stated, in

effect, lhal the ascending limb of the Henle’s loop and the distai convoluted

lubiile were the predominanl sites of lhe degenerative and necrobiotic Iesions.

In the kidneys from the dogs intoxicated with crotoxin, on lhe other hand, the

rnost attained segment of lhe renal tubule was the proximal one. In both cases,

however, the Iesions presented a focal distribution. Amorim and coworkers (3)

also found the ascending limb of the Henle’s loop and the distai convohition to

[>e the prevailing situation of the renal Iesions of dogs Ínjected with the South

American rattlesnake venom: “The histological Iesions of the tubules are prin-

cipally characterized by degenerative and more serious necrobiotic Iesions in the

so-calÍed intermediated segment of the nephron, i.e. lhe ascending branch of

Henle’s loop and of the distai or secondary convoluted tubule predominanting in

lhe boundary zone of the kidney” (3). However, Rodrigues Lima (6) found

in biopsy tissue from human cases of C, d. terrijicus envenomation that degene¬

rative Iesions predominated in the proximal convolutions while necrotic ones were

more frequent in the distai tubules: “Nos nossos casos, o néfron foi lesado

eomo um lodo, embora as lesões degenerativas tenham preferência por túbulos

proximais, enquanto que a necrose e a regeneração ocorreram com mais freqiiên-

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Mem. Inst. Butantan

Simp. Internac.

33(3):1001-1008, 1966

WALTER AUGUST HADLER and

OSWALDO VITAL ERAZIL

1007

degenerativo lesions of lhe tubular cpithelium

as descrihed hy Rodrigues Lima (6), of the

kidneys of dogs injected witli crotoxin:

the

eia no segmento distai” (6). The

of the proximal corivòlutions vvere.

same type of lhose we found in

“Estas lesões (the degenerativo lesions) têm aspectos de microvacúolos que recha¬

çam o núcleo para a periferia, tomando total ou parcialmente o citoplasma celu¬

lar ou apresentam fina

celulares. Èstes aspectos

nados proximais” (6).

Three main faclors

in envenomation caused I

They are (1) nephrotoxic substances,

The haemolytic activities of crotoxin and of lhe

They are due to their phospholipase A activities,

granulação citoplasmáliea com borramento dos limite.-

foram vistos com maior frequência nos túbulos contor-

Thus the factor mentioned

renal histopathologic picture

are prohahly at vvork in generating the lesions found

>y C. d. lerrificus snakebite, and in crotoxin intoxication.

(2) intravascular haemolysis and (3) shock.

venom are nearly lhe same.

i. e. lo lysolecithin formation.

i (2) can not explain the differences observed in the

ol the two conditions.

Lysolecithin besides being haemolytic is toxic to renal epithelial cells. lt is

formed hy crotoxin, which is composed of crolactin (a polypeptide toxin) bound

lo phospholipase A (4), and hy the venom through its content of crotoxin and.

perhaps, free phospholipase A. The local formation of lysolecithin al the surface

of lhe tubular cpithelium could be one of the causes of tubular lesions, mainly

lhose of proximal convolutions. This local formation of lysolecithin seems to be

very probable since crotoxin attains very high concentrations in the kidneys

[a fact which lias been revealed in experiments vvilh Kil I-labeIled crotoxin (un-

puhlished data)]. Thus the nephrotoxic factor wonld play a more important

role in crotoxin intoxication than in the envenomation caused hy C. d. lerrificits

venom. This hypothesis explains why the lesions of proximal convolutions are

prevalent in crotoxin damaged kidneys.

Shock is the main factor responsible for renal ischaemia, which, in tnrn, is

the cause of the lesions typical of tubulorhexis. Crotoxin was found to be much

less potent than the venom in eliciting hypolension and haemoconcentration (8),

i. e., the venom is much more active in producing shock. This fact seems to

explain why lhe renal histopathologic picture of the envenomation is more close

than that of crotoxin, to the histopathologic picture of the acute renal failure

known as lower nephron nephrosis or tubulorhexis.

Summary

An investigation of the renal lesions produced hy crotoxin was carried ont

on the kidneys of twenty dogs intravenonsly injected with this substance. The

kidneys vvere removed for histological section one to nine days after crotoxin

administration from animais under pentoharhital anaesthesia or froin dogs re-

cently died of crotoxin intoxication.

Early lesions which occurred in kidneys of dogs within the first four days

following crotoxin injection, differed somewhat from late lesions observed after

the fourlh day.In early lesions the renal tubules were less attained than the glo-

meruli which showed capillary congestion, thickness of the basement membranc,

deposit of PAS positive material between the cajiillary loops and nuclear pyenosis

of some glomerular cells. I he degenerative lesions of lhe proximal convolutions

reached otdy some epithelial cells in very restricted areas. Damaged cells in

distai convolutions were still less

numerous.

lesions, tubular dai

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10 11 12 13 14 15 16

1008

PHARMACOLOGY OF CRYSTALLINE CROTOXIN.

IV. NEPHROTOXICITY

predominated, the segment of the lubules most attained being the proximal ono.

They showed intonso microvacuolar degeneration as well as nuclear pycnosis and

necrosis of many epitholial cells.

Early or late lesions showed a focal distribntion. Howevor, in lato lesions

lhose of the proximal convolutions had a tendoncy to became diffuse through

confluence of neighboring injuried aroas.

The renal histopathological picture of crotoxin intoxication was comparod

with that presented by kidneys of human beings bitten hy lhe South American

rattlesnako (Crotalus durissus terrijicus ) and of dogs injected with its vonom.

An hypothosis was formulated to explain the differonces observed.

References

1. AMORIM, M. F., e MELLO, R. F., Mem. Inst. Butantan, 24, 281, 1952.

2. AMORIM, M. F., and MELLO, R. F., Avier. J. Path., 30. 479, 1954.

3. AMORIM, M. F., MELLO, R. F„ and SALIBA, F„ Rev. brasil. Biol., 20, 359,

1960.

4. HABERMANN, E„ Biochem. Z., 329, 405, 1957.

5. LISON, L., Histochemie et cytochemie animales, Gauthier Viltars, Paris, 1966.

6. RODRIGUES LIMA, J. P„ Necrose tubular aguda por mordedura de animais

peçonhentos. Tese. São Paulo, 1936.

7. VITAL BRAZIL, O., FRANCESCHI, J. P„ and WAISBICH, E„ Mem. Inst.

Butantan, Simp. Internac., 33 (3), 973, 1966.

8. VITAL BRAZIL, O. et al., Mem. Inst. Butantan. Simp. Internac., 33 (3),

993, 1966.

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10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3) :1009-1010, 1966

C. Y. LEE, C. C. CHANG, T. H. CHIU,

T. C. TSENG and S. Y. LEE

1C09

47. PHAHMACOLOGICAL PROPERTIES OF CARDIOTOXIN ISOLATED

FROM THE VENOM OF NAJA NAJA ATRA *

C. Y. LEE, C. C. CHANG, T. H. CHIU, T. C. TSENG and S. Y. LEE

Pharmacological Institute, College of Medicine, National Taiwan University,

Taipei, Taiwan, China.

Lyophylized venom of Naja naja atra was fractionated on columns of

CM-Sephadex (G-50) into 13 fractions by gradient elution witli ammonium

acetate buffer at pH 5-7. Among them five fractions (V-IX) were found to

be neurotoxic and ihree (X, XII, XIII) were cardiotoxic. Intraperitoneal LD.-,,,

in mice was 0.074 p.g/g for Fr. VIII — the major neurotoxic component (NT)

and 1.48 jug/g for Fr. XIII — the major cardiotoxic one (CT). CT caused con-

tracture, as well as reduction of resting membrane potentials, of the frog’s sar-

torius, chick s biventer cervicis, and rat’s diaphragm. In the absence of calcium,

the contracture was markedly reduced, although the depolarizing effect remained

unchanged. Neither contracture nor depolarization was caused by NT. The

terminal nerve spikes of lhe frog sartorius were abolished by CT but unaffected

by NT. CT caused systolic arrest of isolated frog hearts and rat’s alria by re-

ducing the membrane potentials, whereas NT was almost without effect up to

IO -4 g/ml. CT caused a slow contraction of the guinea pig ileum, which wast

partially antagonized by either atropine or procaine but not by hexamethonium

or antihistaminics. In the presence of CT (10~ 6 — 10 -5 g/ml), the responses

to nicotine and 5-hydroxytryptamine were greatly inhibited, usually preceded by

an initial and transient facilitation. The responses to histamine and acetylcholine

were not, or only slightly, reduced by CT. The vessels of the rabbit ear were

coristricted by CT. In cats, CT caused a fali in systolic pressure more than

diastolic pressure, accompanied by various ECG changes, such as P-R interval

prolongation, inverted T

S-T segment depression, ventricular premature

beats, A-V interference, complete A-V hlock, idioventricular rhythm etc. It is

concluded that cardiotoxin isolated from cobra venom acts on various excitahle

cells, predominantly, if not entirely, by reducing the membrane potentials.

Aided b!y the U. S. Army Med. Res. Develop. Coramand Research. Grant DA — MD

49 193 — 64 — G 108 and by the National Council on Science Development, Repu-

blic of China.

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10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33<3):1011-1016, 1966

K. MASEK and H. RASKOVÀ

1011

48. THE ACTION OF STAPHYLOCOCCAL ALPHA TOXIN AND THE

VENOM FROM R1T1S GARONICA ON RAT STOMACH STRIP

K. MASEK and H. RASKOVÀ

Institute of Pharmacology Czechoslovak Academy of Sciences, Prague

We arc most grateful to bc allowed lo speak before ihis most distinguished

community concerned with the research of animal venoms. We feel a little em-

barrassed as poor relatives concerned only with bacterial toxins. In general two

types of venoms are treated l>y entirely different groups with no close relation

between them. We hope lhat our modest contribution might arouse some interest

lo strenghten the cooperation between workers in the two subjects.

In our lahoratory we are interested in the pharmacology of Staphylococcal

alpha loxin. From your point of view older findings of the group of North and

Doery are very interesting. They demonstrated a protection against Staphylo¬

coccal alpha toxin by pretreating lhe animais with tiger snake venom (1). They

suggested that the snake venom and Staphylococcal alpha toxin occupied the

same type of receptors. Their experiments point to the probability that the actual

protective effect is produced by phospholipase A. Long chain unsaturated fatty

acids are liberated and lysophosphatide is formed. Ganglioside bas the same

protective action against Staphylococcal alpha toxin. The group suggested that

phospholipase A plays a role in animal defence against bacterial exotoxins. This

points to a possible common mechanisms between animal and bacterial toxins

and therefore we looked for suitable models where some similarities and diffe-

rences could be demonstrated. Originally we planned to use for this demons-

tration isolated mast cells, however, according to the results we have up to now,

the correlations on this model were not so easy to demonstrate. We have the¬

refore taken the liberty to discuss our results on another model, which is not in

the original title of our communication.

Material and Methods

Rats of both sexes were fasted over night, killed by decapitation and isolated

ral stomach strip preparations using Vane’s method (2) were set up. The sto-

mach strips were suspended in 5 ml bath with Krebs solution at 37°C, and

bubbled with air.

Two hatches of Staphylococcal alpha toxin were used one crude product

and one of 70% purity (gift from Dr. Bernheimer). Clostridium perfringens

toxin was a product of GLAXO laboratories. Venom from Bitis gabonica was

obtained through generosity of Dr. Kornalík.

Technical assistance: B. Rybová, and O. Fialová.

SciELO

10 11 12 13 14 15 16

1012

THE ACTION OF STAPHYLOCOCCAL ALFHA TOXIN AND THE

VENOM FROM BITIS GABONICA ON HAT STOMACII STRIP

Results

Crude or purified Staphylococcal alpha toxin elicited on isolated rat stomaeh

slrip a contraction which has a two phase character. After thc toxin application

a rapid contraction developed which started to decline ahout 60 sec after and

lhen a new slow, long lasting contraction developed. With repeated toxin appli¬

cation the rapid phase of the contraction did not appear any more and the slow

reaction was of lower intensity than the first time. Fig. 1 demonstrates the

t t t

t t

t f

5HT

5HT sr

5HT

2 4 8

4 0,9

4 4

HU w

ng HU

”9

Fig. 1 -— Effect of Staphylococcal (ST) alpha toxin on isolated rat stomaeh prepa-

ratlon. Doses are In nanograms (ng) or hcmolytical unlts (H.U.) per ml of bath.

results. When the repetition of toxin application was continued, the sensitivity

of lhe organ to toxin or 5HT declined and finally vanished. The organ became

also unresponsive to 5HT administration. In the next series of experiments the

contraction produced hy Staphylococcal alpha toxin was compared with the effect

of Bit is gabonica venom. This venom elicited a rapid contraction which gra-

dually declined even without washing. With repeated doses the organ became

60'

5HT B0

8 5

ng gg

Fig. 2

Effect of snake venom from Bitis gabonica (BG) on isolated rat

stomaeh preparation.

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10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3):1011-1016, 1966

K. MASEK and H. RASKOVÀ

1013

insensitive lo further administration of snake venom. On lhe other hand l>oth

serotonin and Staphylococcal alpha toxin elicited a contraetion vvhen administered

after the snake venom but the character of contraetion differs. The first rapid

phase is not so evident (Figs. 2 and 3). As may be see from Fig. 4, Staphy¬

lococcal alpha toxin administered to the bath rendered lhe organ almost insen¬

sitive to Bilis gabonica venom administration.

i , i > . ■ 11 i~i— ljli i » i t < i i i 11 ixj-J i i 11 ti i.i.i i n 11 i

Fig. 3 — Effect of snake venom from Bitis gabonica (BG) on the contraetion elicited by

Staphylococcal alpha toxin (ST) administration.

5 Hl ST 5HT BG

2 4 Q9 2 5

n 9 HU ,%)• ng gg

Fig. 4 — Effect of Staphylococcal alpha toxin on the contraetion elicited by snake

venom from Bitis gabonica (BG) on the isolated rat stomach.

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10 11 12 13 14 15 16

1014

THE ACTION OF STAPHYLOCOCCAL ALFHA TOXIN AND THE

VENOM FROM BITIS GABONICA ON RAT STOMACII STRIP

When Closlridium perfringens loxin was aclministerecl lo lhe stomach strip

a slow contraction develops after latency period. A second administralion of

ihis toxin was much less effective and the organ lost also its sensitivily lo origi-

nally active doses serotonin (Fig. 5). Also it was impossible to elicit a con¬

traction with Staphylococcal alpha toxin (Fig. 6). The application of Staphylo-

coccal alpha toxin prior to Clostridium perfringens toxin administration caused

insensitivity of tissue to Clostridium perfringens toxin (Fig. 7).

30"

Fig. 5 — Effeet of Clostridium perfringens (CP) toxin on the isolated rat

stomach preparation.

■ ng

30"

ng HU

Fig. 6 — Effeet of Clostridium perfringens toxin (CP) on the contraction elicited by

Staphylococcal toxin (ST) on the isolated rat stomach.

Discussion

From lhe rcsults descrihed above il would seem thal there are two clearly

different phases of the contraction and it might be that they are due to two

different components in the Staphylococcal culture filtrates which contain phos-

pholipases (3, 4). It is not yet clear at presenl to which toxin (alpha or beta)

they belong. We therefore used a snake venom rich in phospholipase A and

Clostridium perfringens toxin rich in phospholipase C to try to contribute at least

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10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3): 1011-1016, 1966

K. MASEK and II. RASKOVA

1015

1 20

r>9 M9

3Q H

‘ ‘ iT . II, - 1 -i-1- i -1 i I I

Fig. 7 — Effect of Staphylococcal toxin (ST) on the contraction elicited on the rat

stomach preparation by Clostridium perfringens toxin.

indirectly lo the question whether the observed spasmogenic activity would he

attributable to phospholipases activity.

Venom toxin from Bilis gabonica does not prevent Staphylococcal alpha

toxin from its activity but the character of contraction is different. The first

rapid phase is not so evident. On the other hand Staphylococcal alpha toxin

does prevent lhe snake venom from its activity completely. This would mean that

part of Staphylococcal alpha toxin action corresponding to the rapid phase on

isolated rat stomach are similar to action of venom toxin from Bilis gabonica

and could he attribute to phospholipase A action. Since Clostridium perfringens

prevents Staphylococcal alpha toxin from its activity as well the Staphylococcal

alpha toxin prevents the action of Clostridium perfringens il would he reasonable

to assume that both toxins might aet on the same place of the phospholipide

molecule. F urther investigations are in progress.

We hope that this small contrihution testifies that comhined studies of hacte-

rial and animal venoms might he of considerahle interest.

Acknowledgments — The authors are most grateful to Dr. A. W. Bernheimer

for the gift of Staphylococcal alpha toxin and to Dr. F. Kornalík for the gift of

venom from Bitis gabonica.

References

1. NORTH, E. A., and DOERY, H. M., Brit. J. exp. Pathol., 41, 234, 1960.

2. VANE, J. R., Brit. J. Pharmacol., 12, 344, 1957.

3. DOERY, H. M., MAGNUSSON, B. J„ GULASEKHARAM, J., and PEARSON,

J. E„ J. gen. Microbiol., 40, 283, 1965.

4. MAGNUSSON, B. J., DOERY, H. M., and GULASEKHARAM, J„ Nature, 19fi,

270, 1962.

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10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(3): 1017-1020, 1966

W. RAAB and E. KAISER

1017

49. NEPHROTOXIC ACTION OF SNAKE VENOMS

W. RAAB and E. KAISER

Department of Medicai Chemistry, University of Vienna, Vienna, Áustria

Experimental investigations have shown that animal venoms exert a nephro-

loxic effect (1, 2). Thus, the clinicai findings in subacute snake poisoning have

been confirmed (3, 4, 5, 6).

The effects of snake venoms on the kidney reveal a most complex pattern.

Several toxic components of the venom act either directly or indirectly on renal

cells. Toxic degeneration of glomerular cells produces nephritis (7, 1, 8), toxic

alteralion of tubular cells evokes nephrosis (9, 10, 11, 1, 12, 13, 14, 15). Toxic

lysis and degeneration of red blood cells or muscle cells (malayan sea snakes)

provoke hemoglohinuric (4, 16) or myoglobinuric (14, 17) nephrosis. Toxic alte-

ration of nerve cells cause an increased susceptibility of kidneys to all kinds of

damages. Thus, an aggravation of the above-mentioned nephrotoxic effects of

snake venom occurs (18, 19). In addition, enzymatic cellular lysis and ihrom-

bosis provoke renal infarct (7, 3, 6). Furthermore, the elicitation of anaphy-

lactoid shock which is regularly found following snake poisoning (20, 21, 22, 23)

causes renal hypoxia by collaps and vasoconstriction (se.e Tahle I).

TABLE I

EFFECTS OF SNAKE VENOMS ON THE KIDNEY

A. TOXIC EFFECTS

Renal glomerular cells

Renal tubular cells

Red blood cells

Muscle cells

Nerve cells

Nephritis

Nephrosis

Hemoglohinuric nephrosis

Myoglobinuric nephrosis

Increased renal susceptibility

B. ENZYMATIC EFFECTS

Cellular lysis t

Thrombosis j

Formation of anaphylatoxin

Mast cell degranulation

renal infarct

anaphylactoid

shock

hypoxic

nephrosis

SciELO

10 11 12 13 14 15 16

1018

NEPIIROTOXIC ACTION OF SNAKE VENOMS

Following administration oí snake venoms. three types of renal damage can

occur: glomerular alterations (nephritis), tubular alterations (nephrosis), and

renal infarct. The glomerular alterations consist in inflammatory and degene-

rative ehanges. The tubular alterations — which seern to be the most important

in loxinologie respeet — are produced by a direct toxic aetion (“intermediate

nephron nephrosis”), by extrarenal cell degeneration (hemoglobinuric nephrosis),

and by vascular hypoxia (Table II).

TABLE II — RENAL LESIONS FOLLOWING ADMINISTRATION OF SNAKE VENOM

1. GLOMERULAR ALTERATIONS (NEPHRITIS)

a. Degenerattve ehanges

b. Inflammatory ehanges

2. TUBULAR ALTERATIONS (NEPHROSIS)

a. direct toxic effects (intermediate nephron nephrosis)

b. seeondary effects by extrarenal cell degeneration

(hemoglobinuric, myoglobinuric nephrosis)

vascular hypoxia

3. RENAL INFARCT

Until now, only pathologic and histologic examinations ol kidneys in snake

poisoning have been performed. In order to obtain information on the degree

of renal damage in vivo and on the duration of nephrotoxic effect, biochemical

investigations (24, 25, 26, 27, 15, 28) of renal function have been carried out

following administration of snake venom.

After determination of normal alkaline phosphalase (AP) and “leucine ami-

nopeptidase” (LAP) aetivities in 24-hours urine specimens (200 rats), 50 rats

reeeived a sublethal dosis of venom of Agkistrodon piscivorus LACEPEDE in sa-

line (10 mg/kg subcutaneously). Daily determinations of enzymatic aetivities

in urine vvere carried out for four days following administration of the snake

venom. The enzymatic aetivities were ealeulated on 24-hours urine excretion.

As shown in Fig. 1, a significant increase in urinary AP- and LAP-aetivilies

occurs following injection of snake venom. On the second day, only a slight

increase in urinary enzymatic aetivities is found. Beginning from lhe third day,

normal aetivities were observed.

In 24-hours urine specimens following administration of snake venom, LAP-

activity increases lo the fivefold, AP-activity ordy to the twofold. AP-excretion

depends upon renal tubular cells: following damage of these cells, an increase in

urinary AP-activity occurs. The same statement applies for urinary LAP-acti-

vity: damage of tubular cells increases FAP-activity in urine. In addition, uri¬

nary LAP-activity is influenced by renal plasmine (see Table III). The acti-

vation of renal |)lasminogen provokes an activation of renal peptidases; this

increases LAP-aetivity in urine.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(3): 1017-1020, 1966

W. RAAB and E. KAISER

1019

TABLE III — MECHANISM OF INCREASE IN LAP- AND AP-ACTIVITY IN RAT

URINE FOLLOWING ADMINISTRATION OF SNAKE VENOMS

SNAKE VENOM

formation of anaphylatoxin,

Mast cell degranulation,

formation of

biologically active peptides

anaphyiactoid shock

constriction of

renal blood vessels

hypoxic nephrosis

cytotoxic effect

on tubular cells

excretion of

eellular

degradation products

activation of fibrinolysis

activation of renal

profibrinoiysin

activation of renal

peptidases

increased

AP-activity

LAP-activity

in urine

venom

Fig- 1 — LAP- and AP-activities in rat urine (24 hours values) following injection

of snake venom ( Apkistrodon piscivorus). (Substrates: L-Leucyl-p-nitroanilide;

Na-p-nitrophenylphosphate).

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10 11 12 13 14 15 16

1020

NEPHROTOXIC ACTION OF SNAKE VENOMS

6 .

8 .

10 .

11 .

12 .

13.

14.

15.

16.

17.

18.

19.

20 .

21 .

22 .

23.

24.

25.

26.

27.

28.

29.

In anaphylactoid shock, an activation of fibrinolytic system occurs(23).

addition, certain snake venoms produce a direct activation of filirinolytic

cm (29). Tubular damage by snake venom increases AP- and I.AP-activities

trine. Activation of renal plasminogen causes a further increase in LAP-acti-

in urine.

Heferences

NOWAK, J„ Ann. Inst. Pasteur, 12, 369, 1898.

TAUBE, H. N., and ESSEX, H. E., Arch. Pathol., 24, 43, 1937.

BOQUET, P., Toxicon, 2, 5, 1964.

CORKILL, N. L„ in E. E. BUCKLEY, and N. PORGES (Editors), Venoms,

Amer. Ass. Advanc. Sci„ Washington, 1956.

DANZING, L. E., and ABELS, G. H„ J.A.M.A., 175, 136, 1961.

ORAM, S., GROSS, G„ PELL, L„ and WINTELER, J„ Brit. med. J., 1, 1647,

1963.

AZEVEDO, A. P., c TEIXEIRA, J. C., Mem. Inst. Oswaldo Cruz, 33, 23, 1938.

PEARSE, R. M„ J. exp. Med., 11. 532, 1909.

AMORIM, M. F., MELLO, R. F. de, Mem. Inst. Butantan, 24, 281, 1952.

AMORIM, M. F., MELLO, R. F. de, Amer. J. Pathol., 30, 479, 1954.

FIDLER, H. K., GLASGOW, R. D., and CARMICHAEL, E. B., Proc. Soc. exp.

Biol. Med. (N. Y.), 38, 892, 1938.

MITCHELL, S. W., Smithsonian Contr. Know., 12, 1, 1860.

MITCHELL, S. W., N. Y. Med. J., 6, 289, 1868.

MITCHELL, S. W., and REICHERT, E. T., Smithsonian Contr. Know., 26,

1, 1886.

RAAB, W., Naturwissenschaften, 53, 43, 1966.

ROSENFELD, G., in Die Giftschlangen der Erde, Marburg/Lahn, Elwert,

1963.

REID, H. A., Brit. med. J., 1, 1284, 1961.

MILLES, G„ MÜLLER, E. F„ and PETERSEN, W. F„ Arch. Pathol., 13, 233,

1932.

MILLES, G„ MÜLLER, E. F., and PETERSEN, W. F„ Proc. Soc. exp. Biol.

Med. (N. Y.), 28, 561, 1931.

KAISER, E., and RAAB, W., Z. antjew. Zoo/., 52, 1, 1965.

RAAB, W., and KAISER, E., Arch. klin. exp. Dermatol., 220, 374, 1964.

RAAB, W., and KAISER, E., Toxicon, 3, 49, 1965.

RAAB, W„ and KAISER, E., Klin. Wschr., 43, 345, 1965.

BERGMANN, H., and SCHELER, F„ Klin. Wschr., 42, 275, 1964.

BERGMANN, H., and TRUSS, F„ Med. Welt, 35, 1760, 1964.

RAAB, W., Klin. Wschr., 78, 364, 1966.

RAAB, W., Experientia (Basel), 22, 91, 1966.

RAAB, W., and KAISER, E., Experientia (Basel), 21, 720, 1965.

KAISER, E., and MICHL, E., Die Biochemie der tierischen Gifte, Wien, F.

Deuticke, 1958.

4 .

6 .

8 .

10 .

11 .

12 .

13.

14.

15.

16.

17.

18.

19.

20 .

21 .

22 .

23.

24.

25.

26.

27.

28.

29.

2 3

5 6

11 12 13 14 15

ÍNDICE DOS AUTORES

— AUTHOR INDEX i

Adam, K. R. — (2) =603-614

Elliott, W. B. — (2) =411-424 |

Amaral, A. do — (1) =293-303

Espinoza, N. C. de — (3) =799-808 |

Amorim, F. 0. — (1):135-150

Fain, A. — (1) =167-174; (3) =835-844 1

Antônio, A. —- (21=453-456; (3) =957-

Fallieri, L. A. — (1) =135-150 1

| 960

Farina, R. — (3) =993-1000 I

í Arvy, L. — (3) :711-724

Franceschi, J. P. — (3) =973-980 1

Augustyn, J. M. —- (2) :411-424

Franco de Mello, R. — (3)761-766 j

1 Ashgar, S. S. — (3) :943-950

Gajardo-Tobar, R. — (1) =45-54; (3): |

i Bachmayer, H. — (3) :903-906

689-698

| Badano, B. N. — (3) :913-920

Gans, C. —- (2) =411-424 I

Bailey, J. R. — (1) :67-72

Ganthavorn, S. — (1) =327-330 1

Barrio, A. — (3) =809-820, 865-868, 869-

Gennaro, J. G. — (1) =175-178; (3): 1

880, 887-892

855-856

Beçak, M. L. — (1) =151-152

Gerken, S. E. — (1) =135-150

Beçak, W. — (1) =151-152; (3) =775-798

Gerschman de Pikelin, B. S. — (3) =667- J

Belluomini, H. E. — (3) =761-766

674, 675-682 j

Betz, T. W. — (1) =115-120

Gillo, L. — (3) =933-936, 937-942 j

Bicher, H. I. — (2) =523-540

Gomes Lomba, M. — (3) =921-928 I

Boquet, P. — (2) =371-378; (3) =929-932

Gomez, M. V. — (2) =453-456; (3) =899-

! Bourillet, F. — (2) =541-554

902

[ Boveris, A. — (3) =913-920

Hadler, W. A. — (3) =1001-1008

Brongersma, L. D. — (1) =73-79

Halstead, B. W. — (1) : 1 -26

] Chang, C. C. — (2) =555-572; (3) =1009-

Henriques, O. B. — (21=359-370

1010

Henriques, S. B. — (2) =359-370

Chapman, D. S. — (1) =213-226

Hoge, A. R. — (3) =735-744

Cheymol, J. — (2) =541-554

Houssay. B. A. — (1) :XIII-XVI

Chiu, T. H. — (3) =1009-1010

Ibarra-Grasso, A. — (3) =809-820

1 Christensen. P. A. — (1) =245-250, 305-

Izard, Y. — (2) =371-378; (3) =929-932

326

Jouannet, M. — (2) =371-378; (3) =929-

I Cochran, D. M. — (1) =61-65

932

Cohen, P. — (1) =339-347; (3) =881-886

Kaiser, E. — (2) =461-466; (3) =1017-

| Corrado, A. P. — (2) =453-456; (3) :

1020

| 957-960

Kieífer, J. — (3) =921-928

i Corrêa, D. D. — (1) =27-30

Kitajima, E. W. — (3) =701-710 |

1 Cruz Landim, C. da — (3) =701-710

Klemmer, K. — (11:101-103 1

I Daly, J. W. — (2) =425-432

Kondo, H. — (1) =193-205, 331-337

1 Darevsky, I. S. — (1) =81-83

Kondo, S. — (1) =193-205, 331-337

K Delgado, A. — (3) =683-688, 829-834

Kornalík. F. — (1) =179-188

1 Del Pozo, E. C. — (2) =615-626; (3):

Krag, P. — (1) =251-280

1 961-964

Kurokawa, M. — (1) =331-337

I Deoras, P. J. — (3) =767-774

Lande, I. S. de Ia — (3) =951-956

E Devi, A. — (2) =573-582; (3) =943-950

Lancini Villalaz, A. R. — (3) =725-734

1 Diaz, M. 0. — (1) =153-154

Latifi, M. — (3) =735-744, 893-898

1 Diniz, C. R. — (2) =453-456; (3) =899-

Lauhatirananda, P. — (D =327-330

1 902, 957-960

Lee, C. Y. — (2) =555-572; (3) =1009-

1 Efrati, P. — (1) =189-191

1010

H Eliazan. M. — (3) =735-744

Lee, S. Y. — (3) =1009-1010 1

SciELO

10 11 12 13 14 15 16

1022

ÍNDICE DOS AUTORES

AUTHOR INDEX

Lello, E. de — (3) :821-828

Lewis, J. C. — (3) :951-956

Lieske, H. — (1) :227-233

Lissitzky, S. — (2) :447-452

Lutz, B. — (1) =55-59

Machado de Lemos, M. — (1):XII

Mandelbaum, F. R. — (2) :359-370

Manhouri, H. — (3) :893-898

Markwardt, F. — (3) :851-854

Masek. K. — (3) :1011-1016

McCollough, N. C. — (1) :175-178; (3):

855-856

Meaume, J. — (2) :371-378; (3) =929-

932

Me bs, D. — (3) :907-912

Melucci, N. B. — (1): 135-150

Mertens, R. — (1) =85-92

Meyer, K. — (2) :433-440

Michl, H. — (3) :903-906

Miranda, F. — (2) :447-452

Miranda, M. E. — (3) :869-880, 887-892

Murata, R. — (11:193-205, 331-337

Nazareth, H. — (1) : 151-152

Ohno, S. — (1): 155-166

Ohsaka, A. — (11=193-205, 331-337

Okada, M. — (2) :589-602

Oliveira, V. A. de — (3) :993-1000

Omori-Satoh, T. — (l):193-205

Pacheco, P. (3) :961-964

Penha, A. M. — (3) :761-766

Pereira Lima, F. A. — (2):627-638

Pesce, H. — (3) :829-834

Pisano, J. J. — (2) .-441-446

Porath, J. — (21:379-388

Puranananda, C. — (1) .-327-330

Raah, YV. — (21:461-466; (3) =1017-

1020

Raskovà, H. — (31:1011-1016

Rathmayer, YV. — (21:651-658

Raudonat, H. W. — (3) :907-912

Rawlinson, P. — (11:235-240

Reed, C. F. — (2) :645-650

Roch, M. — (21:541-554

Rocha e Silva, M. — (2) :457-460

Rochat, C. — (2):447-452

Rochat, H. — (2) =447-452

(2) =477-508

(11 =241-244

— (2) =467-476

(2):583-588; (3) =845-

Rosenberg, P. -

Rosenfeld, G.

Rothschild, A. M.

Russell, F. E. —

850

Saez, F. A. — (1) =153-154

Saint-Girons, H. — (1) =105-114

Salas, M. — (3) =961-964

SanFAna. Y. X. — (1) =135-150

Sarkar, N. K.

950

Sawaya, P. — (1

Schenhenr. S. —

(2) =573-582; (3) =943-

i =31-34

(21 =627-638

1) =207-211

- (3) =667-674, 675-

Schenone, H. —

Schiapelli, R. D.

682

Schildknecht, H. — (1) =121-133

Schreiber, G. — (11:135-150; (3) =761-

766

Seligmann, Jr., E. B. — (1) =339-347

Shulov, A. — (1) =93-99

Stanic, M. — (3) =745-760

Stoppani, A. O. M. — (3) =913-920

Suga, T. — (31:965-972

Suzuki, T. — (2) =389-410, 519-522

Trethewie, E. R. — (1) =235-240; (3):

861-864

Tseng, T. C. — (3):1009-1010

Vallejo-Freire, A. — (1):XVI1-XX

Vannucci, M. — (3) =659-666

Velasco, J. M. — (3) =857-860

Vellard, J. — (1) =35-44; (3) =699-700

Vidal, J. C. — (3) =913-920

Vital Brazil, O. — (3) =921-928, 973-980,

981-992, 993-1000, 1001-1008

Waisbich, E. — (3) =921-928, 973-980

YVeis Bentzon, M. — (1) =251-280

Weiss, Ch. — (21=603-614

Welsh, J. H. — (2) =509-518

Wirtheimer, C. — (3) =937-942

Witkop, B. — (2) =425-432

Witt, P. N. — (21=639-644

Yoshida, L. — (3) =993-1000

Zeller, E. A. — (2) =349-358

Zwisler, O. — (1) =281-291

SciELO

10 11 12 13 14 15