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TRANSACTIONS

OF THE

American Microscopical

Society

VOLUME XXXVII

1918

TRANSACTIONS

OF THE

American Microscopical

Society

ORGANIZED 1878 INCORPORATED 1891

PUBLISHED OQUARTEREY

BY THE SOCIETY

EDITED BY THE SECRETARY

VOLUME XXXVII

NUMBER ONE

Entered as Second-class Matter December 12, 1910, at the Post-office at Decatur,

Illinois, under act of March 3, 1879.

DEcATuR, ILL.

REvIEW Printinc & STATIONERY Co.

1918

TABLE OF CONTENTS

FOR VOLUME XXXVII, Number 1, January 1918

Insects as Carriers of Disease, by Malcolm Evan MacGregor............ Ps

Acanthocephala of North American Birds, with Plates I to V, by H. J.

Wianin Cleauerc cc rintras etl euler a heriocis ig a se oot ta scaMlaley s aleler ere) cya Sretteveh eet 19

Branchiobdellid Worms (Annelida) from Michigan Crawfishes, by Max

Wehbe Seg Set aa le ee MN ee 8 Se EA Leg trae rear 49

Notes and Reviews: A Chart on General Plant Histology and Physiology

(Plate VI), Raymond J. Pool; Method of Mounting Anatomical

Preparations for Exhibition, G. G. Scott; Green Light for Demon-

strating Living Cestode Ova, M. M. Ellis; A New Method of Stain-

ing Tissues Containing Nerves, Fontana’s Spirochete Stain, Simple

Method of Cleansing Old Slides, Menthol for Narcotizing, abstracted

by V. A. Latham; Freshwater Biology (Ward and Whipple); An

Introduction to the History of Science (Libby) ; A Short History of

Science (Sedgwick and Tyler); Biochemical Catalysts in Life and

Read easityy: CEE T ORIG ) shoe ots oh acd wah tence os ae ataecled ae ear at alan 4S Sane ane of fs 53

Minutes ot the Pittsbure Meeting. aie5 2.5 he soe aes ole aig Sas caeraeiaate 7i

PAAR SU ECCDOEE (6 a5, ois 8 oh altel visita kslosaiseoarehai ot Alga Ga monde sielkaitasene saree 72

WEES GEE SUMEDOEEN S: Go)) db nek snob etna son neh Who save EME, ks AS ate age 73

(This Number was issued on March 25, 1918)

OFFICERS

President =) No. ES. (GRIRVING 2 ois. 52! 40,5 Sis cies s amie bole eee sires Pittsburg, Pa.

First Vice President: H.M. WHELPLEY................-0000- St. Louis, Mo.

Secona Vice President: (GC. (0. ESTERLY ..oe sos 33 ove sie ne Los Angeles, Cal.

Secretar ye) (che WoGAEEOWy os ade. Ssidieid Selete weal ete bere ole wie eee one Beloit, Wis.

Dv CRSUFEKS ERS yc VAM OCTBAVE! « S'c'5.8 2 eis Sinks oe Da Ale o oe aie Urbana, IIl.

Gustodian> | MAGNUS (EFEAUME Halas Siete. soviet ee lantels kore se iets Meadville, Pa.

ELECTIVE MEMBERS OF THE EXECUTIVE COMMITTEE

1 ey INTE Dh ee a ee ASS AEG "2 3 Boulder, Colo.

DESPRE RERT Soe aye airs wie ais abla cee 5 50 p siaieis wi a ele Svs sib lelesopginibl arn Sine Manhattan, Kas.

EX-OFFICIO MEMBERS OF THE EXECUTIVE COMMITTEE

Past Presidents Still Retaining Membership in Society

ALBERT McCa.ta, Ph.D., F.R.M.S., of Chicago, Ili.

Geo. E Fett, M.D., F.R.M.S., of Buffalo, N. Y.,

Simon Henry Gace, B.S., of Ithaca, N. Y.,

at Chicago, IIl., 1883

at Detroit, Mich., 1890

at Ithaca, N. Y., 1895 and 1906

A. Currrorp Mercer, M.D., F.R.M.S., of Syracuse, N. Y.,

at Pittsburg, Pa., 1896

at New York City, 1900

at Denver, Colo., 1901

at Winona Lake, Ind., 1903

at Sandusky, Ohio, 1905

A. M. Briere, M.D., of Columbus, Ohio,

C. H. E1icenmann, Ph.D., of Bloomington, Ind.,

E. A. Birce, LL.D., of Madison, Wis.,

Henry B. Warp, A.M., Ph.D., of Urbana, IIl.,

S. lumb hio, 4 (

Herpert Oszorn, M.S., of Columbus, Ohio ai Minacapalie ans gid

at Washington, D. C., 1911

at Cleveland, Ohio, 1912

at Philadelphia, Pa., 1914

at Columbus, Ohio, 1915

at Pittsburg, Pa., 1917

A. E. Hertzver, M.D., of Kansas City, Mo.,

F. D. Heap, Ph.D., of Philadelphia, Pa.,

CHARLES BrooKover, Ph.D., of Louisville, Ky.,

Cuartes A. Kororp, Ph.D., of Berkeley, Calif.,

M. F. Guyer, Ph.D., of Madison, Wis.,

The Society does not hold itself responsible for the opinions expressed

by members in its published Transactions unless endorsed by special vote.

TRANSACTIONS

OF

American Microscopical Society

(Published in Quarterly Installments)

Vol. XXXVII JANUARY 1918 No. 1

INSECTS AS CARRIERS OF DISEASE

By Matcotm Evan MacGrecor

Wellcome Bureau of Scientific Research

So much has been said about insects since the War began that

it is, I think, advisable that some attempt should be made to sum-

marize our knowledge of the more important insect-borne diseases

and their vectors. While insects have long been suspected of being

responsible for the transmission of serious diseases, it may be said

that practically the whole of our knowledge of insects in this rdle

has been acquired within the past twenty years. So rapidly, how-

ever, has the charge of this offence been made out against them

that, although it is common knowledge they have been proved guilty,

it is not generally realized upon how many counts the verdict rests.

It has lately been my good fortune to give class instruction

for the War Office to officers of the R. A. M. C. who are proceed-

ing to the East, and, in order to bring home to my audience the

importance of the connection between insects and disease, I have

compiled the tables which I now publish. These can in no way

claim to be complete, but merely present the more important insect-

borne diseases, including important human diseases that on certain

grounds are suspected of having insect vectors. With these tables

I also publish one (Table VI) which includes the chief insects and

acarina that are directly the cause of disease in man and his domestic

animals. To complete the list of insect-transmitted diseases would

demand the consideration not only of other mammals as hosts,

but also of avian and reptilian hosts. In the present instance this

would be to carry the subject beyond general interest, but it must be

remembered, therefore, that, long as the present list of charges

is, insects are not here arraigned on all the counts that might with

justice be preferred against them.

Reprinted from Journal of Tropical Medicine and Hygiene, Sept. 15, 1917; xx, 18.

8 M. E. MAC GREGOR

During the last few years medical entomology has been rapidly

establishing itself an an invaluable branch of preventive medicine,

and with the outbreak of the present War a great deal of interest

and study has been devoted to this subject in Europe, notably in

connection with the transmission of typhus fever by lice, and the

dissemination of bacteria and other organisms by flies. Moreover,

the importance of insect vectors has been generally realized, and

many of the astonishing interactions between pathogenic micro-

organisms and certain arthropoda have become popular knowledge.

Centuries ago insects were suggested as being possibly con-

cerned in the spread of disease, and from time to time such logical

hypotheses were advanced that it is surprising that the establishment

of the truth was not sooner forthcoming. In 1577 Mercurialis, an

Italian physician, suggested that plague, which was then ravaging

Europe, was spread by flies feeding upon the diseased and dead, and

later depositing feecal matter on food consumed by healthy persons.

This idea held ground, and various suggestions occur as to the

spread of disease by flies in the literature of the eighteenth century.

Edward Bancroft in 1769 advanced the theory that “yaws’” was

transmitted by flies feeding on diseased subjects, and carrying the

contagion by settling on open wounds or scratches on healthy per-

sons.

In 1848 Dr. Josiah Nott, of Mobile, Alabama, published a

remarkable article in which he gave reasons for supposing that

yellow fever was an insect-borne disease. However, although he

mentioned many insects, he did not specify any insect as the particu-

lar vector.

The connection between malaria and the mosquito had long

been held, it is said, by the Italian and Tyrolese peasants, and even

by the natives of East Africa, but the first charge brought against

the mosquito in the spread of disease by scientific authority was in

connection with yellow fever.

In 1853 Dr. Daniel Beauperthuy, a French physician, wrote

ably arguing that yellow fever and other fevers were transmitted by

mosquitoes, but in those days there being no accepted belief in a

contagium vivum, he concluded that the virus was obtained from

decomposing material that the mosquito had consumed, and which

INSECT CARRIERS OF DISEASE )

was later accidentally inoculated into man. MRaimbert in 1869

showed by experiment that anthrax could be disseminated by flies.

Epoch-making in the history of our knowledge of insect vectors

was Manson’s discovery in 1878 that Filaria bancrofti was spread

by mosquitoes, but at first he thought the filarie escaped from the

insect into water, and reached man in this manner. Later work

by Manson and his colleagues determined the exact means of trans-

mission.

It was not until twenty-eight years after Beauperthuy’s theory

that Charles Finlay, an American of Havana, in 1881 definitely

attributed the transmission of yellow fever to a mosquito of a

definite species. He had noticed the connection that seemed to exist

between the presence of large numbers of Stegomyia fasciata and

the prevalence of yellow fever. He then attempted to transmit

the disease experimentally by the bites of this mosquito, and al-

though his experiments are open to criticism, there is no doubt that

he did succeed in doing so.

Three years later, in 1883, another American, A. F. A. King,

advanced the first well formulated mosquito-malaria theory, and in

1898 Ross, in India, demonstrated beyond doubt the important role

played by mosquitoes in the transmission of malaria.

In 1899 the American Yellow Fever Commission (Reed, Car-

roll, Lezear, and Agramonte) were sent to Cuba, and were there

able to demonstrate with certainty that yellow fever is transmitted

by S. fasciata.

It is interesting thus to note the almost parallel development

in time of our knowledge of two of the most important insect-borne

diseases. To deal even briefly with the historical aspects of our

knowledge of other diseases tabulated below would be to consume

a large amount of space, and the foregoing account will have indi-

cated the path that has led to subsequent discoveries whose histories

are readily available.

I will pass, therefore, to a few notes on each of the tables.

Notes To TABLE I.—DIsEASES OF UNKNOWN ORIGIN

The majority of these diseases are doubtless caused by living

viruses; often organisms of ultra-microscopic size, and commonly

referred to as “filterable viruses.”

10 M. E. MAC GREGOR

In the case of pellagra it would appear, however, from the

most recent work that, although it is still considered by many

persons to be a possible insect-borne disease (and, according to

Sambon, having a likely vector in either the Ceratopogonine or

Simulude), Goldberger in America considers it a disease now cer-

tainly attributable to vitamine-starvation through an wnbalanced

diet. If this is the case, there is no causative organism and no vector,

and pellagra should be ruled out of present consideration. The

question, nevertheless, is by no means settled.

The virus of acute anterior poliomyelitis is still not isolated

with certainty. Flexner and his colleagues have been able to culti-

vate a filterable micro-organism which produced the disease in

experimental animals, and more recently Rosenow and his fellow

workers have isolated a polymorphous streptococcus, with which

they were also able to proauce paralysis in certain animals. Nuzum

and Herzog were able to do likewise by a Gram-positive micrococcus

isolated from the brain and spinal cord of persons dead from the

disease.

Poliomyelitis has been very generally suspected of being trans-

mitted by insects, particularly by Stomoxys calcitrans (the stable-

fly), fleas, and Tabanide (gad-flies). Nevertheless, it appears more

likely that it has an eerial transmission, infection being acquired

through the buccal and nasal mucous membranes.

The causative organism of Rocky Mountain spotted fever, Wol-

bach claims to have discovered in the bodies of infective ticks (Der-

macentor venustus ).

Notes To TABLE JI.—DIsEASES OF BACTERIAL ORIGIN

In the majority of cases, diseases of this class have an indirect

transmission by insects—that is to say, instead of the organism

entering the body of the host through inoculation by the bite of an

insect (direct transmission), the organisms are carried in or on the

insect’s body, and are deposited by contact on human food or skin

abrasions, and in this manner cause infection.

Bacillus tuberculosis may be disseminated by house-flies feeding

on infective sputum, as was first shown by Spillman and Haushalter

(1887), and subsequently by the investigations of other workers.

N. B.—Names between

Tasce I,

THE MORE IMPORTANT INSECT-BORNE DISEASE OF UNKNOWN ORIGIN

square brackets = certain vectors; names without square brackets = probable vectors; names followed by ? = possible

vectors.

Organism Host Disease Vector

De reigre fren ro ean aot Dengue (breakbone fever) er [Sandflies (Phlebotomus), Mosquitoes. C. fatigans; S. fas-

Three-day fever, syns. “Dog ciata] ;

Poseurs iets Hare Me ates disease,’ Sandfly fever, Phle- {Sandflies (Phlebotomus), Mosquitoes. C. fatigans; S. fas-

botomus fever ciata]

Ceeraieve etaie cele de AO Yellow fever noe ee [Mosquitoes (Stegomyia fasciata)]

Ree stete ote are we 500 Trench fever SOE rays Lice ?

? Salivary toxin? she dy oBe Tick paralysis (American) ciate [Ticks, (Dermacentor venustus)]

? Salivary toxin? fre ee wae Tick paralysis (Australian) est [Ticks (Ixodes ricinus)]

Papestars atio eee 2 Be Rocky Mountain spotted feve [Ticks (Dermacentor venustus)] :

Petetere teat ate Ud Ae Japenese river fever (shima [Mites (Larval trombidiide)] “aka mushi’”

mushi

Reetatas ware SAG bu ae Acute anterior poliomyelitis siene ? Many insects have been claimed as vectors, notably

Stomoxys calcitrans?

Pees mate ees dg wie Pellagra Save tan acre ? Gnats of the genus Simulium have been claimed?

Poeiais inv eee ? sats Typhus fever site eee [Lice]

- Taare II.

THE MORE IMPORTANT INSECT-BORNE DISEASES OF BACTERIAL ORIGIN

N. B.—Names between square brackets = certain vectors; names without square brackets = probable vectors; names followed by ? = possible

vectors.

The word “flies” includes in the main: Musca domestica, Fannia sps., Calliphora sps., Lucilia sps., and Sarcophaga sps.

Organism Host Disease Vector

Bacillus anthracis els iets .».» Man and animals Anthrax aye ... Flies], Tabanid@? Beetles? F

zy dysenterie ... thes wie’ ot: ere --- Bacillary dysentery dite [Flies] (Musca domestica, Calliphora sps.,

Lucilla _sps.) :

zh lepre aiale eine tele id 400 -.. Leprosy wee ahi LIES Fleas? Bed-bugs? Skin mites?

: : Mosquitoes?

# paratyphosus A arene wane ae ac ae Paratyphoid fever ats [Flies]

a B ofthe “aes ide Fate aes 4 # Ee a eesl

2 pestis a tere arts dire ” and rats ... Plague ACD ..-. [Fleas]

4 tuberculosis aiie aCe ” and animals Tuberculosis ... ... Flies, cockroaches, fleas? Bed-bugs?

Bacillus typhosus Riise ae verste wee Lae pense Dy DHOIantever -»- [Flies]

Bartonia bacilliformis «x-bodies stake ie fee cee Verruga AG ... [Phlebotomus verrucum] :

Spirillum cholere SAD wile ave dd shale -.-- Cholera arts ... [Flies], cockroaches, ants. Although the main

channel of infection is the consumption of

infected food and water

12 M. E. MAC GREGOR

With the high vitality and resistance to drying possessed by the

B. tuberculosis, the possibly long incubation period within the body

and the insidious onset of the disease, the danger from Musca

domestica in this connection is still not sufficiently recognized.

Human infection with plague and typhus has been shown to

be acquired principally by the entrance of the virus through skin

lesions, the insect vector having been crushed either during or

after the act of blood-sucking. The stomach contents or infected

excreta may be rubbed into the lesions or gain entry through

abrasions caused by scratching.

This, however, does not preclude the possibility of direct infec-

tion also occurring, at least sometimes in the case of plague, as the

infected flea has the proventriculus occluded by the plague organ-

isms when the flea infection is at its height. Septiceemia following

mosquito bites occasionally happens, and as likely as not the path-

ogenic organisms are introduced when the mosquito bites. Direct

transmission by blood-sucking insects may possibly also occur in

certain instances in the spread of tuberculosis and leprosy.

If Wolbach’s organism (see Table I and notes thereto) is

proved to be the cause of Rocky Mountain spotted fever, this will

also be a disease of bacterial origin with direct transmission through

a tick, Dermacentor venustus.

Notes To TABLE II]I.—DIsEASES OF SPIROCHZTAL ORIGIN

With these diseases the usual method of transmission is direct

—that is to say, through the bites of the insect vectors.

Exceptions occur in the case of relapsing fever transmitted

indirectly by lice, and yaws where Musca domestica may at times

convey the organism from diseased to healthy persons.

Notes To TABLE IV.—DISEASES OF PROTOZOAL ORIGIN

Both direct and indirect methods of transmission by insects

occur with diseases of this class. With the intestinal parasites,

indirect transmission takes place by the flies feeding on feces con-

taining the resistant stages (cysts), and later depositing them on

human food and drinking water either by regurgitation of the

stomach contents, or more often per anum, as Wenyon and O’Con-

c

Organism Host

Micrococcus melitensts ead Ane

Diplococcus intracellularis —... on Man

fs pemphigi contagiost mies yu.

Taste IJ.—(Continued.)

Tropical impetigo

Tasle III.

Disease

Man and goats... Undulant fever; syns.

Malta fever, Mediter-

ranean fever, Remit-

tent fever

aire ... Cerebrospinal fever ...

Vector

[Flies]. Although the main channel of infec-

tion is the consumption of goat’s milk

Flies?

[Lice]

THE MORE IMPORTANT INSECT-BORNE DISEASE OF SPIROCHATAL ORIGIN

N. B.—Names between square brackets = certain vectors; names without square brackets = probable vectors; names followed by ? = possible

vectors.

The word “flies” includes in the main:—Musca domestica, Fannia sps., Calliphora sps., Lucilia sps., and Sarcophaga sps.

Organism Host

Spirocha@ta carteri ROrhS Soe Man Aen

nde duttont Rec ort " erage

a! gallinarum ateceomeentey Fowls Aa

4 novyi Siraawe tele Man wits

zy pertenuis Seren WY apc

” recurrentis AC eerie, Lu cee

a berbera Steen te aie

Disease

Indian relapsing fever

African 2

Spirochetosis ai

American relapsing fever ...

Yaws (Frambeesia) aie

European relapsing fever ...

North African relapsing fever

.

TaBLeE IV.

eee

Mel (Tick fever) Wes

Vector

[Lice]

[Ticks (O. moubata, O. savigny))

[Argas persicus]

[Lice]

Flies?

[Lice].

[Lice]

Bed-bugs?

THE MORE IMPORTANT INSECT-BORNE DisEAsES OF ProTozoAL ORIGIN

N. B.—Names between square brackets = certain vectors; names without square brackets = probable vectors; names followed by ? = possible

The word “flies” includes in the main:

Organism Host

Entameba histolytica -.. Man ares

Lamblia intestinalis* Pare 3 seme

Plasmodium malarie sere As Noo

4 vivax ae 2 arate

ay falciparum oa =F are

vectors,

Disease

... Ameebic dysentery

... Flagellate ” ere

«+» Quartan malaria ...—

... Benign tertian malaria

... Malignant or subtertian

malaria

Musca domestica, Fannia sps., Calliphora sps., Lucilia sps., and Sarcophaga sps.

Vector

... [Flies]

... L[Flies)

..- [Anopheline mosquitoes]

... [Anopheline mosquitoes]

... [Anopheline mosquitoes]

14 M. E. MAC GREGOR

nor have shown recently.t_ Needless to say, infection also occurs—

and perhaps principally—by mechanical and erial transmission of

the cysts to food and water.

The majority of the protozoal blood parasites have insect

vectors, on which they depend solely for transmission, and in cer-

tain cases these vectors are specific: Malaria, Anophelines; sleep-

ing sickness, Glossine ; European relapsing fever, Pediculi. Other

insect-borne blood parasites are apparently able to be transmitted by

more than one vector, i. e., Kala-azar, bed-bug (Patton) ; kala-azar,

Triatoma rubofasciatus (Donovan); Souma (Trypanosomiasis),

Glossine ; Souma (Stomoxys calcitrans).

Notes To TABLE V.—DISEASES OF HELMINTHAL ORIGIN

With the exception of possible infection with certain helminths,

resulting from the carriage by flies of helminth ova from feces

and subsequent deposition of the ova on food, the insect-borne

helminths all undergo part of their life-history in the body of the

insect vector. Thus the adult Filaria bancrofti live in human

lymphatic glands. The ova find their way into the blood-stream,

where they hatch to the Microfilariz, and some are taken up from

the blood when a mosquito bites a person harbouring the organisms.

These, if they have entered the stomach of Culex fatigans, or other

intermediate host, soon make their way to the thoracic muscles of

the mosquito, where they undergo definite metamorphosis. When

this is complete (usually in from sixteen to twenty days) the worms

make their way into the mosquito’s proboscis, and when next it

pierces the skin of some victim the filariz burst through the pro-

boscis sheath and make their own passage through the skin, from

which they soon travel to some lymphatic gland, where they become

sexually mature, and the cycle is repeated. Similarly, Dipylidium

caninum passes part of its life-history in the rat flea, and becomes

sexually mature in the dog or man. The ova are ingested by the

larval flea, and infection by the cysticercoid stage follows the acci-

dental ingestion of the flea by the definite host.

“The Carriage of Cysts of Entameba histolytica and other protozoa, and eggs of

Parasitic Worms by House-flies, with some Notes on the Resistance of Cysts to Disinfect-

ants and other Agents.” C. M. Wenyon and F. W. O’Connor, Journal of the Royal Army

Medical Corps, May, 1917, p. 522.

TasLe IV.—(Continued.)

Organism Host Disease Vector

Leishmania tropica eee . ae ... Oriental sore re: eat -». Flies? Fleas? Phlebotomus?

Hippobosca?

ee donovani fev Le stare ... Kala-azar Ath Sere ... Bed-bugs? Fleas? Triatoma?

tt sp. incerta naw ste Mere ... Espundia Re ave ..» Probably some tropical blood-sucking

Insect

2 infantum ... Children Nat ... Leishmaniasis aL Rare ..+ Fleas?

Trypanosoma gambiense -». Man aves ..- Sleeping sickness... RG ... [Glossina palpalis (Tsetse flies)]

ae rhodesiense eine 4 moe pare | Leon aad ... (Glossina morsitans) ” u

2 brucei ... Cattle and horses ... Fly sickness (Nagana) ies ... (Glossina morsitans) _” Ps

td lewisi en ate Grave ... Rat trypanosomiasis aleve ... Rat louse? and [Rat fleas (Ceratophyllus

fasciatus), Ctenocephalus canis]

bg evansi .-.- Horses, mules, camels Sutras acs core aie tere io flies (Tabanidew), Tabanus striat-

us

Schizotrypanum crusi --» Man AP --» Chaga’s disease ... sera ... [Triatoma (Conorhinus) megistus]

Babesia bigeminum eee Cattle stale -.. Red-water fever ... nae ... [Ticks (Margaropus annulatus))

td ovis ane «++ Sheep aac ..- Piroplasmosis Seto Ante ..» [Ticks (Rhipicephalus bursa)]

fe! canis mais wre WORE sere ... Malignant jaundice ee ... [Ticks (Rhipicephalus sanguineus and

Hemaphysalis leachi)]

” caballi Bite ... Horses and mules ... Piroplasmosis tiare On ... [Ticks (Dermacentor reticulatus)]

Nuttallia equi Ge cere Us ” atin ot Rae ara ... (Ticks (Rhipicephalus evertsi)]

? Chlamydozoa ? atic Perey Evel AGO .»» Ophthalmia egyptica and other ophthal- [Flies]

mic conditions

*By some authorities Lamblia intestinalis is not regarded as pathogenic,

TaBLe V.

THE MORE IMPORTANT INSECT-BORNE DisEASES OF HELMINTH ORIGIN

N. B.—Names between square brackets = certain vectors.

The word “flies” includes in the main: Musca domestica, Fannia sps., Calliphora sps., Lucilia sps., and Sarcophaga sps.

Organism Host Disease Vector

Dipylidium caninum ... Man and dogs ... Teniasis (tapeworm) ... ... [Dog louse (Trichodectes latus)]; [Dog flea

(Ctenocephalus canis)]; and the [Human flea

(Pulex irritans)]

Ova of certain helminths Man Ati ... Helminthiasis (parasitic worms) [Flies] |

Microfilaria bancrofti ite « at ... Filariasis (Elephantiasis) ... [Mosquitoes (Culex and Anopheles sps.)]

Loa loa trae in Ms ScrC .». Calabar swellings aoe Ah Hees fiies (Chrysops dimidiata and Chrysops

silacea

Filaria immitis. | .». Dogs ie ... Dog filariasis are ... (Mosquitoes (Culex and Anopheles sp.)]

Hymenolepis diminuta Rats, and occasionally Tzniasis ce aes no Lees)

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INSECT CARRIERS OF DISEASE 17

Notes To TABLE VI.—Dtseases DiREcTLY ATTRIBUTABLE TO

INSECTS AND ACARINA

In each case the disease results from the damage done by the

insects and acarina in adopting existence upon the body of the

host and living upon its tissues. The time spent upon the host

may cover the whole life-history of the parasite and many subse-

quent generations, as with Sarcoptes scabiei and the Pediculi, or it

may only embrace part of the parasite’s development, as with the

larve of flies causing myiasis. In either case the injury to the host

may be so extensive as to cause death from the loss of vital tissue,

or the injury itself, although insignificant, may indirectly cause

death to the host by providing a suitable path for invasion by patho-

genic micro-organisms.

CONCLUSION

It should be borne in mind that a large number of diseases

included in the foregoing tables are not confined to being spread by

insects, and insect transmission may in some cases only be occasional.

This fact, however, cannot afford the preclusion of such diseases

from consideration, and where transmission of the indirect type is

possible, it is obvious that we are unable to form any just estimate

of its relative importance. Probably, nevertheless, the dissemina-

tion of pathogenic micro-organisms by flies, for all that has lately

been said in this connection, has not even yet been over-emphasized.

It seems more than likely that Bacillus tuberculosis is spread in this

manner from infective sputum to food (milk particularly) to a

much greater extent than is commonly imagined, and there being

no probability of rapid acute infection, as with B. typhosus, the

part played by the fly is too apt to be overlooked.

Much of our knowledge with regard to insects and disease

is still indefinite, as may be seen from the tables, but to anyone

not particularly conversant with the subject, what we already know

with certainty, even in connection with only the more important

diseases that have been considered, may be sufficient to cause

some little surprise.

THE ACANTHOCEPHALA OF NORTH AMERICAN BIRDS*

VIII.

H. J. Van CLEAVE

CONTENTS

AER OCUIETECIEN IM oh eens 2 ane kaa fed oe otto al fave Reta Urea

ene) Gents) COtyneSOmia.: ois Jaze aye <5 6 odele!-\u rd os Sal piaecal sto lesa

Corynosoma constrictum NOV. SPEC.... 6... cece cere eee e eee eee

(he Genus PlaciothyncHus.. oi. is)i5e alas odes «lec elemee tees sis

Plagiorhynchus rectus (Linton) ..........00cceeseesereeeeee

Plagiorhynchus formosus nov. SP€C...... 0+. ee eee cece ee eeees

hie Gebus \Polymorphes: <<) 5:6)... s\< se-6 6 olsieeretele,sineles vale cwieis

Polymorphus obtusus NOv. SPCC.... 2... cece cece eee eee e renee

LUD GLANS, SOE Oe opis ct le oie vias pie ele: ci biota wisialvisiai eal ore lei oisin)» os /4)5 si

Mie GedusTOentrornyHCHUsy cy vidas sie bac ele ceiw'ereln nae a eer

he Genus Mediorhiynchus’ |... <jjs:e/seice's slecis eo siaerina aime

PPHE GETS TICEELODIIS) | occ cere dials fee chers 4 cic cto alates, elesel esa sialaleia’e

Heteroplus grandis (Van CC.) ........ccccccceeceseesccrcons

MURe WMG emiay HALLICOMIS® (ro ctascise'cieers, a2 vein sible oh die aie </alalemreiatetale Hae

ite Genus Arhythmorhynchus. . (46). cj5 2 \0\s/< a oe ite a caciemiatale

Species Inquirendze and Species of Doubted Determination. .

fEchinorhynchus pict collaris Leidy .............eeeeeeee

?Echinorhynchus caudatus Zeder of Leidy ...........+....

?fEchinorhynchus striatus Goeze of Leidy .............+4..

?Echinorhynchus hystrix Bremser of Leidy................-

Distribution of Acanthocephala of Birds.................---

Occurrence of Genera of Acanthocephala in Families of Eu-

ropean and North American Birds... 00.00.66 056 0000+

Genera of Acanthocephala with the Orders of Birds from

CUT CHIN RECOLGEGIN se clera eee elo Nataberniae teeta ty CTC EN sta Nenel alti ste ail

Rey ito the Genera and Species) 1/2 /).4c-itsewisaiee nae ele t.tiercyalsiains

Seta Vs et tne eter leta rs elias coacarer suis trie cerate ra sieeal ehetausiete enero ate laelaleye

MNECEAPUEG CALC ois g ciarcialtieraid ate ee 4 SIE A ea ARS olathe A: wae

ReMANO OLE NALES 2 0c 2 ae cual ave ele wlels. shel eue/wleversib vain ite s,8) ele.

I. INTRODUCTION

Parasitologists have given little attention to the Acanthocephala

parasitic in North American birds.

The result has been a rather

general belief that infestation with Acanthocephala in this class of

vertebrates is rare.

It is true that heavy infestations are more

commonly found in other groups of vertebrates, but many of the

*Contributions from the Zoological Laboratory of the University of Illinois, No. 104.

20 H. J. VAN CLEAVE

water and shore birds carry very heavy infestations of Acanthoce-

phala. Among the land birds, while infestation is not rare, it is

usually of less frequent occurrence, and the number of parasites

found in a single host individual is usually smaller. Most of the

earlier records of the occurrence of Acanthocephala in North Ameri-

can birds ascribe the forms found to known European species. This

is not surprising when one reviews the extent to which the study of

Acanthocephala in birds has proceeded in Europe. Yet the mere

fact that all earlier writers recognized but the one genus, Echino-

rhynchus, in part explains their lack of appreciation of specific char-

acters within this group. On the whole, most of the earlier specific

descriptions are little more than sufficient to permit of later workers

recognizing the genus to which the species belong. This lack in

specificity of earlier descriptions quite naturally lead the new world

investigators to believe that the species they found on this continent

were the same as the European species, since the descriptions pub-

lished contained insufficient data to permit of a separation.

As indicated by the writer in an earlier paper (Van Cleave

1916 b: 228), the acanthocephalan fauna of North America is in

the main a distinctive fauna with few species identical with those

of the European fauna. The attempt on the part of earlier para-

sitologists to ascribe names of European species to forms found

on this continent has, to a great extent, hindered the appreciation

of this distinctness of the North American fauna, and at the same

time has lead to considerable confusion regarding the geographical

distribution of the genera and species of Acanthocephala. Unfor-

tunately, many of the specimens of the older writers are not avail-

able for restudy to determine the correctness of the original deter-

minations. However, in most cases where further study has been

possible points of difference from the European species have been

found too numerous to permit of including the American forms in

the European species.

The writer has made an extensive study of the Acanthocephala

of birds in which he has had access to the collections of the U. S.

Bureau of Animal Industry, the U. S. National Museum, the Marine

Hospital Service, private collections of Dr. H. B. Ward and of the

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS Z1

writer. Papers covering the species of several genera of Avian

Acanthocephala have already been published by the writer as a

result of the study of these collections. The present article deals

with the descriptions of several new species and a reconsideration

of some forms previously described, but here for the first time

ascribed to the proper genera as recognized by more recent develop-

ments of classification of the Acanthocephala. In all cases the study

has been made from cleared, stained specimens mounted in damar.

Linton (1892: 92) recorded the occurrence of Echinorhynchus

striatus Goeze from the intestine of the black scoter, Oidemia ameri-

cana, collected at Yellowstone Lake, Wyoming. The writer has

reexamined the original material of this collection and finds that

these individuals constitute an undescribed species of the genus

Corynosoma. Linton (page 92) remarked that the two females

constituting the extent of the infestation of one of the hosts differed

from the six males from the other host individual, in the “absence

of spines at the posterior end” of the former. It is true that this

sexual dimorphism is characteristic of the members of the genus

Corynosoma as indicated by the founder of the genus, Luhe (1904

and 1911).

Of the materials from this collection deposited in the U. S.

National Museum, one entire female, a portion of a second and

five of the males, have been studied by the present writer. The

entire female specimen is immature but, fortunately, the fragment

of a specimen is of the anterior end of a fully mature individual

so that the body cavity contains well developed embryos.

II. THe Genus Corynosoma Lihe, 1904

The genus Corynosoma was established by Lithe to accommo-

date two species of Acanthocephala parasitic in fish-eating mammals

and birds. This genus is characterized by the presence of spines

on the anterior end of both sexes while in addition the males bear

spines around the genital opening. The body is swollen anteriorly

and with its coating of spines adheres to the intestinal mucosa of

the host thereby furnishing an accessory means of attachment. Pos-

teriorly the body gradually becomes smaller.

ZZ H. J. VAN CLEAVE

Corynosoma constrictum nov. spec.

Plate I, Figures 1, 2 and 3

Synonym: Echinorhynchus striatus Goeze of Linton 1892.

SPECIFIC DEFINITION. With the characters of the genus. Body

of the males 2.28 mm. to 4.3 mm. long, with a maximum diam-

eter of from 0.5 mm. to 0.6 mm. Linton gives the measurement

of a female 3.3 mm. long with a diameter of 0.8 mm. Proboscis

slightly larger at base than at tip, armed with sixteen longitudinal

rows of ten to twelve hooks each. Hooks near base of proboscis

0.035 to 0.041 mm. long, near middle of proboscis 0.041 to 0.047

mm. long, near tip of proboscis 0.030 to 0.041 mm. long. A con-

striction occurs around the body at about the anterior third (see

Fig. 1). In both sexes the part of the body wall anterior to this

constriction is armed with small cuticular spines about 0.030 mm.

long. Each of these spines is embedded in a triangular elevation

of the cuticula projecting from the general body surface. In the

males there occurs in addition a group of cuticular spines surround-

ing the genital opening (Fig. 2). The genital spines are of the

same size as those on the anterior region of the body, usually with

the tip strongly recurved. Embryos in body of female 80 to 108

long by 12 to 16n wide (Fig. 3).

Type host Oidemia americana, in intestine. Type locality Yel-

lowstone Lake, Wyoming. The cotypes of this species are deposited

in the Smithsonian Institution ; the males under catalog number 5449

and females under number 5439.

III. Tuer Genus PLAGiorHyNcHUS Lithe, 1911

In the same paper mentioned above (1892: 91) Linton described

another species of Acanthocephala from the intestine of a gull taken

at Guaymas, Mexico. To this new species, founded on the study

of one male and one female, he gave the name Echinorhynchus

rectus. The present writer has examined the female of this col-

lection, the only specimen in the bottle of material turned over to

him for study from the collections of the U. S. National Museum.

Facts brought out by the reexamination of the female of this species

and the study of Linton’s description of the male and female clearly

indicate that this species belongs in the genus Plagiorhynchus, which

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 23

up to the present time has not been recorded as occurring in North

America. In the present paper the writer describes still another

species belonging to the genus Plagiorhynchus.

CHARACTERS OF THE GENUS PLAGIORHYNCHUS. Acanthocephala

belonging to this genus are parasitic as adults in the alimentary

canal of birds. The proboscis is cylindrical with numerous hooks

arranged in radial symmetry. The body proper, which is entirely

devoid of spines, is usually short, elliptical, or with a tendency

toward egg-shaped. The proboscis receptacle is a double walled

muscular sac attached at the base of the proboscis. In dealing with

other genera of Acanthocephala the writer has found that in Arhy-

thmorhynchus (Van Cleave 1916:a) the embryos in recently discov-

ered American species differ in appearance from those described for

European species of obviously the same genus. Here again in the

genus Plagiorhynchus Lihe (1911:27) described embryos and fig-

ured one having the middle membrane with a conspicuous spherical

knob at each of its two poles. This he considered characteris-

tic of the entire genus (1911:26). The embryos of P. formosus

(Fig. 6) are elliptical with no polar knobs. Here is evidence, in

addition to that previously brought out by the writer (1916:a) and

mentioned above, regarding the advisability of omitting the shape

of the embryonic shells from the generic diagnosis.

Luhe (1911:26) specified the presence of six closely com-

pacted, thickset, cement glands as characteristic of the genus Plagi-

orhynchus. In earlier papers (1914 and 1916) the present writer has

shown that shape and number of cement glands may both vary

widely among different species of the same genus. Consequently

I propose that a fixed number of cement glands and the shape of

the cement glands be omitted from the list of characters diagnostic

of this genus. Descriptions of two species, one new, and the other

newly attributed to this genus follow.

Plagiorhynchus rectus (Linton, 1892)

Plate I, Figure 7

Synonym, Echinorhynchus rectus Linton, 1892

Described originally from one male and one female ef which

the female only has been reexamined by the present writer. Body

24 H. J. VAN CLEAVE

of female 9 mm. long and 0.8 mm. in diameter. Proboscis cylin-

drical, 19 mm. long; 0.26 mm. in diameter; armed with twenty-

four longitudinal rows of about twenty hooks each. In specimen

examined this last number was calculated on the basis of the num-

ber of hooks on the exposed portion of the proboscis, since the tip

of the proboscis is inverted. Hooks near the base of the proboscis

0.082 mm. long, with a diameter of 0.016 mm. at the point of

emergence from proboscis wall; hooks near middle of proboscis

0.070 mm. long, recurved, with a diameter of 0.020 mm. at the

point where the hooks curve backward; hooks near tip of pro-

boscis 0.053 mm. long. Male 8.8 mm. long and 0.8 mm. in diameter.

Testes oval, approximate, median. Female not fully mature so

measurements of embryos cannot be given.

Type host, Larus (Chroicocephalus) sp. taken at Guaymas,

Mexico. Type female deposited in U. S. National Museum, cata-

log number 5431.

Plagiorhynchus formosus nov. spec.

Plate I, Figures 4, 5 and 6

SPECIFIC DEFINITION. With the characters of the genus. Body

about 10 mm. long, elliptical to slightly ovoid. Proboscis practically

cylindrical, diameter about one-third of length; armed with sixteen

longitudinal rows of thirteen to fourteen hooks each. Cement glands

long, tubular. Hard shelled embryos inside body of female ellip-

tical, 48 to 60u long by 12 to 20u in diameter.

This species is described from four mature individuals, two

males and two females, in the Parasite Collection of the U. S. Bu-

reau of Animal Industry; catalog number 4598. The writer desig-

nates one male and one female as types of the species. Descriptions

of these follow.

TYPE MALE. Body elliptical with anterior and posterior ex-

tremities slightly flexed ventrally; entire length 8.5 mm.; maximum

diameter 2 mm. Body proper devoid of spines. Proboscis sub-

cylindrical, length 1.06 mm., diameter 0.33 mm., armature as men-

tioned in definition of species. The following table of measure-

ments of hooks in a single row from tip to base of proboscis

indicates relative size of hooks in various regions of proboscis.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 25

RET Wa Io 0)c 3ieher'se/'at «16 Tens) Al SRG) Se oS MR BM | EZ ES

Length in w...... Fi FF BS) (8S) SI SSN BS 83): SS Fao), 77), Oo) ease

Proboscis receptacle cylindrical, 1.73 mm. long, 0.42 mm. in

diameter, base of the receptacle rounded, with invertors of pro-

boscis penetrating posterior extremity (Fig. 4). Brain 0.180 mm.

long, in center of proboscis receptacle, between the invertors. Retin-

acula conspicuous at the middle of the proboscis receptacle. Lem-

nisci 0.192 mm. long, 0.058 mm. in diameter. Anterior and posterior

testes form an oblique line of contact; each 1.15 mm. long and 0.6

mm. in diameter. Cement glands long, tubular, extending from the

dorsal margin of posterior testis to region of bursa.

TYPE FEMALE. Body more nearly cylindrical than type male,

9.5 mm. long, 2 mm. in diameter. Tip of proboscis slightly inverted,

size and aramture practically identical with that described for male.

Body filled with developing embryos surrounding and covering

almost all of internal organs. Hard shelled embryos within type

female rather variable in size, 40 to 60% long by 12 to 20» in

diameter.

Type host, the flicker, Colaptes auratus, in intestine. The four

specimens upon which the species is founded were collected at Bowie,

Maryland, October 9, 1906, by Dr. B. H. Ransom.

MORPHOLOGY. The males of this species display a phenomenal

transparency of body structures so that in stained whole mounts

minute internal structures are observable with the greatest ease.

The proboscis in the preserved specimens is so inserted that it

points ventrally at an angle of approximately 60 degrees from the

chief axis of the body. At the base of the proboscis the body proper

is slightly expanded to form a thickened rim. The proboscis recep-

tacle is a cylindrical sac composed of two muscular layers. The

retinacula arise in the brain as two fairly small fibres. Upon pene-

tration of the wall of the receptacle their size increases appreciably.

This increased size is maintained through the remainder of their

course to their insertion upon the body wall. The means of inser-

tion upon the wall of the body is shown in Fig. 4. The lemnisci

are slightly longer than the proboscis receptacle and of smaller

diameter throughout.

26 H. J. VAN CLEAVE

The two testes of the male are roughly oval, lying in the anterior

region of the body. Ltthe (1911:26) described six closely com-

pacted cement glands as characteristic of the genus Plagiorhynchus.

P. formosus appears to have six of these glands (Fig. 4), but their

shape differs radically from the shape of the cement glands of P.

lanceolatus.

The hooks upon the proboscis present a perfect pattern in their

exact arrangement in alternating rows. Roots of the hooks are not

distinct. The base of each hook for a length of about 0.021 mm.

lies embedded in the wall of the proboscis perpendicular to its sur-

face. The free portion of each hook is directed backward at an

angle of about 110 degrees with the basal portion. The foregoing

description of hooks applies to all hooks upon the proboscis except

one or two hooks at the base of each row, which are simple, thorn-

like and one or two of those at the tip of the proboscis which differ

from the remaining hooks in shape and general proportions (see

Fig. 5). In all cases measurement of hooks here as in other papers

by the writer the length of a hook is considered as the straight line

connecting the tip of the hook with its extreme basal part.

IV. THe Genus PotyMorPuHus Lithe, 1911

Acanthocephala parasitic as adults in the intestine of birds.

Posterior tip of the body rather broadly truncated. Anterior end

of body swollen and separated from more attenuated posterior

region by a constriction. Anterior region of the body spined. Pro-

boscis usually cylindrical, frequently smaller at base.

Polymorphus obtusus nov. spec.

Plate II, Figs. 8, 9, 10, 11, and 12

Body with a slightly enlarged anterior region. This enlarge-

ment is set off by a constriction back of which the body again

assumes a diameter equal to that of the anterior enlargement, then

gradually decreases in size to the posterior tip which is more or

less flexed ventrally. Posterior tip terminates bluntly (Fig. 11).

Females 7.7 mm. long, males 4 to 5.5 mm. long. Proboscis in all

specimens examined partially inverted, armed with about sixteen

longitudinal rows of hooks. Anterior body region armed with small

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 27

cuticular spines about 24p long (Fig. 9). Embryos within body of

female 60 to 80u long and 20 to 24» wide with one conspicuous

outpocketing of the middle membrane at each pole (Fig. 10).

Type host Anhinga anhinga (water-turkey), in intestine. Type

locality, Florida. Described from three specimens, one female and

two males. Cotypes in U. S. National Museum, catalog number

2967.

Polymorphus sp?

Plate II, Fig. 13

In addition to Polymorphus obtusus another representative of

this genus has been studied by the writer, but the single specimen,

a male, is insufficient to permit of a description of the species.

Record is given here with a drawing of the proboscis in order to

facilitate comparison in case other specimens are found from the

same host later. The host of this undescribed species in the hooded

merganser, Lophodytes cuculatus. Specimen in the U. S. Bureau

of Animal Industry Collection, catalog number 2808.

V. THe GENUS CENTRORHYNCHUS Lie, 1911

Centrorhynchus spinosus Van Cleave, 1916

Plate III, Figs. 14 and 15

A single species of this genus has been described from North

America. The writer (1916b) described C. spinosus from the intes-

tine of the egret, Herodias egretta. For description see paper cited

above.

VI. THe Genus MepiorHyNCHUS Van Cleave, 1916

Mediorhynchus papillosus Van Cleave

Plate III, Figs. 16, 17, 18 and 19

Mediorhynchus robustus Van Cleave, 1916

Plate IV, Figs. 20 and 21

This genus, which to the present time has been recognized

only from North America, was created by the writer (Van Cleave

1916b) for three new species of Acanthocephala belonging evidently

near to the genus Centrorhynchus, but having characteristics which

28 H. J. VAN CLEAVE

prevented including them in that genus. At the time the description

of this genus was published the writer failed to designate which of

the species should be considered as type. M. papillosus is hereby

designated as type of the genus Mediorhynchus.

Mediorhynchus papillosus has been recorded from Myiochanes

virens (the wood pewee) and Porzana carolina (the sora). Medi-

orhynchus robustus has been found in Icteria virens (the yellow-

breasted chat). |

Mediorhynchus grandis was originally included within this same

genus but a more thorough study of Kostylew’s articles dealing with

the genus Heteroplus has convinced the writer that the species

grandis is, in reality, a member of the genus Heteroplus. Kosty-

lew (1913: 532) considered Heteroplus as near to the genus Gigan-

torhynchus from which he claimed to have separated it. On the

other hand de Marvel (1905: 217) considered Echinorhynchus otidis,

one of the species upon which Kostylew founded his genus Hetero-

plus, as a synonym for E. aluconis. E. aluconis, through the care-

ful work of Lithe (1911), became type of the genus Centrorhynchus.

At the same time Gigantorhynchus mirabilis de Marvel, which Kosty-

lew claimed also belonged to his genus Heteroplus, is clearly, on

the basis of de Marvel’s descriptions and figures, one of the Cen-

trorhynchide. De Marvel’s figures show clearly the constriction

of the proboscis at the line of insertion of the receptacle with the

coincident differentiation of hooks on the anterior and posterior

regions of the proboscis. Kostylew (1913), in describing the pro-

boscis receptacle of Heteroplus, referred to the anterior half as

being twice the diameter of the posterior half. This condition is

typical of the genus Mediorhynchus and serves as an additional

argument for considering the genus Heteroplus as a member of the

family Centrorhynchide and not of the Gigantorhynchide.

VII. THe Genus HeEtTeropius Kostylew, 1914

With the characteristics of the family Centrorhynchide. An-

terior and posterior regions of the proboscis bearing widely differ-

ent numbers of longitudinal rows of hooks.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 29

Heteroplus grandis (Van Cleave, 1916)

Plate V, Figs. 27, 28 and 29

Synonym, Mediorhynchus grandis Van Cleave, 1916

This species, the description of which is given in Van Cleave

1916 b: 226, was described from Quiscalus quiscula (the purple

grackle) and Sturnella magna (the meadow lark). More recently

the writer has examined a specimen from Corvus brachyrhynchus

(the crow) taken in Maryland which proves to belong to this same

species.

Kostylew’s contention that the genus Heteroplus belongs to the

Gigantorhynchide caused the writer at the time of the description

of this species to believe that its superficial and general resemblance

to Heteroplus was not of great significance.

VIII. Tue Genus Firicotiis Lihe, 1911

Filicollis botulus Van Cleave, 1916

Plate V, Figs. 30, 31, 32, 33 and 34

But a single species of this genus, F. botulus, has been recorded

from North America. This species differs widely from F. anatis

of Europe, especially in the shape of the proboscis of the female.

Specific characteristics upon which this species was founded are

given in an earlier paper (Van Cleave 1916). Since the species was

described the writer has discovered younger males than he had seen

at the time of the original description. Figure 30 shows the arrange-

ment of the internal organs of one of these young males. The eider

ducks, Somateria dresseri and S. mollissima, are the known hosts

of the species.

IX. THe Genus ARHYTHMORHYNCHUS Liihe, 1911

Arhythmorhynchus uncinatus (Kaiser, 1893)

Arhythmorhynchus trichocephalus (R. Leuckart, Kaiser, 1893)

Arhythmorhynchus brevis Van Cleave, 1916

Plate IV, Figs. 22, 23 and 24

Arhythmorhynchus pumilirostris Van Cleave, 1916

Plate IV, Figs. 25 and 26

Kaiser in 1893 published a description of Echinorhynchus un-

cimatus Kaiser and E. trichocephalus R. Leuckart. The writer

30 H. J. VAN CLEAVE

(1916a: 172) showed that these two species belong in reality to the

genus Arhythmorhynchus and in the same paper described two addi-

tional species for this genus, A. pumilirostris and A. brevis, both

from the intestine of the American bittern, Botaurus lentigenosus.

X. Species INQUIRENDZ AND SPECIES OF DouBTED DETERMINATION

Echinorhynchus pici collaris Leidy, 1850

Leidy described and recorded the occurrence of four Acantho-

cephalans from North American birds, but no one of these is the

present writer able to locate with any degree of certainty within

the modern system of classification of the group. The descrip-

tion of Echinorhynchus pici collaris from “Picus colaris” is lacking

in details sufficient to permit of speculation as to even the genus

to which it belongs. Consequently E. pici collaris must be regarded

as a species inquirenda. The other three species, given the names

of known European species, probably belong to the respective genera

now recognized as including the European forms.

(?) Echinorhynchus caudatus Zeder of Leidy 1887

This species, which Leidy recorded from Elanoides forficatus

and from Scotiaptex nebulosa, has been considered by the present

writer (1916b:222) as belonging to the family Centrorhynchide,

though the original description is insufficient to permit of a closer

determination. It is extremely improbable that Leidy’s determina-

tion of this species is correct.

(?) Echinorhynchus striatus Goeze of Leidy 1856

From the intestine of Mycteria americana (“Tantalus locula-

tor’), the wood ibis, Leidy recorded the occurrence of Echinorhyn-

chus striatus Goeze. Lthe (1911) was unable to place Goeze’s

E. striatus in his revised system of classification. It seems improb-

able that Leidy was dealing with the species which Goeze described

from central Europe. Many points of detail have caused the pres-

ent writer to consider the form which Leidy recorded as probably

one of the species of Arhythmorhynchus. Its relations to other

species of this genus cannot be determined.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 31

(?) Echinorhynchus hystrix Bremser of Leidy 1887

Leidy’s report of Echinorhynchus hystrix from Anhinga an-

hinga (the water turkey) gives some ground for attributing the

genus Corynosoma as parasitic upon that species of bird, though

here again the specific identity with the European form is to be

seriously doubted.

XI. DistRIBUTION OF THE ACANTHOCEPHALA OF BIRDS

It seems worth while to call attention to the fact that in the

collections from birds examined by the writer the occurrence of

two different species of Acanthocephala within the same host indi-

vidual has never been observed. Furthermore there is no positive

case on record wherein two different genera of Acanthocephala

have been found in the same species of North American bird. The

case of Anhinga anhinga seems to be an exception to this last state-

ment. From this host the writer has described a species of Poly-

morphus while Leidy has described what seems to be a species of

Corynosoma. However in general body form these two genera

resemble one another closely enough to confuse the casual observer.

On this basis the apparent exception to the general condition may

not be a real one.

De Marval, in his significant monographic contribution to the

study of Avian Acanthocephala, has unfortunately failed to furnish

us with any considerable body of data upon the geographical dis-

tribution of the species with which he worked. Evidently many

of his records are compilations from earlier writers. In his treat-

ment of each species of Acanthocephala he has given a host list,

but offers no information as to the locality from which the hosts

were taken. A study of his data shows that he has recorded the

occurrence of Acanthocephala in more than forty families of birds

representing eleven of the seventeen orders of birds recognized

in North America and in addition some families not represented in

the North American fauna.

In the collections available to the writer these parasites have

been found in but seven orders, and a total of but ten families of

birds. Beyond this Leidy’s reports indicate the presence of Acan-

thocephala in at least three additional families. It is a noteworthy

32 H. J. VAN CLEAVE

fact that the geographical distribution of the species known to North

America is restricted almost exclusively to the eastern part of the

continent. In the opinion of the writer this apparent localization

of infestation is probably due to the fact that records from the

West are wanting rather than that the actual distribution is so nar-

rowly limited. Much of the materials studied by the writer has

been the result of the careful work of Mr. Albert Hassall.

XII. DistTRIBUTION OF GENERA OF ACANTHOCEPHALA IN FAMILIES

OF EUROPEAN AND NorRTH AMERICAN BIRDS:

GENERA OF ACANTHOCEPHALA REPORTED FROM

FAMILY OF BIRDS

ACTING AS HOST

CENTRAL EUROPE

(Lithe 1911)

NORTH AMERICA

Colymbide Filicollis

Laridz Filicollis Plagiorhynchus

Anhingide Polymorphus

?Corynosoma

Anatide Polymorphus Polymorphus

Filicollis Filicollis

Corynosoma Corynosoma

?Centrorhynchus?

Ciconiide ?E. striatus?

Ardeide Filicollis Centrorhynchus

?Arhythmorhynchus? Arhythmorhynchus

Rallide Polymorphus Mediorhynchus

Filicollis

Scolopacide Plagiorhynchus

Arhythmorhynchus

Filicollis

Charadriide Plagiorhynchus

Polymorphus

Buteonide ?E. caudatus?

Falconide Centrorhynchus

Strigide ?E. caudatus?

Picide Plagiorhynchus

?E. pici collaris?

Tyrannide Mediorhynchus

Corvide Plagiorhynchus

Heteroplus

Icteride Heteroplus

Mniotiltide Mediorhynchus

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 33

In the above table genera and species marked with a question

mark (?) are of questioned original determination or are species

inquirende. It is quite a striking fact that in only the family An-

atide and probably the Ardeide are any of the same genera of

Acanthocephala found in both North America and Europe.

Lithe (1911), in his register of the Acanthocephala and para-

sitic flatworms from central European hosts recorded the occurrence

of Acanthocephala from hosts representing but eight families of

birds. The accompanying table based upon Luhe’s data and records

of the writer shows in striking manner the differences between the

types of hosts characteristic of the various genera in Europe and

North America. To the writer this table furnishes still farther

evidence of the independence of the acanthocephalan fauna of the

two continents. In the majority of cases even the genera of these

parasites have found hosts in entirely different Families and even

Orders of birds.

XIII. GENERA OF ACANTHOCEPHALA WITH THE ORDERS OF BIRDS

From WHIcH TAKEN

GENUS OF ORDER OF BIRDS SERVING AS HOST

ACANTHOCEPH ALA CENTRAL EUROPE NORTH AMERICA

(Lithe)

Arhythmorhynchus Limicole Herodiones

Centrorhynchus Raptores Herodiones

Anseres

Corynosoma Anseres Anseres

?Steganapodes

Filicollis Anseres Anseres

Pygopodes

Longipennes

Herodiones

Paludicolz

Limicole

Heteroplus Passeres

Mediorhynchus Passeres

Paludicole

Plagiorhynchus Limicolze Pici

Longipennes

Passeres

Polymorphus Limicolz Steganapodes

Anseres Anseres

Paludicolz

34 H. J. VAN CLEAVE

A comparison of infestation within the Orders of birds fur-

nishes a contrast between the two continents even more striking

than that found in the comparison of the Families of birds. The

accompanying table shows among other things, that Corynosoma,

Filicollis and Polymorphus are the only genera having hosts within

the same orders of birds on the two continents.

XIV. Key To THE GENERA AND SPECIES OF ACANTHOCEPHALA OF

; NortH AMERICAN Brirps

(12) Acanthocephala with body proper devoid of spines.............. 2

—

2 (5) Proboscis receptacle inserted at base of proboscis................

bc Pea basse olee gH ee RL easy DD cate SAG batty Genus PLAGIORHYNCHUS.. 3

3). 4): Twenty-four longitudinal rows of hooks: j.\....0....). i245 «00cm

apres ch 2 4 rats. ER Vn ee Plagiorhynchus rectus. Linton

4. (3))-Sixteen) longitudinal sows ‘of ‘hooks. 55... <- 4s. +,«0 ose sree ene meee

ath aie ete fora a poe ere RT Tesret sare cokes Plagiorhynchus formosus nov. spec.

5 (2) Proboscis receptacle inserted at or near middle of proboscis. Pro-

boscis hooks anterior to and posterior to insertion conspicuously

Ney sia od ss ie opts « 3/5) fe ve Glas ol PaaS ie n/a 6.4 sl 6 se 6

6 (7) Invertors of proboscis extending through posterior tip of proboscis

receptacle and continuing as retractors for receptacle...........

BARNS Co AS iS se aie arate avs Rata escete aasle usraan ereneeC ears Genus CENTRORHYNCHUS

Centrorhynchus spinosus Van C., only known species in North America.

7 (6) Invertors of proboscis passing through wall of proboscis receptacle

near its middle or at least considerable distance anterior to the

POSEECION ETI) ooo Peds se ie olam clelerece eieloile wielalni aun he tele nena eee 8

8 (9) Anterior and posterior regions of proboscis bearing different num-

bers of longitudinal rows of hooks............ Genus HETEROPLUS

Twelve longitudinal rows of hooks on the interior region of pro-

boscis, about thirty on posterior. Heteroplus grandis (Van. C.)

only known species in North America.

9 (8) Anterior and posterior regions of proboscis bearing the same num-

ber of longitudinal rows of hooks. Genus MEDIORHYNCHUS. .10

10 (11) Proboscis not covered with conspicuous papillez in which hooks are

embedded. Body usually robust, forms with twenty-four longi-

tudinal rows of hooks on proboscis. Embryos 38u by l6u......

Bh ta iV a a ee Mediorhynchus robustus Van C.

11 (10) Proboscis hooks not conspicuous; embedded in conspicuous papille.

Eighteen longitudinal rows of hooks. Embryos 38 to 47« by

US a tone aoe bo cinerea Mediorhynchus papillosus Van C.

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

(1)

(14)

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 35

Body proper bearing spines, at least in restricted regions......... 13

Body spines on anterior region of the body and also surrounding

ETAT OPEMMN Orem tis tee lao cera nea eevee Genus CORYNOSOMA, males

But one species, Corynosoma constrictum nov. spec., known to North

(13)

(26)

(23)

(18)

(17)

(20)

(19)

(22)

(21)

(16)

(25)

America.

With no cuticular spines around the genital opening............ 15

Anterior end of the body decidedly larger than posterior end..... 16

Anterior and posterior regions of the body of distinctly different

histological structure............ Genus ARHYTHMORHYNCHUS..17

Hooks on mid-ventral surface of proboscis conspicuously larger.

thanvoneany, Other ParilOL PLrODOSCIS =: /5/4-12 6 aele cision ciaieloieiale ciate

Aleta tetatevelotenersiaeae Arhythmorhynchus trichocephalus (R. Leuckart)

Hooks on ventral surface of proboscis not conspicuously larger

SHAM OM ANY OENEE SUTEACES 8 Le Meals lee cblacaid nia ores eels, eats al glGie is 19

Longest hooks more than: 100m lore! 2465 cise «sale cise cle siv'easeis siete eies

IAI OE 1 AO a pe a Arhythmorhynchus uncinatus (Kaiser)

Longest hooks not more’ than 50 Jong o.oo... iio ee jan a7 simvolescole cals 21

Proboscis with sixteen longitudinal rows of hooks. Embryos 65

to 80u long, 184 wide....Arhythmorhynchus pumilirostris Van C.

Proboscis with eighteen longitudinal rows of hooks. Embryos 76

to 100u long, 24 to 30u wide....Arhythmorhynchus brevis Van C.

Anterior end of body larger than posterior end, but body wall of

same histological structure throughout.............0c.c0e000: 24

Anterior region of body set off from posterior region by conspicu-

ous constriction. Proboscis cylindrical, or frequently tapering

siiehtly toward: bases.2o st ese eee eee! Genus POLYMORPHUS

Polymorphus obtusus nov. spec. only species described from North

(24)

America.

Anterior region of body distinctly swollen, but not separated from

posterior by a constriction. Proboscis spindle shaped..........

MRP Pa Nara ne SCN erayie, eaten a Ne taba re ovat Genus coryNosoMA, females

Corynosoma constrictum nov. spec., only known species from North

America.

(15) Body sac-like or sausage shaped, not conspicuously swollen at ante-

rior end. Proboscis spherical or ovate, followed by a neck fully

as long as the proboscis and sharply set off from the body proper.

BHO Lies CS ches ae eS SRC Re ATE EATEN OLE RY RAP NOV Genus FILICOLLIS

Filicollis botulus Van C. is the only species known to North America.

36 H. J. VAN CLEAVE

XV. SUMMARY

1. This article contains the results of a study of the Acantho-

cephala parasitic in birds from the U. S. Government collections

and the private collections of Professor H. B. Ward and of the

writer.

2. LEchinorhynchus striatus taken by Linton from Ozidemia

americana belongs to the genus Corynosoma and here is described

as Corynosoma constrictum nov. spec.

3. LEchinorhynchus rectus Linton, 1892 is shown to belong to

the genus Plagiorhynchus. P. formosus nov. spec. from Colaptes

auratus is described. These two species constitute the first record

of species of Plagiorhynchus in North America.

4. Polymorphus obtusus nov. spec. is described from Anhinga

anhinga.

5. Polymorphus species? is recorded from Lophodytes cucu-

latus.

6. Mediorhynchus papillosus Van C., 1916 is designated as

type of the genus Mediorhynchus.

7. The genus Heteroplus is one of the Centrorhynchide (not

of the Gigantorhynchide as maintained by Kostylew, its creator).

8. Mediorhynchus grandis VanC., 1916 is shown to belong

to the genus Heteroplus.

9. Corvus brachyrhynchus is cited as a new host for Hetero-

plus grandis (VanC.).

10. Among North American birds the occurrence of two

different species of Acanthocephala within the same host individual

has never been recorded.

11. There is no positive case on record of the occurrence of

two different genera of Acanthocephala within the same species

of North American bird.

12. Tables are given to show the comparison of acanthoce-

phalan infestation in the families and orders of birds of central

Europe and of North America.

13. A key to all described species of Acanthocephala from

North American birds is given.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS af

XVI. LiterRAtTuRE CITED

Kaiser, J. E.

1893. Die Acanthocephalen und ihre Entwicklung. Biblioth. Zool. 7:

1-136.

KostyLew, N.

1913. Ein Beitrag zur Anatomie von Gigantorhynchus otidis Miesch.

Centralbl. Bakt. Abt. 1, 72: 531.

1914. Ueber die Stellung einiger Acanthocephalenarten im System.

Zool. Anz., 44: 186-188.

1915. Contributions a la faune des Acanthocephales de la Russie. Ann.

Mus. Zool. Acad. Imp. Sc., St. Petersburg, 20: 389-394.

Lerpy, J.

1850. Contributions to Helminthology. Proc. A. N. S. Phila., 5: 96-98.

1856. A Synopsis of Entozoa and some of their Ectocongeners ob-

served by the Author. Proc. A. N. S. Phila. 8: 42-58.

1887. Notice of some Parasitic Worms. Proc. A. N. S. Phila., 39: 20-24.

Linton, E.

1892. Notes on Avian Entozoa. Proc. U. S. Nat. Mus., 20: 87-113.

LUue, M.

1904. Geshichte und Ergebnisse der Echinorhynchen-Forschung bis auf

Westrumb (1821). Zool. Annal., 1: 139-250.

1911. Die Siisswasserfauna Deutschlands, Heft 16, Acanthocephalen.

Jena. 116 pp.

DE MarvEL, L.

1905. Monographie des Acanthocephales d’Oiseaux. Rev. Suisse Zool.,

13: 195-386.

Van CLEAVE, H. J.

1914. Studies on Cell Constancy in the Genus Eorhynchus. Journ.

Morph., 25: 253-299,

1916. Filicollis botulus, n. sp., with Notes on the Characteristics of the

Genus. Trans. Amer. Micr. Soc., 35: 131-134.

1916a. A Revision of the Genus Arhythmorhynchus with Descriptions

of Two New Species from North American Birds. Jour.

Parasitol., 2: 167-174.

1916b. Acanthocephala of the Genera Centrorhynchus and Mediorhyn-

chus (new genus) from North American Birds. Trans.

Amer. Micr. Soc., 35: 221-232.

38 H. J. VAN CLEAVE

XVII. ExXPLANATION OF PLATES

All figures excepting Figure 7 are drawn by the aid of a camera lucida

from stained specimens mounted in xylol damar. The magnification is indi-

cated by the projected scale accompanying each drawing.

ABBREVIATIONS USED

b. bursa copulatorix 1. lemniscus

br. brain. p.r. proboscis receptacle

c.g. cement glands r. retinacula

e. egg masses r.p. retractors of proboscis receptacle

ins. insertion of proboscis receptacle t.a. anterior testis

i.p. invertors of proboscis t.p. posterior testis

Prate I.

Figs. 1 to 3, Corynosoma constrictum nov. spec.

Fig. 1. Entire male showing general body shape and arrangement of in-

ternal organs.

Fig. 2. Posterior tip of male showing cuticular spines.

Fig. 3. Embryo from body of mature female.

Figs. 4 to 6, Plagiorhynchus formosus nov. spec.

Fig. 4. Entire male showing general body form and arrangement of inter-

nal organs.

Fig. 5. Profile of the proboscis of same individual as shown in Fig. 4.

Fig. Embryo from body of mature female.

Fig. 7. Plagiorhynchus rectus (Linton). Drawing copied from Linton 1892.

coo)

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS

Imm.

Pate J.

H. J. Van Cleave, del.

H. J. VAN CLEAVE

PuateE II.

Figs. 8 to 12. Polymorphus obtusus nov. spec.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

12.

13.

Entire male, poorly preserved specimen, internal organs very in-

distinct.

Cuticular spines in profile, from anterior region of body.

Embryo from body cavity of female.

Posterior extremity of type female showing characteristic blunt-

ness upon basis of which the specific name is given.

Profile of proboscis hooks.

Polymorphus species? from Lophodytes cuculatus.

41

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS

Puiate II.

H. J. Van Cleave, del.

42 H. J. VAN CLEAVE

Prate III.

Figs. 14 and 15. Centrorhynchus spinosus Van Cleave.

Fig. 14. General body form of type female.

Fig. 15. Profile of dorsal surface of proboscis shown in Fig. 14.

Figs. 16 to 19. Mediorhynchus papillosus Van Cleave.

Fig. 16. Entire male showing general body form and arrangement of organs.

Fig. 17. Proboscis and anterior body region of type male.

Fig. 18. Profile dorsal surface of proboscis, same individual as shown in

Fig. 17.

Fig. 19. Embryos from body cavity of fully mature female.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 43

Priate III.

H. J. Van Cleave, del.

44 H. J. VAN CLEAVE

PiatTe IV.

Figs. 20 and 21. Mediorhynchus robustus Van Cleave.

Fig. 20. Male in optical section showing body form and arrangement of

organs.

Fig. 21. Embryos from body cavity of mature female.

Figs. 22 to 24. Arhythmorhynchus brevis Van Cleave.

Fig. 22. Body of entire male.

Fig. 23. Proboscis and anterior region of body of a male.

Fig. 24. Embryos from body cavity of mature female.

Figs. 25 and 26. Arhythmorhynchus pumilirostris Van Cleave.

Fig. 25. Entire male.

Fig. 26. Embryo from body of mature female.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 45

PuateE IV.

H. J. Van Cleave, del.

H. J. VAN CLEAVE

PLATE V.

Figs. 27 to 29. Heteroplus grandis (Van Cleave).

Fig. 27. Proboscis partly invaginated, but showing the hook arrangement

Fig. 28.

Fig. 29.

typical for the genus.

Embryos from body of mature female.

Profile of dorsal surface of proboscis.

Figs. 30 to 34. Filicollis botulus Van Cleave.

Fig. 30.

Fig. 31.

Fig. 32.

Fig. 33.

Fig. 34.

Entire male showing general body form and arrangement of

organs.

Cuticular spines from anterior region of body.

Mature female showing general body form.

Embryo from body cavity of mature female.

Profile, ventral surface, of proboscis of female.

ACANTHOCEPHALA OF NORTH AMERICAN BIRDS 47

H. J. Van Cleave, del.

Ht Ava ir J

niwoe i ih, hy waht Cay

pl : aN 2 ¢

Hie AU doar 6 aN viel ; fess vie

BRANCHIOBDELLID WORMS (ANNELIDA) FROM

MICHIGAN CRAWFISHES

By Max M. EZttis

Through the courtesy of the Michigan Fish Commission the

writer visited several islands in Potagannissing Bay and Lake Huron

on Patrol Boat No. 4, Captain Robert E. Ellsworth commanding,

during August, 1917. The branchiobdellid worms listed below were

taken from crawfishes collected on this trip and from those of two

additional collections made by Captain Ellsworth. Dr. Walter

Faxon of Harvard University kindly identified the crawfish hosts.

The four species of branchiobdellids represented may be dis-

tinguished by the following key.

a. Anterior nephridia opening to the outside through two separate pores

in segment III; segments VIII and IX each bearing a pair of glandu-

lar disks on the ventral surface; dental formula usually 5-4 or 5-5,

the middle tooth of each jaw being the longest tooth in the jaw.

Xironodrilus formosus Ellis in ed.

aa. Anterior nephridia opening to the outside through one common pore in

the dorsal surface of segment III.

b. Body segments without conspicuously elevated portions; accessary

sperm tube present.

c. Upper and lower lips entire excepting a small, median emargin-

ation; dental formula 5-4, teeth approaching a subequal con-

dition, although the middle tooth of the upper jaw is distinctly

longer than the other four teeth of that jaw.

Cambarincola vitrea Ellis in ed.

cc. Upper lip composed of four, subequal lobes, which can be ex-

tended as digitiform processes; lower lip composed of two,

subequal lobes which can be extended; a small, lateral lobe

on each side at the junction of the upper and lower lips;

dental formula 5-4; middle tooth of upper jaw long and

prominent; lateral teeth small, not more than half the length

of the middle tooth.

Cambarincola philadelphica (Leidy)

bb. Dorsal portions of segments elevated; segments VII and VIII with

funnel-shaped enlargements of the dorsal portions of the seg-

ment, more or less completely encircling the segment; funnel of

segment VIII excavated dorsally so that its dorsal margin bears

two, small “horns”; dental formula 5-4.

Pterodrilus durbini Ellis in ed.

50 MAX M. ELLIS

(1) XzRoNnopRiLus FormMosus Ellis

From Cambarus virilis Hagen

1. James Island, Potagannissing Bay, August 4.

2. Three miles up Potagannissing River, Drummond Island, Pot-

agannissing Bay, August 4.

3. Pilot Harbor, Sitgreaves Bay, north side of Drummond Is-

land, Potagannissing Bay, August 6.

4. Little Cass Island, head of Detour Passage, August 6.

5. Churchville Point, head of Lake George, 46° 31’ N., August 7.

6. Harbor Island, Potagannissing Bay, August 8.

7. Winona Slips, Bay City, Saginaw Bay, September (Capt. Ells-

worth).

(2) CAMBARINCOLA VITREA Ellis

From Cambarus virilis Hagen

1. James Island, Potagannissing Bay, August 4.

2. Three miles up Potagannissing River, Drummond Island, Pot-

agannissing Bay, August 4.

From Cambarus propinquus Girard

1. Sault Sainte Marie, St. Marys River, August 7.

2. Echo Lake, Grand Island, Lake Superior, August 17 (Capt.

Ellsworth).

(3) CAMBARINCOLA PHILADELPHIA (Leidy)

From Cambarus virilis Hagen

1. James Island, Potagannissing Bay, August 4. .

2. Pilot Harbor, Sitgreaves Bay, north side of Drummond Is-

land, Potagannissing Bay, August 6.

3. Little Cass Island, head of Detour Passage, August 6.

4. Harbor Island, Potagannissing Bay, August 8.

(4) PTERODRILUS DURBINI Ellis

From Cambarus virilis Hagen

1. James Island, Potagannissing Bay, August 4.

2. Three miles up Potagannissing River, Drummond Island, Pot-

agannissing Bay, August 4.

3. Pilot Harbor, Sitgreaves Bay, north side of Drummond Is-

land, Potagannissing Bay, August 6.

Little Cass Island, head of Detour Passage, August 6.

Churchville Point, head of Lake George, 46° 31’ N., August 7.

Harbor Island, Potagannissing Bay, August 8.

Winona Slips, Bay City, Saginaw Bay, September (Capt. Ells-

worth).

SOK eu

It may be seen from this list that Xironodrilus formosus Ellis

and Pterodrilus durbini Ellis were found in every collection from

BRANCHIOBDELLID WORMS 51

Cambarus virilis Hagen. In each case Xironodrilus formosus was

the more abundant species, represented by hundreds of individuals.

Comparatively few Pterodrilus durbini were taken. The relative

abundance of these two species may be considered as accurate for

these collections as the living crawfish were dropped into the killing

fluid as soon as caught, and all of the branchiobdellid worms carried

by each crawfish preserved. The two species of Cambarincola, if

found, were represented by a fair number of individuals. Cambarin-

cola vitrea Ellis was the only species taken from Cambarus pro-

pimquus Girard in these collections. Both species of Cambarincola

here represented however have been taken from specimens of Cam-

barus propinquus at Douglas Lake, Michigan.

Umiversity of Colorado.

DEPARTMENT OF NOTES, REVIEWS, ETC.

It is the purpose, in this department, to present from time to time brief original

notes, both of methods of work and of results, by members of the Society. All

members are invited to submit such items. In addition to these there will be given a

few brief abstracts of recent work of more general interest to students and teachers.

There will be no attempt to make these abstracts exhaustive. They will illustrate progress

without attempting to define it, and will thus give to the teacher current illustrations, and

to the isolated student suggestions of suitable fields of investigation.—[Editor.]

A CHART ON GENERAL PLANT HISTOLOGY AND PHYSIOLOGY

The valuable teaching aid afforded by charts and diagrams of

various sorts is well understood by most teachers of biology who

are more or less well acquainted with the numerous current sets of

charts offered to the profession. There are a few teachers who

possess the enviable talent of rapidly constructing excellent black-

board sketches during a given lecture or laboratory period to illus-

trate the particular features or phenomena under study at that

particular time. Some instructors have made use of the more

permanent crayon sketches on sheets of drawing paper hung over

an easel. The uses of the various lantern-slide, opaque, vertical

and micro-projection possibilities are also utilized to a very desir-

able degree under certain circumstances.

The principal pedagogical difficulty which all teachers have prob-

ably experienced in the practical application of these or other useful

adjuncts of the same general kind to teaching is, that at best the

student gets a disjointed presentation of the subject in question for

the reason that the subject matter must be presented more or less

disjointedly and interruptedly because of the many other things

which we compel him to study. The student suffers from this regu-

lar lack of continuity. He loses much because of his failure to see

the real position of a given structure or place of a given activity in

the organism as a whole.

I may illustrate my meaning here by a reference to the common

practice in teaching some of the phases of botany, say phytohistology.

Probably students may be found in every class in plant histology

as that subject is currently taught, who, knowing right well the

detailed characteristics of all of the common tissues of the vege-

table kingdom and how to handle the histological technique involved

54 NOTES, REVIEWS, ETC.

in the preparation of such tissues, may fail utterly to acquire an

adequate knowledge of the place and function of those tissues in

the plant as a whole. In fact such students may not even know

where in the plant to find a given tissue if they are thrown upon

their own resources, resources derived from their histology courses.

Surely it is most difficult for them to really understand the function

of the various tissues of the plant if they do not know the position

of the tissues within the plant.

The same general state of affairs sometimes exists in plant

physiology. The student may understand the various fundamental

processes of the plant very well, but he often fails to read through

the whole series of interrelated activities and to visualize, as it

were, the individual plant as a completely equipped and working

entity.

The most fundamental process in all nature is photosynthesis.

Consequently it is quite desirable that the student of general biology,

of general botany, surely of general physiology and general science

should understand at least the fundamental features and the signi-

ficance of that great phenomenon. He should know photosynthesis

as it occurs in the leaf, of course, but he should also know about

many of the other processes and structures which make the photo-

synthetic manufacture of carbohydrates in the leaf possible. He

must know quite well many of the interrelations of the various activi-

ties of the plant if he wishes to really understand photosynthesis.

He must see the plant as a completely constructed mechanism with

its various parts working together in harmony. It is the business

of the teacher to see to it that he does get this notion definitely out-

lined in his mind.

Now to be sure that our students really get all of this and this

point of view of the plant, requires more than a little thought on

the part of the teacher as to the methods of instruction that are to

be followed in the class-room and laboratory. Sometimes I have

thought that some of the common difficulties in this connection might

be lessened if the class were to take a single common plant, say the

sunflower, castor bean or scarlet runner, and work out its entire

structural and physiological life-history from the embryo in the

seed through the development and maturation of the new individual

AMERICAN MICROSCOPICAL SOCIETY 55

including the new fruit and seed. If the exercises on histology

and physiology were properly correlated at every point possible I

believe that such a method would result in giving to the student an

admirable introduction to many of the fundamentals of general bot-

any. At the conclusion of such a course the student would have

secured an admirable and useful insight into the most of the im-

portant plant phenomena. And I believe that he would actually

retain a definite mental picture of what the plant is as a mechanism

and what it does as a living, working organism. From such a course

he would surely obtain a connected view of these matters and he

would not think of the plant as composed of a group of parenchyma

cells here, a strand of fibers there, an irregular patch of epidermis

somewhere else. He would not stop to wonder how and from

whence the root gets the necessary food for its growth or how it is

possible for growth to begin in a tree in early spring before the

leaves have unfolded their chlorophyll-containing tissues.

Something of the same results may be secured by the skillful

teacher from a carefully planned summary of the whole matter in

which the various interrelations and correlations are plainly worked

out. I have found in my own department that generalized draw-

ings aid greatly in connection with such summaries. An illustration

which brings before the student by means of a single chart or page

many of the essentials has been found most useful by myself and

the other instructors in my department. The late Professor Bessey

was a master in preparing such diagrams. His specialty was, as

all know, the preparation of figures to show lines of descent and

evolution of the groups of the plant world. Whether one could

agree as to the derivations and progress of evolution represented

by his diagrams or not did not detract from the fact that his figures

helped greatly to portray the principles and the theories that he

wished to emphasize. Thousands of his students were enabled to

secure a much better idea as to what evolution really is from such

methods even if they went no further in their study of biology

than the Freshman course.

Figures or diagrams of this sort may be studied indefinitely

and the longer they are examined the more illuminating they become

until the average student is enabled to secure a complete and prop-

THE HISTOLOCY AND PHYSIOLOCY OF THE PLANT

REPRODU a

SS

PRIMARY

SS ‘ik TRANSPIRATION

= ‘ il | i, 20

CUTICLE

eecseyenme

CAMBIUM

ROOT HAIRS

GROWING a

WATER

/ AND SOLUTE

MITROGER

>» FIXATION

K

PLATE VI

AMERICAN MICROSCOPICAL SOCIETY 57

erly balanced conception of the chief features of the subject in

question.

Such figures may be constructed in great detail and with much

technical skill or they may be more or less crude and diagrammatic

and still be of great value to the learner. With the information

gained from careful studies of plant anatomy and plant processes

the student should be able to fill in the missing details of a general-

ized drawing and to interpret properly the diagrammatic features

of the figure. This being possible, a diagram of the kind submitted

herewith should be exceedingly helpful and, indeed, illuminating.

It was solely because of the success of these methods in this depart-

ment that I was led to publish these brief notes and the diagram

in the hope that they might be suggestive in some degree to my

fellow workers in biological fields.

The figure as published here has been revised and redrawn

from a similar figure published in 1914 by the writer in a labora-

tory manual of plant physiology. The older diagram was later

enlarged and worked out in the form of a wall chart. The reader

will understand, of course, as he looks at this diagram that the

relative proportions of the various structures exhibited are not

intended to be represented at all as they actually occur in the living

plant. In fact the proportions are mostly so unnatural as to be

grotesque and even misleading if the student or reader does not

understand how to interpret them in the light of what has been said

in the above paragraphs. He is supposed to have acquired this

understanding in his courses of study dealing with the plant. The

figure merely helps him to see the imterrelations of the facts of his-

tology and physiology graphically summarized in the features of the

chart. The chart represents in a more or less diagrammatic man-

ner an epitome of the great facts of histology and physiology.

Many additional entries might be made upon the chart, but the

danger is in so multiplying details that the real purpose of the

sketch may be obscured behind the maze of unessential details.

That would be a serious blunder in the use of this method of

teaching or learning.

I believe that it is one of the chief duties of a teacher to epi-

tomize very carefully his subject as fully as possible by whatever

58 NOTES, REVIEWS, ETC.

methods he may devise. In so far as he is successful in this prac-

tice so far will the students in his classes carry away with them a

definite and compact and possibly useful body of knowledge upon

the subject taught. Such a chart as this has been found very

useful in such an epitome, and the method in general is successful

in application.

Raymonp J. Poot, Pu. D.

The University of Nebraska.

A METHOD FOR MOUNTING ANATOMICAL PREPARATIONS FOR

; EXHIBITION

Oftentimes students in comparative anatomy make excellent

preparations which are worthy of preservation. But this is not done

as a rule because of the trouble in mounting. A glass strip of just

the right size to fit into the exhibition jar must be found. The

preparation is with difficulty tied by thread to the glass plate. If

one could only “pin” into glass! The following method is sug-

gested as a solution. A mixture of hard paraffin, beeswax and

lampblack is melted up and poured into a paper box cover about

the size needed had a glass plate been used. The mixture in the

cover should be between %4 and % inch deep. It should be allowed

to cool somewhat and then on this bed the preparation should be

placed and pressed down into it somewhat. A few small pins can

easily be made to fasten it securely. When the matrix is cooled

they can be clipped off on the back. Labels can also be easily

attached to parts of the dissection. The entire cover can now be

placed in cold water for a few minutes. When hardened the cover

can be cut away with a knife—the paraffin background cut down

to just the size that will fit into the glass jar—care being taken to

make it fit in snugly. The jar can now be filled with formalin and

the cover fastened on. The black background makes the objects

stand out distinctly and the preparation never becomes loosened

from its wax bed. The whole operation takes but a few minutes

time.

G. Gi Scorn

Department of Biology,

College of the City of New York.

AMERICAN MICROSCOPICAL SOCIETY 59

GREEN LIGHT FOR DEMONSTRATING LIVING CESTODE OVA

By projecting the light from a small arc through two screens,

one of Picric Acid yellow and one of Methyl Blue blue, a green

light was obtained which gave a peculiarly sharp definition to the

details of living Cestode ova. The eggs were studied on a slide

under either low or high power. This light has been used in

connection with the demonstration of the eggs of Dipylidium ca-

ninum, Tenia crassicollis and Hymenolepis nana. The color screens

were prepared by staining lantern slide plates, after dissolving out

the silver with Hypo from the unexposed plate, in either a weak

solution of Picric acid or Methyl Blue. In each case the film

was stained until it showed a distinct color when washed in water.

University of Colorado. M. M. ELtIs.

A NEW METHOD OF STAINING TISSUES CONTAINING NERVES

Captain Sydney M. Cone, M. D. (J. Am. Med. Assn., Jan. 19,

1918) proposes the following method for nerves, which he says is

excellent in bringing out axis cylinder and medullary sheaths, being

approached only by Bielschowsky’s Axis Cylinder stain. The latter

requires days, and does not give contrast staining.

(1) Harden in 4% formaldehyde for 6 days; (2) carry thru

graded alcohols to ether and absolute alcohol; (3) embed in cel-

loidin and cut 10-20 microns, washing in water; (4) stain 15 min-

utes in carbol fuchsin, washing rapidly in water; (5) place in 1%

osmic acid 5 minutes; (6) wash rapidly, and stain for 1 minute in

50% aqueous solution of safranin; (7) place in 1% acid alcohol

1 minute; and in 95% alcohol for 2 minutes; (8) place in absolute

alcohol and clove oil alternately until sections appear deep pink and

translucent; (9) place in xylene 2 minutes, and mount in Canada

balsam.

Paraffin sections sometimes stain fairly well, but it is not safe,

as xylene used to remove the paraffin before staining interferes

with the process. The formaldehyde hardening is essential for the

success of the stain.

[I wonder whether terpineol could not be used here, or berga-

mot, or naphtha, or even toluol. It would be a good thing if labora-

60 NOTES, REVIEWS, ETC.

tory workers would give the newer results for clearing agents that

do not decolorize stains, used after imbedding in (a) celloidin or

parlodion, or (b) paraffin.—V. A. Latuam, Abstractor.]

FONTANA’S SPIROCHETE STAIN

Fontana’s method of staining spirochetes involves the use of

the following preparations:—1l. Fixing fluid: acetic acid, 1 c.c.;

formalin, 20 c.c.; distilled water, 100 cc. 2. Mordant: tannic

acid, 5 gm.; phenol solution (1 per cent), 100c.c. 3. Silver solution:

Prepare a 0.25 per cent solution of nitrate of silver, which may be

done with sufficient accuracy by dissolving a small crystal in half a

test tube of distilled water and adding just enough ammonia solu-

tion to cause a slight permanent turbidity. 4. Distilled water.

3. Process: Prepare the slide to be stained by spreading the

material from the syphilitic lesion very thinly on a clean slide, allow-

ing to dry spontaneously ; fix by pouring on the fixing fluid, pouring

it off after a few seconds. Renew immediately, and perform this

process several times. The total duration of this stage should be

not less than a minute. Wash well in distilled water, flood with the

mordant, apply gentle heat until steam arises, and allow the process

to continue for half a minute; wash thoroughly in distilled water

(15 to 30 seconds), flood with the silver solution, again warm gently

for half a minute, wash, blot and dry. Mount in balsam for per-

manent specimens. The spirochetes are stained jet black, and appear

larger than when stained by ordinary methods. Cedar oil causes

the spirochetes to pale. Vi AL

SIMPLE METHOD OF CLEANING OLD USED SLIDES

Johnson (J. Am. Med. A., Dec. 8, 1917) suggests soaking slides

indefinitely (24 hours at least) in full strength commercial (house-

hold) ammonia, followed by rinsing with water and wiping clean.

Stained smears of all kinds, immersion oil, balsam mounts are

equally well cleaned. The same supply may be used repeatedly if

kept in tightly closed receptacle. V.-AL UE:

MENTHOL FOR NARCOTIZING

Don’t forget that menthol is an effective reagent in narcotizing

or anzsthetizing lower forms of life such as Rotifers, Infusoria, or

even small crustacea. Vo Aris

AMERICAN MICROSCOPICAL SOCIETY 61

FRESH WATER BIOLOGY

Teachers in America have been eagerly awaiting this volume

for several years. The knowledge of the plans of the editors and

publishers, including as they have the collaboration of a large num-

ber of active biologists, has inevitably become wide spread. The

need of such a work, planned on a scale that would be at once

liberal and feasible, has been so genuine that it was assured in

advance of a very wide use among teachers and working biologists.

The unanimous verdict will be that every reasonable expecta-

tion has been met by Professors Ward and Whipple and their

helpers, in spite of the fact that the delays inevitable in getting

such a synthetic task before the public will cause many of the

contributors themselves to feel the need of revising their work by

the time of its first appearance. All teachers of Biology, all

advanced students of any group, all amateurs who use the micro-

scope on living things, will find “Fresh Water Biology” a necessary

part of their equipment.

The volume is, in many ways, very close to the kind of work

which has long been fostered and advanced by the American Micro-

scopical Society and its Transactions. An organized interest in

limnological work was manifest as early as 1899, at which meeting

a Limnological Commission, consisting of Professors Birge, Eigen-

mann, Kofoid, Ward and Whipple, was appointed to “unify, extend,

and stimulate limnological work in this country.” The following

year this Commission made a report which anticipated much of

the ecological work done since with the fresh-water forms of this

country, and unquestionably gave inspiration and impetus to the

studies on which this book is based. While the volume cannot be

listed among the annual “Transactions” of the Society, certain it

is that much of the contributory work leading to this fine showing

in American fresh-water Biology has been done by members of

this Society and published in one form or another in its Transac-

tions.

So close is this enterprize to what this Society has been encour-

aging in every possible way for many years, that the pages of the

Transactions are now freely offered the editor and collaborators,

62 NOTES, REVIEWS, ETC.

pending new editions of the book, for such supplementary and

revisional statements as may be necessary from time to time to

keep the accounts and keys of the various groups up to date. Such

a cooperative arrangement would contribute greatly both to the

convenience of our membership and to the most effective use of

this manual.

The work is much too compendious and condensed to allow an

adequate statement even of its scope, much less to bring to our

readers any of its specific contents. In general the material pre-

sented is to be classified under three headings: (1) General dis-

cussion of the conditions of life and of the effective study of organ-

isms; (2) the biological conditions, method of collection, culture

and preservation of the special groups; and (3) systematic keys,

with descriptions and illustrations of the classes, orders, families,

genera, and representative American species of the groups treated.

Under the first head may be included the introductory chapter

by Professor Ward, the chapter on “Conditions of Existence” by

Professor Shelford, on “Methods of Collecting and Photograph-

ing” by Professor Rieghard, and the concluding chapter by Pro-

fessor Whipple on ‘“‘Technical and Sanitary Problems,” as related

to fresh waters. The biological features of the special groups are

treated at the beginning of the appropriate chapters. The chap-

ters on Bacteria, Larger Aquatic Vegetation, and Aquatic Verte-

brates are confined to this aspect, making no effort at systematic

display.

The following experts furnish the systematic chapters: Ed-

gar W. Olive, Blue-green Alge; Julia W. Snow, Other Fresh

Water Alge; C. H. Edmondson, Amceboid Protozoa; H. W. Conn

and C. H. Edmondson, Flagellate and Ciliate Protozoa; Edward

Potts, The Sponges; Frank Smith, Hydra and Other Fresh Water

Hydrozoa; Caroline E. Stringer, The Free-living Flatworms ; Henry

B. Ward, Parasitic Flatworms; Wesley R. Coe, The Nemerteans;

N. A. Cobb, Free-living Nematodes; H. B. Ward, Parasitic Round-

worms; H. S. Jennings, The Wheel Animalcules; H. B. Ward,

Gastrotricha; Frank Smith, Aquatic Chetopods; J. Percy Moore,

The Leeches; A. S. Pearse, The Fairy Shrimps; E. A. Birge, The

AMERICAN MICROSCOPICAL SOCIETY 63

Water Fleas; C. Dwight Marsh, Copepoda; R. W. Sharpe, Ostra-

coda; A. E. Ortman, Higher Crustaceans; R. H. Wolcott, The

Water Mites; James G. Needham, Aquatic Insects; Charles B.

Davenport, Moss Animalcules; Bryant Walker, The Mollusca.

Two devices in the arrangement of the systematic matter call

for comment. The guide numbers in the artificial keys are arranged

in accordance with a plan developed by Professors Forbes and

Smith at the University of Illinois. Each guide line begins with

a number. In addition to its own appropriate number which leads,

there follows in parentheses the alternative number (or numbers)

which indicates the contrasted line to which the seeker must go if

that particular legend is not diagnostic. This is true both of the

earlier and the later guide lines in a given series. If a given key

line is acceptable the further guiding number is at the close of the

line. The device thus gives a perfect system of cross references

both forward and backward between categories of a given grade.

This is unnecessary in brief keys; but where there are scores of

intervening subordinate categories it is a great convenience. The

name and description of a species, all the supplementary biological

facts concerning it and the illustration are included in a solid panel

between its own key line and the next. This gives a convenient

compactness which is very satisfying.

The general impression which follows examination of the book

is the perfectly enormous amount of material condensed into its

somewhat more than 1000 pages. This means, of course, great

brevity, and yet no one interested in these groups can feel that the

interesting and important matter has been left out. To one whose

studies are confined largely to a single group there must come

a renewed and enlarged sense of the representative character of

the fresh-water organisms. One has brought home to him also

the vast incompleteness of our records of the American distribution

of even the better known fresh-water species. It ought to be pos-

sible in connection with the extended use and further revision of

such a work as this to get a better account of specific range in this

country.

It seems ungenerous to mention slight imperfections where

so much has been brought to our aid. However, the appearance

64 NOTES, REVIEWS, ETC.

of the chapters on Protozoa and Oligochztes is marred by the use

of occasional cuts too heavy and opaque to give any true idea of

the delicacy of the organisms. Figure 982 of Chetogaster is an

example of this.

A list of important references, in no case purporting to be a

complete bibliography, concludes each chapter. An adequate index,

including important descriptive terms and all of the scientific names

used in the keys, concludes the book.

Fresh Water Biology, by Henry B. Ward and G. C. Whipple, with a staff of

Specialists collaborating. Pages ix and 1111, with 1547 text figures. John Wiley and

Sons, New York and London, 1918. Price, $6.00.

AN INTRODUCTION TO THE HISTORY OF SCIENCE

Nothing which has come to the attention of the reviewer puts

more convincingly the meaning of the history of science than the

preface of this little book by Professor Libby. “The history of

science has something to offer to the humblest intelligence. It is a

means of imparting a knowledge of scientific facts and principles

to unschooled minds.

“The history of science is an aid in scientific research. It

places the student in the current of scientific thought, and gives

him a clue to the purpose and necessity of the theories he is required

to master. It presents science as the constant pursuit of truth

rather than the formulation of truth long since revealed; it shows

science as progressive rather than fixed, dynamic rather than static,

a growth to which each may contribute.

“It is only by teaching the sciences in their historical develop-

ment that the schools can be true to the two principles of modern

education, that the sciences should occupy the foremost place in

the curriculum and that the individual mind in its evolution should

rehearse the history of civilization.

“The history of science should be given larger place than at

present in general history. History of science studies the past

for the sake of the future. It is a story of continuous progress.

It is rich in biographical material. It shows the sciences in their

interrelations, and saves the student from narrowness and pre-

mature specialization. It affords a unique approach to the study

AMERICAN MICROSCOPICAL SOCIETY 65

of philosophy. It gives an interest in the applications of knowledge,

offers a clue to the complex civilization of the present, and renders

the mind hospitable to new discoveries and inventions.

“The history of science is hostile to the spirit of caste. It

reveals men of all grades of intelligence and of all social ranks

co-operating in the cause of human progress. It is a basis of intel-

lectual and social homogeneity.

“Science is international,—English, Germans, French, Italians,

Russians—all nations—contributing to advance the general interests.

[The teaching of it] cannot fail to enhance in the breast of every

young man or woman faith in human progress and good will to all

mankind.”

In method, this introduction takes up certain great scientific

relations and applications, and treats these largely in connection

with the personality of the men who have contributed their solu-

tions. Some of the graphic chapter headings will carry the sug-

gestion of method and of content:—1. Science and Practical Needs

—Egypt and Babylonia; 2. Influence of Abstract Thought—Greece:

Aristotle ; 3. Scientific Theory Subordinated to Application—Rome:

Vitruvius; 4. The Continunity of Science—the Medieval Church

and the Arabs; 6. Scientific Method; 7. Science as measurement;

8. Cooperation in Science; 9. Science and the Struggle for Lib-

erty; 10. Interaction of all the Sciences; 11. Science and Religion;

12. Reign of Law; 14. Scientific Prediction; 16. Science and War;

17. Science and Invention; 19. The Scientific Imagination; 20.

Science and Democratic Culture.

The presentation is simple, direct, vivid, untechnical, and well

suited to the intelligent reader with general interests.

An Introduction to the History of Science, by Walter Libby. [Illustrated; 288

pages. Houghton, Mifflin Company, Boston, 1917. Price, $1.50, postpaid.

A SHORT HISTORY OF SCIENCE

Evidently the stay and the work of M. Sarton in this country

is helping create an atmosphere in which we may prophesy an exten-

sion of interest in the history of science. In this atmosphere our

own American teachers, who have been doing something in this field

for their students, are being encouraged to bring their work to the

66 NOTES, REVIEWS, ETC.

more general audience. All this is very much worth while and will

stimulate the giving of similar courses in many of our schools and

colleges, both to culture the general student and to unify the scien-

tific consciousness of the student of science.

The book under review is by Professors Sedgwick and Tyler

and embodies very largely the well known course of lectures on

the subject begun by the senior author in Massachusetts Institute

of Technology more than twenty-five years ago. The purpose is

expressed by the authors thus,—“To furnish a broad general per-

spective of the evolution of science, to broaden and deepen the

range of the students’ interests, and to encourage the practise of

discriminating scientific reading, . . . by furnishing the student

and the general reader with a concise account of the origin of that

scientific knowledge and that scientific method which, especially

within the last century, have come to have so important a share in

shaping the conditions and directing the activities of human life.”

The general treatment is broadly chronological and geographic,

—following the origin and rise of the wonderfully varied civiliza-

tions of the near-Mediterranean peoples and their distinctive marks

upon the progress of knowledge, of its applications, and of the

method and spirit which its right pursuit demands of its followers.

The chapter headings indicate this phase of the treatment: Early

Civilizations; Early Mathematical Science in Babylonia and Egypt;

Beginnings of Science; Science in the Golden Age of Greece; Greek

Science in Alexandria ; Decline of Alexandrian Science; The Roman

World,—The Dark Ages; Hindu and Arabian Science; Progress

to 1450 A. D.; A New Astronomy and the Beginnings of Modern

Natural Science ; Mathematics and Mechanics in the Sixteenth Cen-

tury; Natural and Physical Science in the Seventeenth Century ;

Beginnings of Modern Mathematical Science; Science in the Eigh-

teenth Century; Modern Tendencies in Mathematical Science; Ad-

vances in Science in the Nineteenth Century.

Within these general headings, further analysis and presenta-

tion are based upon a combination of biography, the rise and solu-

tion of problems, and the discovery of the principles which have

proved significant and fruitful. Topics like the following raise

the expectations of the reader and indicate the emphasis: Primitive

AMERICAN MICROSCOPICAL SOCIETY 67

Interpretations of Nature; Astrology; Primitive Counting and

Geometry ; Mathematics in Egypt; the Calendar and Measurements

of Time; Greek Mathematics; Pythagoras; Beginnings of Rational

Medicine; the Hippocrates; the Sophists; Circle Measurements ;

Aristotle; Euclid; Archimedes; Earth Measurements; Beginnings

of Human Anatomy; Mathematics and Astronomy at Alexandria;

Ptolemy ; Hindu Astronomy; Arabian Contributions to Mathematics

and Astronomy; Renaissance and Sciences ; Alchemy ; the Compass ;

Clocks; Textiles; Printing; the New Astronomy,—Copernicus,

Tycho Brahe, Kepler, Galileo; Medicine and Chemistry, Anatomy ;

Vesalius ; Higher Algebraic Equations and Symbolic Algebra; Gre-

gorian Calendar; Harvey and Blood Circulation; Studies of the

Atmosphere, Barometer, gases; Phlogiston; Beginnings of Chem-

istry ; Bacon and Descartes; and thus on to the great wave of mathe-

matical and natural science discoveries of the eighteenth and nine-

teenth centuries which cannot even be enumerated here.

The book is enlivened thruout by appropriate quotations from

the men who did the work and from appreciative commentators on

that work. In a series of appendices are more lengthy documents,

—as, the oath of Hippocrates, Dedications by Copernicus and Har-

vey, Gallileo before the Inquisition, and the like. Appendix I

enumerates and discusses briefly some leading inventions of the

last two centuries.

The volume closes with a table of the important dates in the

history of science and of civilization, a brief list of reference books,

and an index. Each chapter closes with a list of references.

The book is attractively made up and printed.

A Short History of Science, by Sedgwick and Tyler. Illustrated, 474 pages.

The Macmillan Co., New York, 1917. Price, $3.50.

BIOCHEMICAL CATALYSTS IN LIFE AND INDUSTRY

This volume discusses only the proteolytic enzymes, being the

second volume by the author on enzymes and their uses. A pre-

liminary chapter discusses the nature of the transformations that

take place in the living cell, the inorganic catalysts, the biochemical

catalysts, the theories as to their mode of operation, and a classifi-

cation of proteolytic enzymes based on the number of molecules of

68 NOTES, REVIEWS, ETC.

water they are capable of fixing in a molecule of albumin. Follow-

ing Schutzenberger’s conception of the structure of the polypeptide

molecule, the author presents a very attractive and cogent statement

of the mechanism of progressive hydrolysis of these molecules under

ferment action.

The general discussion proceeds under these heads:—The

Coagulating Catalysts,—thrombin, myosinase, and rennet; Pepsin;

Trypsin, both pancreatic and from various animal and vegetable

sources; Erepsins, including those secreted in the intestines, the

poorly defined peptolytic enzymes which act on so-called peptones,

nucleases which transform the phosphoric nucleo-proteins, argin-

ase, and a small group of creatin-destroying catalysts; and the Amz-

dases, the group of enzymes which aid in the final decomposition

of the amino-acids,—the last stages of the reduction of the protein

molecule before assimilation or excretion.

The statement of the nature, origin, mode of isolation, prop-

erties, and physiological role of these vital substances is extremely

lucid, and meets the need of the general biologist who has not

the opportunity to keep abreast with the more technical aspects of

this department of biochemistry.

Most general readers will be especially attracted to Part VI,

which deals with the applications of these organized catalysts to

medicine and industry, together with the grounds upon which such

applications are possible. The author traces the use and abuses of

pepsin in therapeutics, and progress made in standardizing tests of

its efficiency both as to dissolving and in actual peptonizing power.

Reference is made to peptones, both peptic and pancreatic, offered

as an easily assimilable diet for greatly debilitated patients. Sim-

ilar preparations are used in making culture broths in bacteriolog-

ical laboratories.

In a similar way diagnosis of stomach states is made by analysis

of the gastric contents at different stages of test meals, with a view

to obtaining the amount of chemical change, the acidity, and the

enzymic contents. The author holds that the disrepute into which

this determination has fallen is due to poor methods of application

rather than to any fault of the principle itself.

In preservation and use of grains and flours native proteolytic

AMERICAN MICROSCOPICAL SOCIETY 69

enzymes, and those produced by micro-organisms on the surface

of the grains or placed in the flour purposely, bring changes that

must be considered. So in brewing and in grain distillation, these

biological catalysts play an essential role. The same processes are

seen in the milk ferments and in the ripening of cheeses. In the

latter some of the enzymes are native to the milk, some are pro-

duced by micro-organisms, and rennet is added artificially.

There is an interesting discussion of the relation of the pro-

teolytic milk ferments to intestinal putrefaction. The writer him-

self has done work with the Bulgarian ferment, and his views

as to the cause of the benevolent intestinal action of the various

clotted milks are contrasted with those of Metchnikoff and others.

Other topics discussed are :—putrefaction, enzymes operative in

tanning, biocatalysts of the soil, assimilation of atmospheric nitro-

gen, fertilizers, recovery of nitrogenous wastes, and artificial nitro-

genous foods.

As the outcome, largely, of his own experiments the author

sums up his conclusions in respect to the last item thus:—“It ap-

pears that there is ample proof that the organism draws all its

nitrogenous constituents from the hydrolysis of proteins. These

may result either from the actual process of digestion, or from

artificial means, like the action in vitro of proteolytic enzymes or

the action of concentrated acids. In all events, these [artificially

reduced nitrogen molecules] are directly assimilable substances and

should be considered as food materials of great nutritive value.

In fact, it has been established that a mixture of amino-acids,

containing qualitatively and quantitatively all the principal products

of the complete hydrolysis of proteins, can replace the albuminoid

foods, and as such maintain the animal organism in nitrogenous

equilibrium.” The writer is convinced that nutrition can ultimately

be effected more economically and rationally by the substitution of

some of these artificially produced nitrogenous foods for the com-

plex natural ones, such as meat.

Each chapter is followed by a bibliography ; and an index closes

the book. The mechanical part is well done.

Biochemical Catalysts in Life and Industry, by Jean Effront. Translated by

Samuel C. Prescott, 752 pages. John Wiley and Sons, New York, 1918. Price, $5.00,

postpaid.

PROCEEDINGS

of the American Microscopical Society

MINUTES OF THE PITTSBURG MEETING

The thirty-sixth annual meeting of the American Microscopical Society

was held in affiliation with the A. A. A. S. at Pittsburg, Pa., Dec. 29, 1917.

In the absence of President Guyer, Vice-President Griffin acted as

chairman.

The report of the Custodian was presented and was accepted, ordered

printed and referred for audit to a committee consisting of Professor Grif-

fin and any other Pittsburg members whom he might select.

The Treasurer’s report for the years 1916 and 1917 was accepted and

referred to an auditing committee consisting of Drs. Latham and McCalla

of Chicago.

The Society approved the recommendation of the Treasurer that the

fiscal year be regarded as extending from Dec. 1 to Nov. 30.

The following officers were duly nominated and elected for the con-

stitutional periods: President, Professor L. E. Griffin, University of Pitts-

burg; First Vice-President, Dr. H. M. Whelpley, St. Louis; Second Vice-

President, Professor C. O. Esterly, Occidental College; Secretary, T. W.

Galloway, Beloit College (for two years); Custodian, Magnus Pflaum, Esq.

In connection with the re-election of Mr. Pflaum it was noted that he

has been custodian for eighteen years continuously since the formation of

the office, having been Treasurer for three years before. A cordial vote

of appreciation was extended him for this long and efficient service.

Professor Max M. Ellis of University of Colorado and Professor

J. E. Ackert of Kansas State Agricultural College were chosen as the elective

members of the Executive Committee for 1918.

Professors Grifin and Galloway were appointed a committee to approve

and print the minutes. Adjourned.

T. W. Gatitoway, Secretary.

72 MINUTES

SPENCER-TOLLES FUND

Custodian’s Report for the Years 1916 and 1917

Amount reported in TG soi cide eee oe eee $4489.32

Sane Oly 1916) Dividends ee ioe Tay aia $ 134.67

ely) 45 1SIG: SDivademasnite s). ade sais tet rordina we ties Miele eee 138.69

Pec.) 6, 1916: (Salei pti ransactions :))s ))s/.6 bonis eaaicexeene 60.00

Bec:/15, 1916) Sale jor Mransactions 3.024 issn fee emote 60.00

CU ST TOG. So vaAeTES Hs) lee 6 Sys cis Wis ccioere ulead wivwele etree 142.86 536.22

$5025.54

etl ds OL7 A ONVIGEMUS Cen RU Wl tno menhe Gesu a ralg $ 150.75

PCOS HADET. EMVIGEMES ) le odie ois Netemieiniojctetale eee cetes aieereee 155.28 306.03

$5331.57

GRAND TOTALS

AU CONETIBUHORS ae ite Nieisd Sa uitlo miaalie eis a eee a dwrmcaiereere $ 800.27

ATE Sales Of TransSactecarsy ye ee Ne te ate le 878.38

ATE Te KMEmbEnSHigs eee snot s cet ole ole eemretel aie tele lee 300.00

AMD Taterest and (Dividends' (253.0. sess assay ein oe ecient 3542.92 $5521.57

LESS

PAU MaraMIPS eee. iota el wa mbliclen teldian vad cheisiets eke ale muntomtetetm mete $ 150.00

WlbyDites on) tafe ‘Members tick. ocias oc ce sae cae cule wits 40.00 190.00

$5331.57

Life Members: (Robert Brown, dec’d); J. Stanford Brown; Seth

Bunker Capp; Henry B. Duncanson; A. H. Elliott; John Hately.

Contributors of $50 and over: John Aspinwall; Iron City Microscop-

ical Society; Magnus Pflaum; Troy Scientific Society.

Macnus Prraum, Custodian.

Pittsburgh, Pa., Dec. 29, 1917.

We, the undersigned committee, hereby certify that we have carefully

examined the above account for the years 1916 and 1917 and found the same

correct.

L. E. Grirrin, Chm. Auditing Committee.

MINUTES : 73

ANNUAL REPORT OF THE TREASURER OF THE AMERICAN

MICROSCOPICAL SOCIETY

December 24, 1915, to December 24, 1916

RECEIPTS

Bates on, band)tromy TODS i oe Ae aU crt) er et $ 476.65

Merabershigi QUES: | sos oe s.2 ai ce Re AEROS Pe AAR EEE Ur 532.10

LGREESEE NB LG S15 ex stop NA UE Rees DIG Br cay Pies NE Le Ns AN ae nN IR OS ae a 129.00

STI SC DCL Space erat Feat tene aS RUA a Epa a lhe as ayreria an equ uN Enh Sma a 302.20

Salesvo fee DEATISACHONS a Va seer alee ical eae chat ieraih cpa nia atsaees Sieitepall 150.40

RAV CEEISEE Sie Meee ats eee ene tee Nene Ete Le oy aL) Mtn ata aaa toreta 184.90

MEAGRE nei S ok Aha Sata eer ra GTA «ele chaser erate: Se tahets hoa 2.68

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EXPENDITURES

Prntine Transactions, volume 34, nov 4. o0.06 vcd ios cece samiiees $ 269.28

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PMMESRENS MICH ATEC ETEIRE cr ooo ists Wiel es eae titers Ee uae ene ae She ee 37.00

Office expenses; stenography, supplies, etc.—

SSE CE CEE Wine sch eters ta coc Sica teas aCe aoe = are eC tS ce ee eee era 134.59

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Postage and express—

CS ECSUTLE 9 5 pr NRE MERE oe UL A ea UR NC Pr REP BT 2 SR St ah 109.78

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Ra ated E ALISACTIONS) iaig(s nosed at en ne eae ee eae he pee 16.16

Spencer-Tolles Fund from sales of sets of Transactions to U. of Pa.

ANGTEOS NOEs Dames Ue oe aceaaan rd beth st UL e anes Sethe mS 120.00

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Expenses of Secretary at Columbus meeting, 1915................... 31.65

SVEST EAA Be eh NPE RY iP lW SRA aS e a LEN ACEO Och ALOR A 5.70

woe. Eheaes Tae tell 231 imate PB ante SR ih Cpe AA ce CAN PO Rae SE 273.71

$1777.93

Respectfully submitted,

H. J. Van CLeAvE, Treasurer.

Feb. 15, 1918.

We hereby certify this statement corresponds with the Treasurer’s book.

V. A. LatHaM,

GEORGE Epwarp FELL,

Auditing Committee.

74 MINUTES

ANNUAL REPORT OF THE TREASURER OF THE AMERICAN

MICROSCOPICAL SOCIETY

For the year beginning Dec. 25, 1916, and ending Dec. 21, 1917

RECEIPTS

Balance on hand ‘from 1916. 0/0/20. Sarees at eee eee ee $ 273.71

Membership iGUesie rari sie nate vss es ele Ae ANCL a ss Seed at a 880.00

MIEIATION s TEES it artite ct Cates isisle eos oes yar e e cle a means Caan ane a ee 42.00

BSISGTIDELS OAR hee ep con ake dice Sic shatters tie Sieieiu oho Me wld eine Ea aa 119.80

MIIGUOE SL TARSACTIONIS. ollctslosciatass ito Bele de ak hhc coe elas et ae 174.00

DROLET RISET SNe Casey eh RCo gece See tl Ul UR tel a 435.00

Partial mavinentutor Cuts, 2. ccc cee sand) ion Sabe vei cas cine oa ee eee 23.14

MRIEAN CVERENPES ei eitcin es otan Galstad eels bie wrens eater blo son gt ne $1947.65

EXPENDITURES

Printing) transactions. -voOlime (5. t10.04 s mntoesa sae aes aml oe eee $ 259.28

Prntine Transactions, volume 36, nos; 1,:2,'and 3 2)..4.....0 Gaon 543.74

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Postace arid iepressy ak ole iss etic cheeks okeniss Wid ns oe teen ae ree 75.42

Office expenses—

SS ECTELAEY |)’ aes c chon araie ecb gS wide e ocala oleh do oun sn ee eR ance atin aie ee eno 73.37

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In part payment of Secretary’s expenses to Pittsburg meeting...... 50.00

Binding set’ of; 0 ransactions fOr Sale’. cic eicisie eters sole wists) ng <u os 20.00

FISECIIAREOUS ITEMS s,s Wioislece ewddie va bid nes detec ca ies Oe ee 16.02

Babatice-on Hands: <.sceg Sh eeeioseanhccead eee oe SRG ata ee 770.57

$1947.65

Respectfully submitted,

H. J. Van CLeAvE, Treasurer.

Feby. 15, 1918.

We hereby certify this statement corresponds with the Treasurer’s book.

V. A. LatHam,

GEORGE EDWARD FELL,

Auditing Committee.

Feb. 12th, 1918.

I hereby certify that I have examined the vouchers and accounts of

H. J. Van Cleave, Treasurer American Microscopical Society, for the period

ending Dec. 21, 1917, and I find the balance of $770.57 as shown on the books

to be correct.

F. S. Jonnson, Public Accountant.

TRANSACTIONS

OF THE

American

Microscopical Society

ORGANIZED 1878 INCORPORATED 1891

PUBLISHED QUARTERLY

BY THE SOCIETY

EDITED BY THE SECRETARY

T. W. GALLOWAY

BELOIT, WISCONSIN

VOLUME XXXVII

NUMBER Two

Entered as Second-class Matter December 12, 1910, at the Post-office at Menasha,

Wisconsin, under act of March 3, 1879.

The Culleniate Press

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eam Vice-president: ©. QO: WSTERTY: 20: os eee en ee Los Angeles, Cal.

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EX-OFFICIO MEMBERS OF THE EXECUTIVE COMMITTEE

Past Presidents Still Retaining Membership in the Society

ALBERT McCALLA, PhD., F.R.M.S., of Chicago, IIl.,

at Chicago, Ill., 1883

Gero. E. FELL, M.D., F.R.M.S., of Buffalo, N. Y.,

at Detroit, Mich., 1890

Smion Henry Gace, B.S., of Ithaca, N.Y.,

at Ithaca, N. Y., 1895 and 1906

A. CLIFFORD MERcER, M.D., F.R.M.S., of Syracuse, N. Y.,

at Pittsburg, Pa., 1896

A. M. Biere, M.D., of Columbus, Ohio,

at New York City, 1900

C. H. E1GENMANN, Ph.D., of Bloomington, Ind.,

at Denver, Colo., 1901

E. A. Birce, LL.D., of Madison, Wis.,

at Winona Lake, Ind., 1903

Hnery B. Warp, A.M., Ph.D., of Urbana, IIL,

at Sandusky, Ohio, 1905

HERBERT Oszorn, M.S., of Columbus, Ohio,

at Minneapolis, Minn., 1910

A. E. HErTz1ER, M.D., of Kansas City, Mo.,

at Washington, D. C., 1911

F. D. Heatp, Ph.D., of Pullman, Wash.

at Cleveland, Ohio, 1912

CHARLES BROOKOVER, Ph.D., of Louisville, Ky.,

at Philadelphia, Pa., 1914

Cuartes A. Koron, Ph.D., of Berkeley, Calif.,

at Columbus, Ohio, 1915

M. F. Guyer, Ph.D., of Madison, Wis.,

at Pittsburg, Pa., 1917

The Society does not hold itself responsible for the opinions expressed

by members in its published Transactions unless endorsed by special vote.

TABLE OF CONTENTS

For VoLuME XXXVII, NuMBER 2, Aprit 1918

Three New Species of Amebas, with Plates, by Asa A. Schaeffer

Spermatogenesis of the Dog, with Plate, by Julian Y. Malone.......................

Thigmotactic Reactions of the Fresh Water Turbellarian, Phagocata Gracilis,

Leidy, by Bernol 4. "Weimer .--0i5.:52.2..0.-0. oe ote ee

Additions to Our Knowledge of Unionicola Aculeata (Koenike), with Plate, by

1 OPA GA Or: | Rem nS A Dr gtd e. YG Pn Sera nel ae Gat Mlb hrs i Bocce

Notes and Reviews: Methods for Studying Living Trematodes, William Walter

Cort; A Substitute for Euparal, E. S. Shepherd; Chromosomes of Ranatra

Sp?, A. M. Chickering; Notes on Collecting and Mounting Rotifers; Methods

of Preserving Certain Marine Biological Specimens; The Silverman Ilum-

MmAtor Lor WVECOSCOPES: -.<oiees cots osacsens sees cota e reese eee ot cnc ses ec ea nae ee a

125

TRANSACTIONS

OF

American Microscopical Society

(Published in Quarterly Instalments)

Vol. XXXVII APRIL, 1918 No. 2

THREE NEW SPECIES OF AMEBAS: AMOEBA BIGEMMA

NOV. SPEC., PELOMYXA LENTISSIMA NOV. SPEC.

AND P. SCHIEDTI NOV. SPEC.

Asa. A. SCHAEFFER

The classification of the amebas' is peculiar in that two methods of

species determination are followed. The larger amebas are classified

according to the characteristics possessed by the cytoplasm and the

general character of the vegetative stage, while the smaller amebas

are being described according to the method of nuclear division pre-

vailing during reproduction. In the larger amebas a study of nuclear

division is extremely difficult and for many species impossible, owing

for one thing to our ignorance of culture methods by means of which

these species could be raised in abundance. On the other hand the small

amebas exhibit so little cytoplasmic differentiation that specific deter-

minations on this basis seem impossible.

A specific determination is interesting, however, from at least two

points of view. One is the viewpoint of establishing blood relationships

of descent between the different species, or systematics proper. The

other point of view is a purely practical one, i.e., quick identification.

The physiologist or the experimentalist wants a quick and correct method

for identifying the organism he is working with. It is obvious therefore

that if an ameba possesses characteristic cytoplasmic differentiations

which may be observed at any time, the ameba will come to be recognized

by these characteristics rather than by a complicated series of nuclear

events which occur only occasionally and are frequently made out only

with difficulty. In short, as a means of identification, cytoplasmic char-

1 The word ameba is used as a common name for naked rhizopods lacking internal

skeletons such as Amoeba, Pelomyxa, Protamocba, Endamoeba, Négleria, Hyalodiscus,

Dinamoeba, Vahlkampfia, etc.

80 A. A. SCHAEFFER

acters are preferable where they exist; where these do not exist, recourse

may be had to the nuclear division process.

All the larger amebas possess cytoplasmic differentiations in sufficient

number and conspicuousness to serve as a ready means of recognition.

Many of these characters are subject to very slight variation as a result

of changes in the environment. Individual isolation pedigrees carried

on for upwards of a hundred linear generations together with many

collateral lines under varying food conditions, showed that most of the

cytoplasmic characters are hereditary and practically uninfluenced by

what might be said to be the most common environmental changes

(Schaeffer, Science, 1916, p. 468). Amebas are therefore in this respect

like all other groups of animals and the method of classifying them ac-

cording to cytoplasmic differentiations is therefore sound.

These considerations should convince especially our younger micro-

scopists that the investigation of our larger amebas is not nearly as diffi-

cult or forbidding a field as might be imagined from the great amount

of labor that has been expended on the study of the life histories and

nuclear phenomena of some soil and parasitic amebas during the past

decade. The fifty or so species of aquatic amebas thus far described

represent beyond any question only a very small fraction of the number

of species in existence, and this number of known species could probably

be doubled within a few years by careful examination of our marshes

and ponds.

AMOEBA BIGEMMA NOV. SPEC.

Diagnosis. Size in locomotion, 100 to 300 microns long. Form very changeable.

Pseudopods, numerous, tapering, blunt, never with sharp points. Surface smooth,

no fine folds or ridges. Endoplasm usually containing numerous small twin crystals;

crystals attached to ‘excretion spheres.’ Movement rapid, about 125 microns per

minute. Nucleus single, spherical or slightly ovoid, about 12 microns in diameter;

chromatin in small masses clumped loosely together in the center of the nucleus in a

nearly spherical mass about 6.5 microns in diameter. Contractile vacuoles small

about 15 microns in diameter; numerous; no coalescence among them; systole slow.

Endoplasm filled with small vacuoles. Food: flagellates, ciliates, diatoms, rhizopods,

nematodes, vegetal tissue, etc.

This ameba, for which the specific name bigemma is proposed, re-

sembles to some extent the figures and descriptions of Parona’s digitata,

Mereschkowsky’s angulata, Gruber’s spumosa and Penard’s ves pertilio.

In fact I regarded it at first as the angulata of Mereschkowsky or the

ves pertilio of Penard, which it occurred to me might possibly be synony-

THREE NEW SPECIES OF AMEBAS 81

mous. But after further study of the characters of this ameba I began

to suspect that my earlier conclusions regarding its specific reference

might be mistaken. I accordingly investigated the specific characters

of this ameba in connection with some experimental work, under widely

varying conditions for about three years, and compared my observations

with the published reports of earlier investigators of amebas with the

result that I am unable to confirm the specific descriptions by any of the

authors named from this ameba.

In the first place, Mereschkowsky’s (’79) description is extremely vague

(pp. 203-204) Mereschkowsky says the plasm of angulata is trans-

parent, that it contains two kinds of grains: a few large ones and numer-

ous small ones. About three contractile vacuoles and a small round

nucleus are present. Few, ‘“‘am ende zugespitzten (doch nicht wie bei

A. filifera mit welcher A. angulata viel Aehnlichkeit hat) und die gestalt

dicker, breiter Kegel habenden, vom K6érper ausgehenden Pseudopodien

characteristisch. diam. 0.0235.” Movements very rapid. The figure

illustrating this description is very crude. With the exception of size,

this description as far as it goes might apply to a number of species of

amebas. The size, 23.5 microns, is very much smaller than that of

bigemma.

Parona’s description of A. digitata (1883. Essai d’une Protistologie

de la Sardaigne. Arch. des Science physiques et naturelles. T. 10. Troi-

siéme periode. p. 225-243. 1 plate) is somewhat more definite than Mer-

eschkowsky’s of angulata. A. digitata possesses a very granular endo-

plasm, a rounded and conspicuous nucleus, a large contractile vacuole,

““pseudopods longs, conique et aigus,’’ pseudopods always in small num-

ber. Movement is rather slow. Size, 63 microns (p. 228). The only

three characters which may be considered distinctive are the size, the

conical and pointed (the figure shows needle points) pseudopods, and

the number of contractile vacuoles. None of these characters however

are found in digemma. Parona makes no mention of vacuoles in the

endoplasm, which, if he had seen a bigemma, he could not have helped

seeing, since these vacuoles are quite as conspicuous as the nucleus.

There can be little question, I think, that Parona described another

ameba than bigemma under the name digitata.

Leidy(79) figures several amebas resembling bigemma, vespertilio

and digitata more or less closely, but he regarded them all as varieties

of proteus, or as forms of uncertain specific reference.

82 A. A, SCHAEFFER

In his description of A. spumosa Gruber is no more explicit than

Mereschkowsky in the instance mentioned. Spumosa has broad flat

pseudopods, a vesicular nucleus, an endoplasm filled with vacuoles, no

granules, a size of 25 microns, according to Gruber. As emended by

Penard (’02) spumosa possesses these characteristics: A length of from

fifty to one hundred and twenty-five microns; form resembling the foot

of a goose, with very fine longitudinal lines on the surface; numerous

vacuoles; contractile vacuole as much as thirty microns in diameter;

a great many bicuspid granules of very small size in the endoplasm;

nucleus deformable like that of A. limax; a compact nucleolus with a

narrow margin of nuclear sap between it and the nuclear membrane.

Althought I am inclined to accept the emendation of Penard because of

its making for greater definiteness and stability in this difficult genus,

yet it appears to me that instead of really emending or elaborating

Gruber’s description, he actually describes a new and different species.

It is evident that another ameba than bigemma was under observation

by both Gruber and Penard when these authors wrote their descriptions

of A. spumosa.

Penard’s description of A. verspertilio (1902, Faune Rhizopodique du

bassin du Leman. Geneva, pp. 714) is as follows: size, about seventy

microns length; pseudopods have always a conical form, their extremities

being usually sharp pointed although the point may be slightly rounded

occasionally for a moment’; posterior end sticky, dragging debris along

as it moves forward; a profusion of extremely small green grains, and

sometimes large excretion grains, in the endoplasm; a sperical nucleus

with a compact nucleolus which is often covered with fine points; con-

tractile vesicles one, two or three; almost always a considerable number

of vacuoles appearing and disappearing as if they were contractile in

the endoplasm (pp. 92-95). Penard’s figures (p. 94) resemble in a

general way the figures of bigemma, but in the important points such as

the number and character of vacuoles, the shape of the pseudopod

extremities, the relative diameters of the nuclear membrane and the

nucleus, the character of the “nucleolus,” stickiness of the posterior

end, inclusions, etc., there is little resemblance between ves pertilio

and bigemma.

2. . . les pseudopodes ont toujours une forme conique, anguleuse; leur extermite

est en principe acérée; mais parfois la pointe peut s’arrondir pour un instant (p. 93).

His figures all show sharp needle points on the extremities of the pseudopods.

THREE NEW SPECIES OF AMEBAS 83

The Amoeba bigemma is of medium size, being usually from 100 to 300

microns long in locomotion. Occasionally the size is very much greater.

In several old cultures the amebas frequently arrived at a length of 500

microns in locomotion. As a rule, the average size in new cultures is

about 150 microns.

10:37 10:373

10:38% FA aS = a 8 ‘

/0:394 ESS 40:38Z = 0:39%

Fig. 1. Camera lucida drawing of an A. bigemma in locomotion during the process

of fission. The figures indicate the time in hours and minutes. The drawings from

10:3714 on were moved up from their true position the length of the vertical line below;

likewise for all drawings from 10:36 on. Note slowing down in the rate of locomotion

as the fission crisis was approached, and the gradual increase of speed after division.

Although the general shape of this ameba is subject to very great

variation, yet the various transformations are very characteristic. Per-

haps the most characteristic feature of these transformations is the

tongue like pseudopods, usually short, sometimes very long, which are

continually being thrown out at the anterior end and on the free surface

during locomotion (PI. VII, fig. 2). These projections are frequently more

or less conical, though more often perhaps they have somewhat the shape

84 A. A. SCHAEFFER

of a hart’s tongue in outline. These projections are not pseudopods in

the same sense that the projections in dubia or in proteus are pseudopods.

In the latter species a pseudopod gives direction to locomotion, that is,

the whole ameba frequently flows into a pseudopod. In bigemma, how-

ever, the whole ameba almost never, if at all, flows into a pseudopod;

but the tips of the pseudopods advance at about the same rate of speed

as the anterior end of the ameba as a whole advances. The pseudopods

are, therefore, to be regarded rather as “toes” on the single pseudopod

of which this ameba usually consists. During active locomotion the

general shape of the body is most frequently triangular, with the broad

base advancing, and more or less compressed. When the animal is dis-

turbed it assumes a spherical shape. When suspended in the water for

some time the ameba assumes a shape closely resembling that of the

rayed state of a radiosa. From a central spherical mass of about thirty

or forty microns diameter, long, slender, tapering pseudopods are thrown

out which are of a much more permanent character than the pseudopods

thrown out during locomotion. These projections are sometimes per-

fectly straight and of equal size and disposed opposite to each other on

the spherical mass. More often however they are curved and of irregu-

lar shape and size. Their number is usually from six to eight. All the

pseudopods formed during locomotion or while suspended are blunt,

very definitely blunt. Of all the thousands of individuals which I have

examined in all the different stages and cultural conditions, I have never

seen any but definitely obtuse pseudopods. The photographs of live

amebas, figs. 4 and 5 indicate the degree of obtuseness characteristic of

the pseudopods of this species (Pl. VII).

The streaming of protoplasm during locomotion presents some points

of interest. One does not observe the endoplasm flowing slowly in a

definite direction in this species as one may in proteus or dubia, for

example, but the streaming is jerky and irregular. The endoplasm

seems to drain from the sides and posterior end toward the anterior end

against numerous obstructions which often give way. Thus there are

developed momentary counter or cross currents and eddies. The peculiar

character of the streaming is due to several causes. In the first place

the numerous pseudopods are formed and retracted without definite

reference to the direction of locomotion. Then again the upper surface

of the ameba is not level but extremely irregular; one observes a con-

fusion of high ridges and deep depressions thrown together without

THREE NEW SPECIES OF AMEBAS 85

observable order. From the depressions more or less permanent pillars

of stiffened ectoplasm pass down to the lower surface obstructing the

flow of endoplasm. When these pillars give way, as they frequently

do, the bottom of the depression is suddenly pushed upward as if the

endoplasm were under considerable internal pressure. Another cause

of the irregularity of endoplasmic streaming is due to the fact that the

anterior end does not advance steadily over the whole front, but by a

series of waves here and there. Such a wave consists usually of a web

of ectoplasm flowing out between and connecting two or more of the

pseudopods; then the web halts momentarily while the pseudopods push

out again or while new pseudopods form.

The rear end of the ameba usually plays little part in locomotion in

a special way; usually the rear end is smooth and free from any pseudo-

podial projections. Occasionally, however, a long thin flat pseudopod

may be rapidly thrown out near the posterior end which fastens itself

to the substratum so well that considerable force is required to dislodge

it (fig. 2). The ameba sometimes becomes stretched out to several times

its usual length before the pseudopod is pulled loose. This behavior

also results often in another phase of movement that I have much more

frequently observed in this ameba than in any other, viz., some of the

endoplasm begins to flow toward the posterior end until the ameba is

cut almost in two in the middle. The connection is so thin that one

looks every second for the connecting strand to break, but it does not

break. Sooner or later one or the other portion of the endoplasm flows

back and the whole mass again becomes unified in streaming.

The rate of movement is rapid, being about 125 microns a minute.

Although the anterior end advances very unevenly and irregularly, there

are, nevertheless, long segments in the path of an ameba moving on a

plane surface that are straight. There is present in this ameba, there-

fore, the same tendency to keep on moving in the direction in which it

started to move as there is in proteus (Schaeffer, ’12, ’18; this point will

also be discussed at length in a paper soon to be published).

There is present in this ameba a layer of very thin protoplasm on

the outside of the ectoplasm, as usually defined, which moves forward

over the ameba in the same direction as that in which the ameba moves,

but at a more rapid rate. This is clearly shown by the forward move-

ment over the ameba of small particles which cling to this layer of pro-

toplasm (Schaeffer, ’17).

86 A. A. SCHAEFFER

During locomotion there is usually a broad zone of clear ectoplasm

at the anterior end. Not only are the smaller pseudopods in this region

free from granules but the intervening spaces also consist of ectoplasm.

The nucleus is easily seen. Unless obscured by food masses photo-

micrographs of the living amebas usually show it (fig. 4.). It is spherical

or very slightly ovoid (fig. 3). In an ameba of about 200 microns length

the nucleus is about twelve microns in diameter. The chromatin con-

sists of very small ovoid granules collected together in the centre of

the nucleus in a slightly irregular oval mass of about six or seven microns

diameter. The color of the chromatin is a pale bluish yellow green.

Between the chromatin mass and the nuclear membrane, which is per-

fectly transparent, there is a zone of clear nuclear sap. The nucleus is

single, though occasionally an ameba is found with two nuclei, which

statement is true of course for practically all amebas. The nucieus is

deformable though it is not often that one observes striking deformations

as it is swept along by the endoplasm.

In one culture of iarge amebas of this species the nuclei were about

twenty-eight microns in diameter, and the chromatin mass of irregularly

spherical shape was about fourteen microns in diameter. When these

amebas were slightly squeezed under the cover glass one or two masses

of mostly perfectly homogeneous pale bluish yellow green material was

pressed out from the chromatin mass. There were present here and there

large spheres of denser material of the same color as the homogeneous

masses. Both the granular mass and the homogeneous masses rounded

themselves up and collected near the centre of the nucleus. The material

making up the masses seemed to be of the same sort, though I did not

employ staining methods to determine whether the interior of the gran-

ular chromatin mass was really chromatin or some other substance.

It is, however, interesting to know that at least some of the material

inside the chromatin mass is not granular while the outside material

is.

There seem to be three kinds of vacuoles in this ameba in so far as

their functions are concerned. The endoplasm contains scattered about

in it everywhere numerous (100 or more) small clear vacuoles of various

sizes, mostly under ten microns in diameter, which may be called per-

manent vacuoles. What the function of these vacuoles is, or what con-

ditions are necessary to their origin, remains unknown. That all of

these may become contractile is hardly possible, unless they retain their

identity in the ameba’s body for many hours.

THREE NEW SPECIES OF AMEBAS 87

The second kind of vacuoles are the contractile vacuoles. These have

‘the same general appearance as the permanent vacuoles, excepting that

they are more refractive to light. These vacuoles arrive at a diameter

of about fifteen microns before they contract. It is very seldom that

a larger size is attained. The diastole of these vacuoles is rather slow.

The systole is also very slow, occupying from two to six seconds. There

are a number of contractile vacuoles present at one time. Under favor-

able conditions as many as four may be observed to be in the process

of diastole at one time. The general appearance of the later stages of

a contractile vacuole, that is, higher refractive index of its contents,

possibly indicates that these vacuoles are different from the permanent

vacuoles from their beginning.

The third kind of vacuole, which may be called the fecal vacuoles,

are not frequently met with in amebas. These are large spherical vacu-

oles containing in proportion to their size a very small amount of fecal

matter. These may reach a diameter of twenty or twenty-five microns

and in occasional large specimens a diameter of from forty to sixty

microns. I have not been able to ascertain whether the vacuole ori-

ginates around the fecal matter or whether after the vacuole is partly

formed the fecal matter is voided into it. Since however, the vacuole

usually becomes very large, it is evident that the later stages of the

increase in size is due to the contained solid matter. The systole of these

vacuoles is very slow. The liquid contents is first expelled and then

after a pause the solid matter is thrown out in the way common to amebas

generally.

None of these different kinds of vacuoles seem to grow by coales-

cence; at least from extended observation with this point in view I have

never seen a single case of coalescence, although vacuoles frequently

remain in close contact for a minute or longer.

Another very interesting element in the endoplasm is the crystals.

These are usually very numerous and conspicuous, ranging in size from

one and one-half to two and one-half microns in length. The general

shape is like that of an hour glass or dumb-bell. They seem to be formed

of two bicuspid crystals attached to each other by their apices (figs.

2,3). Under ordinary light they appear dark gray in color; but in po-

larized light they show up very brightly, and then their twin structure

becomes very evident. This is the only ameba known in which such

a twin structure of crystal formation isfound. The polariscope shows an

88 A. A. SCHAEFFER

unmistakable twin structure, however, in only about half the cultures

I have so far investigated. In the others the two points of light are

joined by a bar of light so that one sees a band of light not constricted

in the middle. It may be inferred, however, that in these cultures the

earlier stages of crystal formation are also on the twin pattern. It has

been found that the character as well as the amount of food influences

the size and to some extent the character of the crystals formed. Per-

haps the most striking thing about the crystals in this ameba is the fact

that they are always attached to the so-called excretion spheres when

these are present, as they nearly always are (fig. 3). There is never

but one crystal attached to a sphere. The size of the sphere bears no

relation to the size of the crystal, the spheres being in some cases just

barely visible, while the crystals may be two microns long. When the

the spheres are small the crystals are always attached to them at their

middle. The sphere and crystal bear a remarkable resemblance to a

fish embryo lying on its egg yolk, and they form interesting objects

for observation as they tumble along in the streaming endoplasm. Occa-

sional twin crystals are observed apparently free from attachment to

spheres, but it is possible that the spheres are extremely minute in such

cases, too small to be seen. The excretion spheres rarely exceed a dia-

meter of three microns. In some cultures a few bicuspid crystals with

irregular sides are observed occurring singly. The maximum size of

these is about two and one-half microns. Occasionally also two twin

crystals are found crossed, (fig. 3). Altogether, the crystals form the

most definite specific character of this ameba, and the presence of such

crystals attached to spheres in an ameba may be regarded as definitely

proving its specific identity.

This species is a voracious feeder. Flagellates, shelled rhizopods

ciliates, rotifers, nematodes, diatoms, etc., and especially bits of vegetal

tissues and masses of bacteria, are readily devoured. The body is fre-

quently stuffed with food.

This ameba is one of the hardiest known to me. I have kept numer-

ous and continuous cultures, after being well established, for several

years without much difficulty. The species is subject to very little

variation excepting size. In nature this species must be considered

rare, though it is found frequently where large masses of vegetation are

undergoing decay in quiet water.

THREE NEW SPECIES OF AMEBAS 89

PELOMYXA LENTISSIMA NOV. SPEC.

Diagnosis. Length in locomotion, 75 to 125 microns. Body usually very much

compressed and applied closely to substratum. Changeable in shape, general out-

line oval with few pseudopods. Quiescent stage with numerous pseudopods of various

shapes. Color of body brownish; of protoplasm, pale bluish. Uroid of fine or large

root like projections. Rate of movement very slow, from 1 to 2 microns per minute.

Nucleus spherical, about 14 microns in diameter. Chromatin in a spherical layer of

granules about 11 microns in diameter, with spherical body about 2.5 microns in the

centre of the nucleus. Two nuclei frequently present. Contractile vacuoles numerous,

40 or more; maximum size about 10 microns; systole sudden; diastole very slow. Num-

erous or few small irregular crystalline masses present. Numerous bacterial rods of

about 4 microns length present. Only very few refractive (starch) bodies present.

This pelomyxa is readily recognized by its small size and its very

slow rate of locomotion (PI. VIII, fig. 6). It is, in fact, much the slowest

moving pelomyxa thus far reported, and I, therefore, propose the specific

name of /entissima for this species.

I have found this organism in large numbers in old cultures from the

marshes of Lonsdale on several occasions. But on account of its small

size and its habit of flattening itself out on and sticking close to the sur-

face on which it moves, it more readily escapes detection than other

amebas of the same size. The color of this species is a dull brownish,

somewhat like that of P. belevskii, but not so deep a shade, owing to its

smaller size. This color is, of course, due to the contained food materials

and the indigestible remains of food objects. It seems to be a habit of

this and other pelomyxas to carry for a long time indigestible materials

from food objects before excreting them, if indeed some of this material

is ever excreted while the animal is in the vegetative stage. The color

of the protoplasm is of a bluish violet tint.

This pelomyxa as distinguished from the other species, flattens itself

out and sticks very close to the surface during locomotion. At such a

time it is thickest in the centre and gradually becomes thinner as the

periphery is approached. Around the entire animal there is a clear zone

of protoplasm which is hyaline and very thin and of which the exact

outer limit is very difficult to see. Pseudopods are continually being

extended and retracted from the entire periphery of the animal except

from the posteriorend. These pseudopods are of clear protoplasm except

for a small number of very pale bluish granules which are frequently

found at their bases. The pseudopods are broad or narrow and always

blunt. They do not usually determine the direction of locomotion.

90 A. A. SCHAEFFER

The posterior end terminates in a uroid or group of root-like pseudopodial

projections attaching the organism to the substrate (fig. 6). They play

a part in locomotion but just what their function is has not been carefully

determined. It is certain, however, that the uroid is not necessary to

locomotion to the same extent as in P. schiedti.

Locomotion in this form is so extremely slow that it is difficult to

tell just how it is accomplished. Movement occurs at the rate of from

one to two microns a minute, so that it takes from 30 minutes to an hour

and a half to move the distance of its own length, or three and one-half

months to creep around a baseball. There is a slight but continual and

irregular movement of the endoplasm of an oscillatory sort, which,

together with slight changes of body shape and the retraction and pro-

jection of pseudopods, masks the definite forward movement of the

pelomyxa. Figure 9 shows that this movement undoubtedly exists.

But under ordinary circumstances it is impossible to detect definite

and continuous forward streaming of the endoplasm. It seems as if

forward movement was the sum of all the separate local streamings.

Fig. 9. Camera lucida sketch of a moving P. lentissima to show rate of, and form

changes during, locomotion.

When suspended in the water or when loosened from the substratum

this ameba is remarkable for the number of pseudopods which it throws

out from all sides (fig. 7). This habit may indeed be regarded as a

distinguishing characteristic of this species while in this stage. The

pseudopods are usually more or less conical in shape though they always

have blunt ends. The majority are of simple form, others are branched,

Se eee

THREE NEW SPECIES OF AMEBAS 91

while some are of very odd shapes. Their length is variable, the maxi-

mum being about 50 microns. As might be expected from their rate of

locomotion, the transition from a spherical shape in the suspended stage

to the locomotive stage consumes much time. As the organism again

begins to move, a broad wave of clear ectoplasm appears at some point

near the substrate. The wave slowly enlarges as the pseudopods are

withdrawn, and gradually the locomotive form reappears.

The nucleus of this species presents several points of interest. In the

first place there are usually two. In perhaps three out of four indivi-

duals examined two nuclei were found. In no case did I see more than

two. It seems that in this species it is the normal condition for a con-

siderable interval of time to supervene between division of the nucleus

and the division of the organism. The two processes do not seem to be

directly dependent on each other. In the case of most other amebas cell

division must closely follow nuclear division, or else in the majority

of cases it will become impossible for cell division to take place and the

animal in consequence dies (Schaeffer, ’16). So that what is normal

in division sequences for Pelomyxa lentissima is pathologic for Amoeba

proteus, for example. A P. lentissima with two nuclei may be looked

upon as an individual whose cytoplasm has not yet divided.

Another point of interest with regard to the nucleus of this species

is that a central body is clearly observable in the living condition. The

composition of this body has not been investigated. The diameter of

this body is from two to two and one-half microns. Its appearance is

similar to that of the chromatin granules.

The general appearance of the nucleus is somewhat like that of P.

belevskii. It is spherical, about fourteen microns in diameter, and con-

tains a spherical layer of chromatin granules of about eleven microns

diameter. Not very much can be said about the physical character of

the nuclear membrane owing to the very slight movement of the endo-

plasm. The nuclei are usually found somewhere near the centre of the

animal.

The contractile vacuoles are numerous. In one individual there

were at least sixty, but whether all were contractile or not could not be

determined. The average maximum size of these contractile vacuoles is

about ten microns, though many contract when only five microns or so

in diameter. Occasionally one may reach fifteen microns before con-

traction. Consonant with the slow rate of locomotion is that of enlarge-

92 A. A, SCHAEFFER

ment of the vacuoles. A vacuole of five or eight microns diameter may

take fifteen minutes or longer to enlarge to ten microns, followed by

contraction. The systole is, however, relatively rapid, occurring usually

in about one second.

The spectroscope shows some very irregular small crystalline masses

in the endoplasm sometimes numerous, usually comparatively few. Of

“refractive bodies” (starch grains) there are only a few small ones

present. Other inclusions in the ectoplasm are the bacterial rods, dis-

tinctive of the genus, reaching a length usually of four microns, very

rarely of eleven microns; and the very numerous brownish masses of all

sizes and shapes so common in several species of pelomyxas. In addi-

tion to these bodies there are also numerous very small greenish blue

granules found in the endoplasm.

Besides an occasional diatom or shelled rhizopod, I have found no

recognizable food bodies in this species.

PELOMYXA SCHIEDTI NOV. SPEC.

Diagnosis. Length in locomotion about 75 microns. Usual shape ovoidal. Color,

brownish olive green, almost opaque. ~Pseudopods very rarely formed. Protoplasm

fluid. Movement by eruptive waves of endoplasm partly reflected back along the

sides. Rate of movement about 95 microns per minute. Nucleus sometimes single,

usually double; spherical, about 7 microns in diameter. Chromatin granules in

the form of a hollow sphere immediately under the nuclear membrane or at a slight

distance from it. Contractile vacuoles small, numerous; maximum size about 4

microns; diastole rapid; systole instantaneous. Starch grains very numerous, irregular

in shape, olive green in color, maximum size about 6 microns. Numerous bacterial

rods present of 3 to 4 microns length. Small uroid always present during locomotion.

This species, the smallest of the pelomyxas has been found on several

occasions in large numbers in old cultures of material brought from the

marshes of Lonsdale (Pl. VIII, fig.8). Because of its dark color and rapid

rate of locomotion it at once attracts attention. Although three or four

of my cultures were very rich in this form, it must, nevertheless, be

classed as a rare species. The environment must be of a very special

kind apparently in order that it may develop in numbers. It remained

in my richest culture for about four weeks from the time it was first dis-

covered. I propose for this pelomyxa the specific name sciedti in

honor of my friend Professor R. C. Schiedt.

Under low magnification this organism appears quite black excepting

at a few small places along the sides where the color is temporarily gray-

ish owing to the accidental presence of but few of the starch grains so

THREE NEW SPECIES OF AMEBAS 93

abundant in this ameba. The posterior end also usually is light gray.

Under high powers however, the color is of a dull brownish olive green,

due to the brownish endoplasmic inclusions and the numerous starch

grains. The protoplasm is bluish green.

This species passes through very slight transformations in shape

during locomotion or at other times (fig. 10). Its general shape during

locomotion is ovoidal with the anterior end broad, while the posterior

end is narrow. Never at any time does the organism flatten itself out

to any extent on the substratum. The details of the process of locomo-

tion have not been observed carefully, but the following details may be

mentioned. Owing partly perhaps to the fluid nature of the proto-

plasm, no pseudopods are formed for the purpose of locomotion, but

broad eruptive waves of endoplasm break out somewhere near the anterior

end into which then flows a part of the animal’s endoplasm. These

waves are usually partly reflected back along the sides of the animal,

leaving a more or less clear space at the farthest point reached by the

reflected wave. Occasionally for a short period the pelomyxa may also

advance by endoplasmic streaming as is commonly observed in amebas

generally, but the larger part of its path is negotiated by eruptive waves

as described.

Another important factor in locomotion is the uroid. The animal is

not attached to the substratum anywhere except at the uroid. This is

readily observed when they are taken up with a capillary pipette. The

anterior part of the body is readily displaced by slight currents in the

water but the posterior end is not affected in this way. It seems that

the thin pseudopodium like projections of which the uroid is formed

are for the purpose of holding the organism in place and at the same

time allow it to move forward. But just how this is done could not

readily be determined owing to the fact that these uroidal projections

are very small and very transparent. It has been possible to determine

however, that the projections may be thrown out very rapidly, almost

instantaneously, so that it is possible that new projections are continually

being formed as old ones are being retracted. The alternative view is

that the uroidal projections are dragged along over the substratum

attaching themselves temporarily and locally as they pass over the

substratum. But whatever maybe the exact réle the uroid plays in

locomotion, it is evident that the organism is prevented from rolling over

by reason of its attachment to the substratum.

94 A. A. SCHAEFFER

Although there is, as has been stated, considerable uncertainty in

the direction which the waves of endoplasm take at the anterior end, the

path of the organism may nevertheless, because of the activity of its

prehensile uroid, be straight for a considerable distance. In a sense

therefore the guiding agency in locomotion is located at the posterior

end of the ameba.

The rate of locomotion in schiedti is rapid, being about 95 microns per

minute (fig. 10).

The nucleus of this species presents nothing unusual. Its shape is

spherical. The chromatin occurs in rather large masses arranged in

the shape of a hollow sphere immediately underneath the nuclear mem-

brane or at some distance from it. Usually two nuclei are present as

in the case of lentissima. The binucleate condition therefore represents

an intermediate stage between nuclear division and cell division. This

stage in these two species (schiedti and lentissima) is much longer than

in most typically uninucleate unicellulars, so that the binucleate stage

is much more common. These pelomyxas are therefore to be looked

upon not as binucleate organisms, but as typically uninucleate. The

size of the nucleus is about 7 microns. Owing to the numerous endo-

plasmic inclusions, the nuclei are difficult to see in the living condition.

The contractile vacuoles are made out only with the greatest difficulty

in normal individuals. They are seen only in the small clear areas

which are observed occasionally along the sides during locomotion. With

very attentive examination the vacuoles may then be seen. The maxi-

mum size of the vacuoles is about four microns. Nothing very definite

can be said about their number which is certainly not less than ten, but

is probably very much greater. The diastole is rather rapid. The

systole is practically instantaneous, almost as rapid as the bursting of

a bubble on the surface of water. There is a readily observed character-

istic rush of protoplasm from the immediate vicinity to the place where

the vacuole has just burst, which may be taken advantage of to locate

a bursting vacuole without actually seeing the vacuole. In this way

it is possible to determine that there are several systoles a minute in

different parts of the animal. Doubtless the fluidity of the endoplasm

is in some way connected with the small size and sudden contraction

of the vacuoles.

This species is full of what are probably glycogen grains (Stole, A.,

1900. Zeit. f. wiss. Zool. Bd. 68). Their color is a shade of olive green.

THREE NEW SPECIES OF AMEBAS 95

The shapes of the bodies are varied, mostly irregular, angular with

rounded corners and edges. The maximum size commonly met with

is about six microns. Most of them are only two or three microns long.

They are not evenly distributed throughout the body, but there seems

to be a tendency for them to collect very near the surface in what is

called the ectoplasm. In focussing along the edge one observes a serrated

outline, the teeth being represented by the protruding starch grains.

I presume that a layer of protoplasm at all times covers these bodies

when lying at the surface, though one cannot observe such a layer in

the living organism.

Besides the starch bodies there are found considerable numbers of

very small spherical bodies of a bluish green color. These are met with

in nearly all amebas, but of their nature nothing is definitely known.

The bacterial rods, the presence of which characterizes the genus

Pelomyxa, are found in considerable numbers in schiedti. The length of

these rods is about three or four microns.

The number of brownish colored inclusions which are so commonly

found in pelomyxas, is small in this species. Sometimes only two or

three masses of appreciable size are found. Very little food has been

observed in the bodies of these animals. Occasionally a diatom or a

flagellate was seen, but in the great majority of individuals no recogniza-

ble food objects were found.

When the cultures began to die out, the glycogen bodies began to

disappear gradually. In the last few surviving individuals almost no

glycogen grains could be seen. The organisms were very pale yellowish

and sluggish. Numerous large permanent vacuoles appeared. The

nuclei also changed in appearance. The chromatin receded further from

the nuclear membrane and collected itself in much larger but fewer

granules than normally. From these observations we may conclude

that the starch grains are reserve food stores ashas been shown by Stole

to be the case in P. palustris, and that the cultures died out chiefly

because of lack of food.

Zoological Laboratory, University of Tennessee.

96 A, A. SCHAEFFER

BIBLIOGRAPHY

Leidy, J., 1879. Freshwater Rhizopods of North America. pp. 324. 48 pls. Washing-

ton.

Mereschkowsky, 1879. Studien ueber Protozoen des nérdlichen Russland. Archiv.

f. Mikros. Anat., Vol. 16, pp. 153-248. 2 pls.

Parona, 1883. Essai d’une Protistologie de la Sardaigne. Arch. des Science physique

et naturelles. T. 10. Troisieme periode. pp. 225-243. 1 pl.

Penard, E. 1902. Faune Rhizopodique du Bassin du Leman. pp. 714. Numerous

figures. Geneva.

Schaeffer, A. A. Contributions to the feeding habits of ameba. Trans. Tenn. Acad.

of Science. Vol. 1. pp, 35-43. 2 figs.

1916. Concerning the species Amoeba proteus. Science, Vol. 44.

pp. 468-469.

1917. On the third layer of protoplasm in ameba. Anat. Record.

Vol. 11. p. 477.

1918. Functional inertia in the movement of ameba. Anat. Record.

Vol. 14. p. 93.

Stolc, A. 1900. Beobachtungen und Versuche iiber dié Verdauung und Bildung der

Kohlenhydrate bei einem amébenartigen Organismus. Zeit. f. wiss. Zool.

Vol. 68. pp. 25-668. 2 pls.

EXPLANATION OF PLATES

Prate VII

Fig. 2. Photograph from water color drawing of bzgemma in characteristic attitude

during locomotion. C, nuclear chromatin mass; C-S, crystals attached to spheres;

F, food mass; M, nuclear membrane; V vacuoles.

Fig. 3. Photograph from water color drawing of nucleus and excretion spheres

and crystals of bigemma. CR, chromatin granules of nucleus; C, twin crystals at-

attached to excretion spheres; M, nuclear membrane; S, spheres; Occasionally two

twin crystals are attached to each other as shown.

Fig. 4. Photomicrograph from unretouched negative of several live bigemmas

among diatoms and arcellas. The nucleus, N, is faintly shown in two of them. The

arcellas measured 58 microns in diameter.

Fig. 5. Photomicrograph from unretouched negative of several live bigemmas

in characteristic attitudes during locomotion. Note especially the blunt character

of the ends of the pseudopods. Same magnification as figure 4.

Pirate VIII

Fig. 6. Photograph from water color sketch of a P. lentissima in locomotion.

N, nucleus; CV, contractile vacuole; U, uroid.

Fig. 7. Water color sketch of a quiescent stage of lentissima. Note the numerous

and bizarre shaped pseudopods.

Fig. 8. Water color sketch of P. schiedti in locomotion. CV, contractile, vacuole;

N, nuclei; U, uroid. Note the numerous irregularly shaped starch bodies.

Fig. 10. Camera lucida sketch of a moving P. schiedti showing the slight changes

in body shape during locomotion.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

SOCIETY VOL. XXXVII

LENKA)

SI

4

2

)

Fic. 4 Fie. 5

PLATE VII SCHAEFFER

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

SOCIETY VOL. XXXVII

Brea Fic. 8

PLATE VIII SCHAEFFER

SPERMATOGENESIS OF THE DOG

BY

Jutian Y. MALONE

The problem of spermatogenesis has been extensively worked on in

insects and various other invertebrates. Recently several vertebrates

have been shown to possess the so-called X-chromosome which is asso-

ciated with sex-determination. Two hetero-chromosomes have been

described in most of the mammals, and they probably correspond to the

X- and Y-elements as described by Wilson (712) in the Hemiptera. Dif-

ferent members of the latter class show a variation of these elements

from forms which have only the X-element, thru those which have the

X- and Y-element of unequal size, to those in which these elements are

of equal size.

In the dog I find only one such element, the history of which I have

tried to follow in this paper. The present study has been carried on at

the suggestion of Professor M. F. Guyer, to whom I am especially in-

debted for kindly help and criticism. I wish also to acknowledge my

obligation to Dr. Elizabeth A. Smith for valuable criticism.

MATERIALS AND METHODS

Thru the courtesy of the Department of Physiology of the University

of Wisconsin, I was enabled to get the material in the living condition.

Eleven of the animals from which the tissues were taken were mongrels;

the twelfth was a thorobred bulldog. No especial differences were

noted in the material except occasional minor discrepancies in the size

of the cells.

At first samples from different regions of the living testes were placed

directly into the fixing reagents in the preliminary preparations, but the

best results were obtained when the tissues were allowed to stand for

twenty minutes before being immersed in the fixative. As experienced

by all other workers on mammals the great problem is to get the chro-

mosomes to stand out as individual elements, the tendency being for

them to mass-up before the fixative takes effect. For this reason the

following fixatives: Gilson’s, Carnoy’s, Flemming’s strong, Bouin’s,

Tower’s, Herrmann’s, Haner’s modification of Flemming’s strong, and

Allen’s modification of Bouin’s, were tried out several times in an effort

98 J. ¥Y. MALONE

to overcome this difficulty. It was found that the latter reagent gave

the best results. As urea in this solution presumably increases the

rate of penetration of the fixative, the amount of urea was varied from

one to five percent in order to determine the optimum concentration

for this tissue. A four percent solution was found to give the most

satisfactory results. The formula for this fixative is as follows: to

100 cc. of Bouin’s fluid add slowly 1.5 gm. of chromic acid and 4 gm. of

urea just before using. Heat to 37° C., add the fresh material and leave

it in the fixative for one to two hours keeping the temperature up to

37° C. For good cytoplasmic fixation put the fresh material in the

fixative at 60° C. allow to cool to 37° C. and keep at this temperature

as before. The fixative is then replaced gradually by 70 percent alcohol

until entirely removed. A convenient apparatus for this part of the

technic is described by Allen (16). The tissues are then dehydrated,

placed in xylol or chloroform, xylol-paraffin and imbedded in paraffin.

As the material fixed better after standing 20 minutes the experiment

was tried to see if allowing the tissues to autolyze from one to six hours

would improve the fixation. It was found that there was no perceptable

difference in the appearance of the nucleus altho the cytoplasm showed

evidence of being digested and the chromosomes showed a tendency to

clump up more after the first hour of autolysis.

Flemming’s strong and Haner’s modification of Fleming’s strong

gave good nuclear figures but the chromosomes did not stand out very

clearly. Carnoy’s fixative gave good spindle figures but always distorted

the cells.

The sections were cut 5 microns and 7 microns thick and were stained

with: (1) Haidenhain’s iron-haemotoxylin and counter-stained with

eosin, Bordeaux red, acid fuschin, or orange G; (2) Saffranin-lichtgrun;

(3) Saffranin-gentian violet; (4) Benda’s mitochondrial method. The

most satisfactory stain was the iron-haemotoxylin counter-stained with

acid fuschin, altho the saffranin preparations were valuable in studying

the resting stages, different phases of the growth period, and the shape

and number of the chromosomes.

Smear preparations were fixed in Allen’s modification of Bouin’s

fluid and stained with Haidenhain’s iron-haemotoxylin and Benda’s

mitochondrial stain. These preparations gave as good results in all

stages as was found in the sections and were of advantage in so far as

the cells were isolated, thus removing the danger of any confusion due

the surrounding cells or to part of the cell being cut away.

SPERMATOGENESIS OF THE DOG 99

ARRANGEMENT OF THE GERM CELLS IN THE TESTIS

The main bulk of the testis is made up of the coiled seminiferous

tubules which contain the germ cells. The seminiferous tubules are held

together by connective tissue which contains a small number of inter-

stitual cells, blood and lymph vessels. Practically all stages of develop-

ment of the germ cells may be found in a single tubule. In general the

spermatogonia and the Sertoli cells are at the periphery, then comes the

primary spermatocytes, the secondary spermatocytes, the spermatids

and the spermatozoa. ‘There is no definite seriation of the stages in any

one tubule, such as is seen for example, in the insect testes. Some

tubules are filled practically with cells in the growth stages, others with

spermatids and some with nothing but a few Sertoli cells and fibers left

by the discharged spermatozoa.

It is thus apparant that the great problem in Mammalian spermato-

genesis is to determine the seriation of the stages of development. The

criterion employed was, mainly, resemblance to the stages in forms which

had already been worked out. The size of the different cells and the

number of chromosomes were the chief guides.

SPERMATOGENESIS

S permatogonia

The spermatogonia usually lie at the periphery of the tubules, but

occasionally when rapid multiplication is taking place, they are found

in the deeper portions. The spermatogonia are hard to distinguish

from the Sertoli cells but usually they can be identified by the small

amount of their cytoplasm. The nuclei of the Sertoli cells present the

same general appearance as those of the resting spermatogonia and as

far as I can determine are the same. According to some investigators

they both have a common origin from the primordial germ cells (Heg-

ner 714),

There are present in the resting spermatogonial nuclei from one to

four large, deeply staining nucleoli which always fuse when activity

commences. This process of fusion is seen best in the saffranin prepara-

tions, which show that these bodies approach each other but before com-

ing in contact seem to be connected by thin threads of chromatin on each

side. They then completely fuse forming an oval nucleolus (figs. 1-3).

Even in the earliest periods when these bodies are well separated they

seem to be connected by thin fibers or strands of chromatin. A number

100 J. Y. MALONE

of interlacing linin threads along which small granules of chromatin are

deposited radiate from the nucleolus (fig. 4). As activity commences

in the nucleus there is a marked increase of chromatin granules about the

nucleolus as though they were threading out from it (fig. 5). At the

same time the amount of chromatin along the linin fibers increases until

the linin is practically obscured from view. Undoubtedly some of this

chromatin along the threads is from the nucleolus but the increase is

so great that some must either be synthesized or is present in the resting

nucleus in such a chemical structure that it does not react to the stains

used The cytoplasm which is made up of short interlacing fibers con-

tains in all stages a spermatosphere. This stains more intensely with

cytoplasmic stains.

As development continues the separate threads of chromatin shorten

and become thicker. These finally condense into the chromosomes of

the spermatogonial metaphase, as shown in figs. 7 and 8. It appears

as though a single thread does not become a single chromosome but

that parts of threads condense about different “centers”? which are

connected to each other by the linin fibers. The nucleolus does not

lose its identity thruout these stages but remains a large, dark body with

threads radiating from it. It does, however, approach the chromosomes

in size in the early prophase (fig. 7), but it soon becomes more darkly

stained than they (fig. 8). Cells showing the nucleolus as in fig. 7 are very

few hence it indicates that this stage is of very short duration. This so-

called nucleolus is probably the X-chromosome as it can be traced as such

throuout all the later stages by its staining reaction. The newly formed

chromosomes now take up their usual position on the spindle as twenty-

one oval-shaped bodies of which one is the X-chromosome. Information

as to the origin of spindle fibers and of the centrosomes was not obtainable

but they were very. distinct in all cases. During the formation of the

spindle, the nuclear wall disintegrates. The spermatogonial metaphase

is probably of short duration as stages where none of the chromosome

had started to divide, as in fig. 9, were very few. Counts were made

from polar views (Fig. 10), and oblique side views of the metaphase

spindles. In material obtained from a female foetus an attempt was

made to determine the somatic count in tissues taken from the pancreas,

liver, mesonephros, metanephros and kidney. A few clear spindles were

secured from liver material in which 22 chromosomes appeared (fig. 69).

In cases where the autosomes had just started to divide (figs. 11, 13,

14), it was obvious that the X-chromosome was dividing ahead of them.

SPERMATOGENESIS OF THE DOG 101

In the middle anaphase (figs. 15 and 16) the X-chromosome can be seen

still slightly ahead of the autosomes, which are now clumping together.

The early telophase (fig. 17) shows the chromosomes clumped with a

spermatosphere near each mass. The cell wall (fig. 18) constricts in

the middle of the long axis and finally divides the cell into two complete

daughter cells. The spindle fibers are still present but soon disappear

leaving the cells with a mass of chromatin, and the spermatosphere

imbedded in the cytoplasm. Thus the cells are ready for the growth

period as primary spermatocytes.

GROWTH PERIOD

Stage A-preleptotene (figs. 19 and 20). In this stage the cells from

the spermatogonial telophase have the chromosomes clumped together

in an irregular granular mass. The nuclear membrane has not yet

formed. This stage possibly represents that described by Wilson (’12)

in Oncopeltus and Lygaeus as an uncoiling of the individual chromosomes

to form separate leptotene threads, but as the chromosomes are so massed

the actual processes cannot be determined. A large black body which

is present in this mass of chromatin retains its identity throughout

the growth period and from its subsequent behavior will be called the

X-chromosome. Fig. 20 shows the leptotene threads emerging from

the chromatin mass.

Stage B-leptotene (fig. 18). The nuclear wall is now present and is

seen to enclose several thin, beaded threads of chromatin and the X-

chromosome. These leptotene threads do not form a continuous spi-

reme but appear as independent threads in both the smear and section

preparations. While the threads show no definite polarization such as

Wenrick (716) found in the Phrynotettix magnus, they form a network

which makes them very hard to count. They have not been seen to

exceed twenty in number which further indicates that they take their

origin in the spermatogonial telophase chromosomes.

Stage C-synapsis and synizesis (fig. 22). The leptotene threads of

Stage B drift toward one pole of the nucleus where they condense into

a mass. The parts of the nucleus not occupied by these threads is

clear. This stage has been studied in several forms of mammals by

von Hoff (12) who concludes that synizesis is the result of the action

of the fixative but as pointed out by Wilson (’12), Fasten (’14) and others

it occurs in the living material and thus cannot be the result of the action

102 J. Y. MALONE

of the fixative. In cases of poor fixation, however, the leptotene threads

are so contracted that their individuality cannot be made out, while

with proper fixation it is very clear that the threads do not lose their

identity during this contraction. They appear to arrange themselves

in pairs for they emerge from the mass in parallel strands.

Stage D-pachytene (fig. 23). The leptotene threads which paired

up in the previous stage now fuse side by side; that is, undergo parasynap-

sis. This is conclusively shown in this figure as in many others where

two leptotene threads can be seen fused at one end and separated at the

other. The line of fusion of these threads is not obliterated until a

much later stage. Thus the dog is another form which shows parasynap-

sis such as described by von Winniwarter (’09) Gregoire (’04), Schreiners

(04), Wilson (’12) Smith (’16), and others.

Stage E-diplotene (fig. 24). This stage is hard to distinguish from

stage D except that all the leptotene threads have fused to form the

thicker diplotene threads. In these the line of fusion of the leptotene

threads cannot be seen except in well destained preparations. The

ends of the threads appear thicker than the rest of the thread. This

stage might be called the beaded stage for each thread has the appearance

of a string of beads. They approximate the haploid number. It will

be noted that thruout these stages the X-chromosome does not lose

its identity and that the spermatosphere is present.

These diplotene threads now contract gradually into somewhat oval-

shaped chromosomes. As this contraction progresses, linin threads

connecting them appear. In the late prophase the nuclear wall breaks

down and the chromosomes take their places upon the primary sperma-

tocyte spindle. Since twenty-one chromosomes entered the primary

spermatocyte from the spermatogonial division, the leptotene threads

paired and the X-chromosome did not lose its identity, it is obvious that

the spermatocyte autosomes must be bivalent; that is, each one is made

up of two univalent chromosomes, and the X-chromosome is univalent.

This material contained no indications that the leptotene threads twisted

about each other to form chiasmas such as observed by Janssens (’09) and

Smith (16). Heterotypic tetrad figures in the late prophase stages

are not apparent although preparations stained favorably with iron-

haemotoxylin reveal a quadrapartite appearance of the bivalent chro-

mosomes as tho they were preparing for the following maturation divi-

sions.

SPERMATOGENESIS OF THE DOG 103

The actual growth period might be considered to be from stage

C to Eas there is very little change in volume up to the time of synizesis

but from there on the increase is very marked. The diplotene stage

probably lasts longer than any other as cells in this stage of development

are found in large numbers in the majority of the tubules.

REDUCTION DIVISION

When the primary spermatocytes are ready for division they reveal

ten large bivalent chromosomes, and one large X-chromosome. As the

X-chromosome lies, in the metaphase, in very close proximity to the

autosomes it is often difficult to determine its shape. But it can be

distinguished from the others as a longer, slightly curved body, (figs.

28, 29, 32, 35, 38, 39) similar to that noted by Guger (’12) and (’16).

However, a curved body from the concave or convex side would appear

as an oval body (fig. 30 and 34). Fig. 31, a polar view, presents clearly

the reduced number of chromosomes. All the chromosomes of the equa-

torial plate were not usually in the same plane.

As division starts in the autosomes the X-element can be seen to pass

slightly ahead of them to one pole. The dividing chromosomes are long

and thin giving the appearance of overlapping each other (fig. 36 and

37). This division is probably the reduction division as the autosomes

divide longitudinally and the X-chromosome passes unchanged to one

pole. In fig. 41 there could be counted at one pole, ten ordinary chromo-

somes plus the accessory while only ten autosomes passed to the other

pole. It will also be noted that the spermatosphere is still present.

INTERKINESIS

After the primary spermatocyte division no resting stage occurs.

The secondary spermatocyte metaphase is formed by the rearrangement

of the chromosomes present in the primary spermatocyte telophase.

In the late anaphase of the primary spermatocyte the centrioles with

short spindle fibers between them and the chromosome masses (fig. 44)

are apparent, indicating that the chromosomes do not reach the poles

as in the other divisions. The division of the centrioles and the forma-

tion of the new astral system cannot be followed but from the appear-

ances of such cells as figs. 46 and 47 one might infer that there is not a

new astral system formed but that the remnants of the previous spindle

are re-organized to form the secondary spermatocyte spindle. The

104 J. Y. MALONE

chromatin mass is imbedded in a more or less clear space surrounded

by cytoplasm.

As two types of cells enter this stage two kinds must result from their

division, one with ten univalent autosomes, (fig. 49), and one with ten

univalent autosomes plus the X-chromosome, (fig. 48). When the auto-

somes divide they pull apart in the center, the X-chromosome dividing

slightly ahead of them and passing to each pole, fig. 52. In the telophase

of this division the chromosomes are usually massed up but in such a

figure as 54 approximately ten chromosomes plus the accessory can be

distinguished passing to each pole whereas in others no trace of an

accessory can be found, fig. 55. Thus two kinds of spermatids which

will develop into mature sperm result from this division.

SPERMIOGENESIS

The chromosomes of the second maturation division break up into

an open reticulum composed of linin threads and chromatin granules.

The nucleus thus goes into a resting condition which apparently lasts

for some time. In approximately half of the nuclei there can be seen a

definite round body, fig. 60, which possibly corresponds with the X-

chromosome It can be seen in the nucleus after condensation of the

chromatin has occurred and the nucleus has migrated to one side of the

cell, fig. 62 and 63.

The spermatosphere, which takes the cytoplasmic stains, is present

in all of the spermatids in the cytoplasm and either imbedded in it or

closely associated with it can be seen the centrosome. In the same

region of the cytoplasm, fig. 57, is found the idiozome or remnant of the

previous spindle.

The centrosome and the idiozome are differentiated from one another

by the fact that the centrosome remains in close apposition to the sperma-

tosphere. It is a single, regular body while the idiozome is usually

lobular and irregular. As the idiozome comes in contact with the nuclear

wall an oval, clear space as described by Leplat (’10) in the cat appears

between it and the nuclear wall (fig. 58). It is apparently caused by

some repulsive force between the nuclear membrane and the idiozome

for the nuclear wall is definitely depressed. The wall of the cavity

opposite the nuclear wall is probably formed by material from the

idiozome. The nuclear wall then gradually returns to its original posi-

tion, fig. 60, the nucleus becoming longer than it is wide and the idiozome

SPERMATOGENESIS OF THE DOG 105

forming a cap over about two-thirds of its length. Later this cap be-

comes the acrosome of the mature sperm fitting closely over the anterior

end of the head. There seems to be little change in the idiozome threads

during this transformation but the dark mass at the tip disappears. In

sections threads running between the Sertoli cells and the acrosome

were usually noted but no indication of their origin could be found unless

they come from the acrosome. Fig. 68 shows a tubule from which all

of the sperm have been discharged and has nothing in it but a few Sertoli

cells with these threads running to them.

While this development of the acrosome has been going on the sper-

matosphere and the centrosome have migrated to the opposite side of

the nucleus. The spermatosphere becomes closely applied to the

nuclear wall and the centrosome divides into an anterior and a pos-

terior portion, fig. 61. At this stage the nucleus gets smaller and the

chromatin material appears as an indefinite granular mass in which a

dark body, possibly the remnant of the X-chromosome, is seen in approxi-

mately half of the cells, fig. 62. The nucleus migrates to the side of the

cell toward the acrosome apparently carrying with it a definite amount

of cytoplasm enclosed in a denser wall, fig. 62. The migration of the

nucleus is usually toward the tubule wall. At the base of this cytoplas-

mic neck which is destined to become the sheath of the middle piece of

the mature spermatozoa, is found the spermatosphere. It retains this

position until the nucleus, now the sperm head, has broken thru the

cell wall. In the “giant cells” (fig. 74) in which a number of spermatid

nuclei are present in one cell, there is a spermatosphere associated with

each nucleus.

The process of formation of the acrosome and middle piece is similar

to that found by McGregor (’99) in Amphiuma, except that he finds part

of the centrosphere or idiozome also going to form the middle piece.

After division of the centrosome the anterior one comes in contact

with the nuclear wall causing a temporary depression in the latter and

then flattens out into a disc between the walls of the cytoplasmic neck.

This disc, which forms the end knob of the sperm, has extending back

from it a thin filament which extends to the spermatosphere fig. 63.

Attached to this filament by a fine stalk is the posterior centrosome.

This condition differs from that described by Leplat (’10) in the cat and

Wodsedalek (’13) in the pig, in that the posterior centrosome in these

animals forms a ring which migrates along the axial filament and is

cast off with the cytoplasm.

106 J. Y. MALONE

As the sperm head breaks thru the cell wall the axial filament becomes

much longer. The flattened head is composed of three regions of dif-

ferent staining reaction, fig.64. The point of attachment of the acrosome

corresponds with about the posterior margin of the anterior two-thirds

of the head and the point of attachment of the cytoplasmic neck cor-

responds with the anterior margin of the posterior third of the head’

Thus it appears that the difference in the density is due to the presence

of these membranes and that the lighter middle portion is due to the

absence of these membranes. Further evidence of this is seen in fig.

65 in which the acrosome is becoming applied to the nuclear wall. Here

there are only two regions of different color. This observation may be

carried to the mature sperm, fig. 67, which shows in a side view that

there is a layer of dark staining material covering the posterior third

of the head, which also appears darker than the rest of the head when

viewed from above, fig. 66.

When the cells reach the stage shown in fig. 65 they become attached

in groups to the Sertoli or nurse cells by long filaments. At this time

the cytoplasm is cast off and the sperm continue to develop. The sur-

rounding cytoplasm is found to disappear gradually and as the sperm do

not leave the tubule until this is about complete, it is possible that they

derive some of their nourishment from this bed of cytoplasm. In the

Benda preparation there was found large and small globules of fatty

substance in this cytoplasm, in the Sertoli cells and a few in the inter-

stitual cells. These globules stain black with iron-haemotoxylin but

do not stand out as well as in the Benda preparations.

In the final changes of the sperm the acrosome becomes closely

applied to the sperm head and is no longer distinguishable. The cyto-

plasm of the middle piece contracts and slightly elongates to cover al-

most the anterior half of the sperm tail or axial filament. During this

contraction the posterior centrosome breaks thru the cytoplasmic wall

and is seen lying outside the middle piece (fig. 66). There is no evidence

of the sperm head enlarging during this process as described by Wad-

sedalek (713).

Thus the mature sperm is seen to consist of: a head formed from the

entire nucleus of the spermatid, the cytoplasm of the middle piece from

the spermatosphere and the axial filament together with the anterior

and posterior centrosomes from the centrosomes. The spermatozoa

now lose their attachment to the Sertoli cells and pass into the lumen of

SPERMATOGENESIS OF THE DOG 107

the tubule leaving behind the Sertoli cells with large bundles of threads

attached to them as shown in fig. 68.

Further evidence of the dimorphism of the sperm was obtained by

camera lucida measurements. Three hundred measurements were made

at 2,000 magnification of mature sperm, obtained from the vas deferens.

It was found that these sperm, selected at random from the preparation,

could be grouped into two classes on the basis of size. The heads of

one hundred and sixty-one at this magnification measured five to six

millimeters when projected onto paper with a camera lucida while the

balance of the three hundred measured seven to eight millimeters.

This difference in size is probably due to the presence of the X-chromo-

some.

SUMMARY

1. The five typical cells ordinarily found in spermatogenesis:

spermatogonia, primary spermatocytes, secondary spermatocytes, sper-

matids, and sperm occur in the dog in an unseriated arrangement.

These are enclosed in long, thin, winding tubules which are held together

by connective tissue and interstitutal cells.

2. Large numbers of spermatogonia and Sertoli cells are present

around the periphery of the tubules and may contain one or more large,

deeply staining bodies or nucleoli. In case of the spermatogonia these

nucleoli always fuse before activity is marked. This body is possibly

associated with or is the X-chromosome.

3. The spermatogonia show twenty-one oval shaped chromosomes,

the X-chromosome usually not being distinguishable until the early

anaphase where it divides and passes to the poles slightly ahead of the

autosomes.

4. Following the spermatogonial division the chromosomes weave

out into separate leptotene threads, while the X-chromosome remains

as a rounded or slightly oval dark-staining mass.

5. The leptotene threads undergo parasynapsis.

6. Eleven chromosomes appear in the primary spermatocyte, ten

are bivalent autosomes and one the X-chromosome. The X-chromosome

passes undivided to one pole while the autosomes divide by longitudinal

splitting. Thus there are produced two kinds of secondary sperma-

tocytes. This division is réductional.

7. There is no resting stage between the primary and secondary

spermatocyte divisions, the chromosomes retaining their identity al-

though they increase in size slightly.

108 J. Y. MALONE

8. The two kinds of secondary spermatocytes upon division give

rise to two kinds of spermatids, one with ten univalent autosomes and

the other with ten univalent autosomes plus the X-chromosome. This

dimorphism is further evidenced by the resting spermatids as approxi-

mately half of them show a large, deep staining body which is probably

the X-chromosome.

9. The chromatin of the spermatid nucleus condenses into an indif-

ferent mass, the nucleus contracts, becomes narrower and flattened. It

passes to one pole of the cell, breaks thru the cell wall and leaves most of

the cytoplasm of the cell behind. It then attaches itself to a Sertoli

cell by a thin fiber and shapes up into a mature sperm.

10. During spermiogenesis the centrosome gives rise to the end knob,

axial filament and the posterior centrosome; the sphere substance of

the secondary spermatocyte division to the acrosome; and the sperma-

tosphere to the sheath of the middle piece.

11. Measurements of mature sperm give a distinct bimodal curve,

also indicating their dimorphism.

Zoological Laboratory, University of Wisconsin.

LITERATURE CITED

ALLEN, EZRA

1916. Studies in Cell Division in the Albino Rat (Mus Norvegicus). Anat.

Rec., Vol. 10, No. 9.

FASTEN, NATHEN

1914. Spermatogenesis of the American Crayfish, Cambarus virilis and Cam-

barus dumnnics (2) with Special Reference to Synapsis and the Chroma-

toid Body. Jour. of Morph., Vol. 25, No. 4.

GREGOIRE, V.

1905. Les resultats acquis sur les cineses de maturation dans les deux regnes. La

Cellule, T. 22.

Guyer, M. F.

1912. Modifications in the Testes of Hybrids from the Guinea and the Common

Fowl. Jour. of Morph., Vol. 23, No. 1.

1916. Studies in the Chromosomes of the Common Fowl as Seen in the Testes

and in the Embryos. Biol. Bull., Vol. 31, No. 4.

Heecner, R. W.

1914. The Germ Cell

JANSSENS, F. A.

1909. La theorie de la chiasma typie. Nouvelle interpretation des cinese de la

maturation. La Cellule, T. 25.

LEPLAT, GEORGES

1910. La spermatogeneses chez la chat. Archiv. de Biol., T. 25.

SPERMATOGENESIS OF THE DOG 109

McGrecor, H.

1899. The Spermatogenesis of Amphiuma. Jour. of Morph., XV suppl.

ScHREINERS, A. and K. E.

1906. Neue Studien uber die Chromatinreifung der Geschlechtszellen. Archiv.

devbrolen ha 22-

SmiTH, E. A

1916. Spermatogenesis of the Dragon Fly Sympetrum Semicinctum (Say) with

Remarks upon Libellula basilis. Biol. Bull., Vol. 31, No. 4.

Van Hoor, LUCIEN

1912. Synapsis dans les Spermatocytes des Mammifers. La Cellule, T. 27.

von WryniwaTer, H. and Sarymont, G.

1909. Nouvelles recherches sur l’ovogeneses et l’organogeneses de l’ovarie des

Mammifers (chat). Archiv. of Biol., T. 24

WoODSEDALEK, J. E.

1913. Spermatogenesis of the Pig with Special Reference to the Accessory

Chromosomes. Biol. Bull., Vol. 25, No. 1.

WEnrRIcH, D. H.

1916. The Spermatogenesis of Phrynoteitix magnus with Special Reference to

Synapsis and the Individuality of the Chromosomes. Bull. of the

Museum of Comparative Zoology of Harvard College, Vol. 60.

WItson, E. B.

1912. Studies in Chromosomes. Jour. of Exper. Zoology, Vol. 13, No. 3.

EXPLANATION OF PLATES

All drawings were made at 1600 magnification with a camera lucida. The plates

were reduced about one-fourth.

PLATE IX

Figs. 1, 2 and 3. Resting spermatogonial nuclei.

Fig. 4. Resting spermatogonia.

Figs. 5 and 6. Spermatogonia in early stage of activity showing the increase in

chromatin along the linin fibers.

Figs. 7 and 8. Spermatogonia showing the condensation of the chromatin to

form the chromosomes of the metaphase. Fig. 8 is from a smear preparation.

Fig. 9. Side view of a spermatogonial metaphase. j

Fig. 10. Polar view of a spermatogonial metaphase showing 21 chromosomes.

Figs. 11, 12, 13 and 14. Early spermatogonial anaphase showing the chromo-

somes starting to divide.

Figs. 15 and 16. Spermatogonial anaphase showing the X-chromosome dividing

ahead of the autosomes.

Fig. 17. Early telophase of the spermatogonial division.

Fig. 18. Late telophase of the spermatogonial division.

Figs. 19 and 20. Preleptotene stage or beginning growth period. The dark

staining compact mass is the body which later is seen to be the X-chromosome.

Fig. 21. Leptotene stage showing the X-chromosome.

Fig. 22. Synapsis and synizesis showing the drifting of the leptotene threads

~ and their contraction to one side of the nucleus.

110 J. Y. MALONE

Fig. 23. Pachytene stage showing the chromsomes pairing by parasynapsis.

Figs. 24 and 25. Diplotene stage showing the beaded appearance and the unfused

threads.

Figs. 26 and 27. Late primary spermatocyte prophase showing the condensation

of the diplotene threads to form the chromosomes.

Figs. 28, 29, 30, 32, 33 and 35. Side views of primary spermatocyte metaphase,

figs. 28, 29, 32 and 35 showing the curved X-chromosome.

Fig. 31. Polar view of a primary spermatocyte metaphase showing ten autosomes

and the X-chromosome.

Figs. 34, 36, and 37. Early primary spermatocyte anaphase showing longitudinal

division of the autosomes.

Figs. 38 and 39. Parts of primary spermatocyte anaphases showing the curved

X-chromosome going to one pole.

Figs. 40 and 41. Early primary spermatocyte telophase.

Figs. 42, 43 and 44. Late primary spermatocyte telophase.

Figs. 45, 46 and 47. Secondary spermatocyte prophase.

Figs. 48 and 50. Secondary spermatocyte metaphase showing ten autosomes

plus the X-chromosome. Fig. 50 from a smear preparation.

Fig. 49. Secondary spermatocyte metaphase showing ten autosomes.

Fig. 51. Early secondary spermatocyte anaphase showing equational division.

From a smear preparation.

PLATE X

Figs. 52 and 53. Early secondary spermatocyte anaphase showing equational

division.

fe ~. Fig. 54. Secondary spermatocyte telophase showing approximately ten auto-

somes plus the X-chromosome at each pole.

Fig. 55. Same as fig. 54 except that the X-chromosome is not present.

Figs. 56 and 57. Spermatid showing the spermatosphere with the centrosome

imbedded in it and the remnants of the secondary spermatocyte spindle (idiozome).

“ 5 Figs. 58, 59, 60 and 61. Spermatids showing the formation of the acrosome from

the idiozome, the migration of the spermatosphere plus the centrosome to the other

side of the nucleus, and the division of the centrosome.

. » Figs. 62,63 and 64. Spermatids showing the migration of the nucleus to one side

of the cell, the formation of the middle piece, end knob, posterior centrosome and the

axial filament.

Fig 65. Immature sperm which has cast off its cytoplasm except that destined

to’ become the sheath of the middle piece and the fibre which seems to connect it with

the Sertoli cell.

Fig. 66. Mature sperm viewed from the broad side.

Fig. 67. Mature sperm viewed from the side.

Fig. 68. Cross section of a tubule the sperm cells of which have all matured

leaving only a few Sertoli cells and fibres.

Fig. 69. Polar view of a somatic cell of a female foetus showing 22 chromosomes.

Figs. 70 and 71. Anomalies showing heterogenic division.

Figs. 72 and 74. Giant cells showing more than one nucleus and their associated

spermatospheres.

Fig. 73. Two spermatids side by side.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

SOCIETY VOL. XXXVII

PLATE IX MALONE

AMERICAN MICROSCOPICAL

TRANSACTIONS OF THE

AX XVII

SOCIETY VOLE:

MALONE

THIGMOTACTIC REACTIONS OF THE FRESH WATER

TURBELLARIAN, PHAGOCATA GRACILIS, LEIDY

BY

BERNOL R. WEIMER

The Turbellaria have ever been popular subjects for experimen-

tation, yet in looking over the literature, I find that very little has

been done upon the thigmotactic reactions of these animals. Thinking

that here was a problem that might yield some interesting results, I col-

lected a number of the animals in Falling Run, Morgantown, West Vir-

ginia, and these were used in the following experiments. Five specimens

were sent to Dr. H. S. Pratt of Haverford College, and were identified

by him as Phagocata gracilis.

Six rocks of varying degrees of roughness were collected and cemented

together with Portland cement in a circular glass dish 7 cm. high. The

rocks were so arranged as to form a surface as nearly flat as possible.

The outer walls of the dish were covered with heavy black paper in order

that light could fall only vertically upon the surface of the rocks.

After allowing water, which was changed frequently, to stand in the

dish for three weeks, some animals were placed in the dish. All the

animals were in good condition, but those animals which came in contact

with the cement between the rocks, secreted a slimy substance and died.

The cement was then coated with paraffin, and at the same time a piece

of glass of nearly the same area as the different rock surfaces, was waxed

in.

After carefully washing the paraffin for two days in running water,

some animals were again placed in the dish and after two days were alive

and in good condition. Forty animals were then put into the dish which

was placed beneath the skylight in a room so darkened that all the light

came from this source. The surface of the rocks in the experiments

was covered by water to an average depth of 1.5cm. The temperature

of the water varied from 21° -25° C.

In collecting the rocks used in the experiments, care was taken that

the surface should have different degrees of roughness and that each

rough surface should be as nearly uniform as possible. The rocks and

the glass plate were each numbered with India ink. These numbers

are used in the tables given, to designate the rock ard, obviously, the

surface. Rocks numbered 1, 2, 3, and 4 were of weathered sandstone

and rock number 5 of weathered lime-stone.

112 B. R. WEIMER

The surface of rock number one was composed of rounded quartz

crystals of about the same coarseness as granulated sugar. Rock number

two had a surface somewhat smoother than number one, the particles

of sand were about as coarse as table salt. Rocks numbered four,

three and six approached, in the order mentioned, rock number five, a

smooth lime stone. Thus the rock surfaces varied in coarseness or

roughness of surface from particles the size of granulated sugar to a

comparatively smooth surface in the following order: one, two, four,

three, six, and five. For a surface a grade smoother than the rocks

described, there was the glass plate and the paraffin coating the cement.

Practically the only surface difference noticed between the glass and the

paraffin was, that the paraffin had a slightly greasy feeling.

The following explanation refers to tables 1 and 2. The total number

of animals counted on a given surface during a set of observations was

found and this number, divided by the number ofobservations in the set,

gave the average observation for the set. This average observation for

each set was added and the average of this total gave the average ob-

servation for all the sets. Since only one set of observations was taken

in tables 3 and 4, the second average of the sets, was not necessary. The

area of the paraffin and the vertical glass sides of the dish covered by

water were averaged as one surface (para-glass in tables). This para-

glass surface was used as the common unit area and from this the relative

number of animals resting on a common unit surface, was computed.

Since the paraffin and the vertical glass surface was so nearly alike, both

were averaged as one. With this brief explanation, the tables given

should not be difficult to understand.

The data set forth in table 1 was procured in the following way.

The observations were taken at either ten or fifteen minute intervals

and usually after each observation the animals were stimulated, i.e.

caused to move from the place they were resting. No distinction was

made between an animal moving across and one at rest on a surface.

This probably accounts for the high averages for the various surfaces

in table 1. The dish was frequently moved around to counteract any

light reaction, tho the light apparently made no difference since it came

vertically from above. A tendency to gather in groups was also noticed.

These groups were usually found on the paraffin. The animals in these

groups were always stimulated after an observation.

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 113

TABLE 1

“hog wnt | 8} 10 86) 74 tee

av. lav. jav. lav. lav. lav. jav. | av. of | No. animals | Total no. of

set1 |set2 |set3 |set4 |set5 |set6 |set7 all per unit animals

obs. surface counted

Rock

1 1.70] 1.80} 2.75} 0.70} 2.00) 4.30} 1.60} 2.13 25.53 145

2 2.40} 1.20] 1.50] 0.40} 2.55} 3.66] 0.46) 1.73 15.65 108

3 2.10} 3.40} 2.70) 1.10) 2.44) 2.50} 1.05} 2.19 18.79 249

4 0.90} 4.80} 8.25} 0.50} 2.20) 1.16) 1.90) 2.81 34.59 192

5) 2.30} 2.40} 1.66] 0.80) 1.60} 2.30) 1.90) 1.85 USA) 126

6 1.40} 1.20} 1.40] 1.30} 1.70) 3.60) 1.26} 1.66 17.66 113

Glass 7 | 1.70} 0.90} 1.00) 0.70} 1.30} 2.10) 1.20} 1.27 16.25 86

iPara-

glass |32.70|28.50/25.12|34.50/28.30)/30.30|31.86| 30.18 30.18 2053

Observations showing number of animals seen on each surface with vertical

illumination only. The time between sets varied from a day to a week. The sur-

faces in order from roughest to smoothest are as follows: one, two, four, three, six,

five and glass.

From table 1 it is seen that when all the surfaces have been reduced

to the same unit area, the three highest averages of animals resting,

are as follows, rock four, 34.59, paraffin-glass, 30.18 and rock one 25.53.

The other surfaces have much lower averages and do not vary much

among themselves. One peculiar fact to be noticed is that rock one has

the roughest surface, the glass-paraffin has the smoothest surface, and

intermediate between these two is the surface of rock four. It would

be well to remember that after each observation the animals were stimu-

lated. Since so few animals were found resting, they were averaged

with those moving.

The data in table 2 differs from that found in-table 1, in that all the

observations were taken in a photographic dark room. An electric

light was used only long enough to record the observation. The observa-

tions were taken at intervals varying from 30 minutes to an hour and the

animals moved from their resting position after each observation. As

a result of the longer interval elapsing, most of the animals were found

resting when the observation was recorded.

114 B. R. WEIMER

TABLE 2

No. of Total

obs. ; 6 6 4 20

av. av. av. av. av. of | No. of animals | Total no. of

set 1 | set 2 | set 3 | set 4 | all obs. per unit animals

surface counted

Rock 1 | 0.00 1.50 1.60 | 0.75 96 11.41 192

Dale O0n\eas:00 1.16 | 0.25 1.60 14.38 32.00

3 1009) 92.330 1 O80 2250 1.65 14.15 33.00

4 E50 et SON OlGN ee OO 1.04 12.80 21.00

Bl Os 1.66 | 6.10 1.00 2.22 18.20 45.00

GA ee SOR O50 Mrs OOU pants 2.18 23.06 44.00

Glass 7 1.50 | 2.30 5000225 1.38 17.66 28.00

Para-

glass | 32.75 | 25.60 | 27.60 | 30.75 29.17 29.17 584

Observations taken in photographic dark room showing number of animals

counted on each surface. The time between sets varied from a day to a week.

The surfaces in order from roughest to smoothest are as follows: one, two, four,

three, six, five and glass.

The averages of these observations in table 2 show, that when all

the surfaces have been reduced to the same unit area, the surfaces having

the most animals resting upon them are the paraffin-glass, 29.17, and

rock six, 23.66. Rock six has a comparatively smooth surface of weather-

ed lime-stone. The averages for the other surfaces are about the same.

After conducting the first set of experiments, the results of which are

found in tables 1 and 2, the glass was broken from around the cement

block containing the rocks. The cement necessarily exposed was covered

with a coating of bees wax. The whole block, surface downward, was

then placed upon three glass supports, 2.5 centimeters high, in an en-

ameled pan. The pan was then filled with water until it covered the

surface of the rocks. This apparatus was first put in a photographic

dark room, and observations taken each hour by lifting the rocks. The

results were so decidedly negative that only a few observations were

taken. See table 3. The animals not only did not rest upon the rock

surfaces but were found scattered in average numbers over the entire

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 115

bottom and sides of the pan. Only an average number was found on

the bottom of the pan underneath the rocks.

TABLE 3

No. animals Total no.

Surface Observations Av. per unit animals

surface counted

Rock 1 Av.

2 1 0.20 1.19 1

3 2 0.40 3.43 2

4

5

6

Glass

Para.

Pan 39 | 38 | 40 | 40 | 40 | 39.90 39.90 237

One set of five observations taken in photographic dark room with the rocks

inverted, showing the number of animals counted on each surface.

The surfaces in order from roughest to smoothest are as follows: one, two, four,

three, six, five and glass.

TABLE 4

No. animal | Total no.

Surface Observations Av. per unit | animals

surface counted

ee ees ee

Rock 1

2 1 0.09 | 0.80 1

3

4

5 1 0.09 | 0.73 1

6

Glass

Para. 1 1 0.18 0.18 2

Pan 40 | 40 | 40 | 40 | 39 | 40 | 39 | 40 | 38 | 39.5 | 39.50 435

One set of nine observations taken with vertical illumination only and with

rocks inverted, showing the number of animals counted on each surface.

The surfaces in order from roughest to smoothest are as follows: one, two, four,

three, six, five and glass.

116 B. R. WEIMER

The same apparatus was then placed underneath the skylight in a

room so darkened that all the light came from this source. The same

negative results were obtained as in the photographic dark room, though

the difference was noted in that the animals in this experiment, gathered

on the pan underneath the rocks, whereas in the dark room this preference

was not shown.

According to Pearl (’02), the ventral surface of Planaria maculata

is strongly positively thigmotactic; but this does not explain the ten-

dency of the animals to rest in angles. This resting in angles he calls

goniotaxis. The physiological condition of reduced tonus helps to

determine whether or not the animal will rest on a smooth surface.

The increased resistance to movement may be one cause for stop-

ping on the uneven surface tho the most important factor is probably

light. Nevertheless, instances are known where the animal may stop

in a bright light, tho such instances are rare.

From table 1 it is seen that rock four has an average of 34.59 animals

per unit area. The surface of this rock in degree of roughness, is mid-

way between the rough granular surface of rock number one (average

25.53) and paraffin-glass (average 30.18). Table 2 shows that the

paraffin-glass surface (average 29.17) is the highest per unit area, fol-

lowed by rock six (average 23.06) whose surface is of smooth weathered

limestone. In tables 1 and 2, it will be noticed that surface number

seven is of glass. This surface is horizontal, not vertical as is that

averaged with the paraffin. If the average of this surface (16.25 in

table 1 and 17.66 in table 2) were added to the surface average of the

paraffin-glass (average 30.18 in table 1 and 29.17 in table 2), the total

average of these surfaces would be 46.43 in table 1 and 46.83 in table 2.

This is much higher than any of the rough surfaces. From this the con-

clusion may be drawn that the animals prefer a smooth surface.

Pearl (’02), in describing the method of locomotion of Planaria macu-

lata says that the ventral surface of the body constantly secretes mucus

in greater and lesser quanities. This is very sticky and under normal

conditions adheres to the surface on which the animal reposes. Thus

between the animal and the surface on which it moves there will be a

constant layer of mucus. The beating of cilia in this mucus pushes the

animal forward. Considering this explanation, then, more mucus may

be secreted by an animal when passing over a rough surface than when

passing over one that is smooth. Likewise a regular granular surface

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 117

might not have quite so much effect as a very angular surface. Pearl

(02), also suggests that the reduced physiological tonus of the animal

might cause it to rest on the rough surface.

To find whether or not more mucus was extruded and secreted when

passing over a rough surface than over a smooth one, the following

experiment was tried. A piece of ordinary window glass was scratched

or grooved by a diamond glass cutter. The glass with the grooved

surface was then placed in a large petri dish and covered with water.

Some of the animals were then placed in the dish and allowed to move

over the grooved surface. After an hour the animals were removed

and the glass “developed” by immersing in Delafields’ hematoxylin,

which stained the slime blue. This gave better results than the method

used by Pearl (’02), who used a solution of carmine. This was tried

and the carmine particles by adhering to the mucus, showed the tracks

but the mucus was not stained and hence a close study of it could not

be made. The glass was then broken so that cross sections of the grooves

could be examined with the microscope. A number of sections were

thus examined but at no place could a greater slime secretion be noticed

where the slime tracks crossed a groove.

Thinking perhaps that the width and depth of the grooves in a sur-

face might cause some difference in the amount of slime secreted, the

bottom of a large petri dish was covered by a layer of paraffin to an

average depth of 5mm. In this paraffin were cut grooves varying from

0.20 to 3 mm. in width and the same in depth. The dish was then filled

with water and in it were placed some animals. The smaller grooves

did not seem to reduce the rate of locomotion of the animals. How-

ever, when an animal approached one of the wider grooves, it would

hesitate, raise up the anterior end of the body and move it to and fro,

then pass across without touching the bottom of the groove.

After the animals had been undisturbed for two hours, they were

removed and the water replaced by a solution of Delafields’ hematoxylin.

On examination of the stained slime tracks it was found that in few cases

was there a greater amount of slime secreted. The slime strands did

not descend to the bottom of the grooves but in most cases were con-

tinued on across as a sort of bridge. In some cases these strands were

broken but even then no abnormal amount of slime was found. At

the edges of the grooves, however, there were places where there seemed

to be a greater amount of slime. These were the places, probably, where

the animals hesitated.

118 B. R. WEIMER

From these results it would appear that there is the same amount of

slime on all surfaces and that no abnormal secretion is caused by a rough

surface. Thus a change in the physiological condition of the animal

as caused by the difference in mucus secretion, would not effect the

thigmotactic reaction to surfaces of varying roughness and smoothness.

Pearl (’02), suggests that perhaps the rough surface offers more

resistance to movement than does a smooth surface. This naturally

would cause a slower rate of locomotion when an animal was passing

over a rough than when passing over a smooth surface but since an equal

amount of slime is secreted on all surfaces, there should be approxi-

mately the same rate for all surfaces. Accordingly the rate of locomo-

tion was found for two surfaces, the one of rough sandstone, somewhat

coarser than rock one, and the other glass.

A piece of white paper was marked off in 1 cm. squares and placed

underneath a large petri dish filled with water. The surface of the

rock was marked off in 1 cm. squares with waterproof India ink and

around the edges were placed sides of pasteboard to keep the animals

from wandering off the surface. The rock was placed in a galvanized

pan which was filled with water to such a height that the surface of the

rock was covered to a depth of 2 cm. The two surfaces were placed

underneath the skylight in a room from which all other sources of light

were cut off.

The four animals used in these experiments were kept in separate

dishes carefully marked. When the animals were transferred from these

dishes to the surfaces, a camel’s hair brush was used in order not to

injure them. In the course of these experiments, animal number one

died, so that most of the tables show the results of the reactions of

numbers two, three and four, all of which were active, healthy, indivi-

duals. The animals were placed on the surface singly, and a number of

observations taken by means of a stop watch, as to the length of time

required to move over 1 cm. of surface. No allowance was made for an

approximate deviation of 2 mm. from a straight line in passing over 1 cm.

of surface.

The data recorded in table 5 was procured thus. A number of

observations of the rate of locomotion of each animal was taken on two

consecutive days for the glass surface and on the two following days for

the rock.

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 119

TABLE 5

Av. Av.

yen Rock ist day | Rock 2nd day | rate in || Glass Ist day |Glass 2nd day | _. Bae

a mm. per) }——______________|mm. per

no. of |av. rate |no. of jav. rate san no. of jav. rate |no. of |av. rate

obs. | in mm.| obs. |in mm. : obs. |in mm.| obs. |in mm.| ‘S°

1 15 0.92 15 1.23 1.07 10 0.74 0.74

2 15 0.80 15 1.10 0.95 16 1.43 18 12 1.31

3 15 1.05 15 i LAIY/ wala 17 1.47 18 1.46 1.46

4 15 0.97 15 1.24 1.10 18 1.38 18 1.47 1.42

Observations showing the rate of locomotion. Observations taken on one

surface only each day

Table 5 shows that the average rates per second of animals number

three and four are the highest, being 1.11 mm. and 1.1 mm. on the rock

and 1.46 mm. and 1.42 mm. on glass. This shows a difference in rate

of speed per second between the glass and the rock of 0.35 mm. and 0.32

mm. Animal number two shows a difference per second of .36mm. In

these three instances the animals moved more rapidly on the glass.

However, a further examination shows that an animal in twenty-four

hours time will vary in rate of locomotion on the rough surface .31 mm.

and on the smooth .21 mm. per second. Walter (’02), who tested the

rate of speed on glass only, found that the rate varied from 1.22 mm. to °

.96 per second. So the variation in rate between the two surfaces can

scarcely be attributed wholly if at all, to the difference between the two

surfaces but to some other factor. This is further proven by a study

of tables 6 and 7.

The data in tables 6 and 7 differs from that in table 5 somewhat.

A number of observations on the rate of locomotion of an animal was

taken first on one surface and immediately on the other. Several hours

later another set of observations was taken. This lessened the chance

for so great a change physiologically as to effect the speed. The results

are seen in tables 6 and 7.

Table 6 shows at 8:00 the maximum difference of speed of all the

animals, between the two surfaces to be .16 mm. per second. At 2:00

the maximum difference is .1 mm. per second.

120 B. R. WEIMER

TABLE 6

Rock Glass

Animal time no. of obs. av. rate in mm. no. of obs. ay. rate in mm."

per sec. per sec.

2 8:30 8 NEES 7 1.21

3 until 7 0.98 y 0.92

4 11:00 9 1.17 7 1.01

2 2:00 7 0.99 9 0.99

3 until 7 1.02 9 1.12

4 4:00 7 1.01 5 1.11

Observations showing rate of locomotion. All the observations were taken on

one day.

TABLE 7

Rock Glass

Animal time { :

Nae APS ee av. rate In mm. ovo oe av. rate In mm.

per sec. per sec.

2 9:00 5 1.06 7 1.36

3 until 7 1.01 8 0.98

4 11:00 ii 1.19 8 1.24

2 1:30 5 1.03 Gf Lae

3 until Z 1.01 7 1:17)

4 3:00 7 1.19 4 1.33

Observation showing rate of locomotion. All the observations were taken

on one day.

Table 7, a day later, shows the maximum difference in rate at nine

o’clock to be .3 mm. per second and at 1:30 o’clock to be .16 mm. per

second. Further, in some cases the rate of speed is greater on the rock

than on the glass.

The difference, then, in rate of locomotion between a smooth surface

and a rough surface is so small as to be almost negligible. This further

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS iVA\

shows that the amount of slime extruded and secreted is not increased

for a rough surface since this would probably reduce the speed of the

animal.

According to Pearl, (02), the method of locomotion in Planaria

maculata is by means of the cilia beating in the mucus strands. Think-

ing that perhaps a study of the cilia and hypodermis might throw some

light upon the thigmotaxis and locomotion of these animals, some work

was done upon the histological structure of Phagocata gracilis.

Planarian tissue is one of the most difficult of animal tissues to study

because it is so hard to fix properly and to stain. A fixative may give

good results in one case and not in another. Five different fixing fluids

were tried, hot Gilson’s, hot and cold corrosive-acetic, twenty-five per

cent solution of nitric acid, hot corrosive sublimate and a fixative recom-

mended by Woodworth, (91), made up oi a saturated solution of cor-

rosive sublimate in fifty percent nitric acid; of these, corrosive sublimate

gave the best results. Of the various stains, borax-carmine, picro-

carmine, neutral red, picric acid and carmine (aqueous), Delafields hema-

toxylin, Ehrlich’s hematoxylin and eosin gave the best results. The

sections were cut 6.6 microns thick. In all about thirty animals were

studied.

The study was made principally of the hypodermis. On the ventral

side, figure 2, it is made up of strongly ciliated columnar epithelial cells

These contain large subcircular nuclei, nu, with evenly distributed

chromatin granules. Definite cell walls between the cells could not be

distinguished. Between the cells are spindle shaped, homogeneous

bodies, the rhabditi, rb, which (Woodworth, ’91) are developed in sub-

cutaneous flask-shaped cells which are ectodermic in origin. These were

supposed to be homologous to the nematocysts of the Coelenterata but

later were supposed to be gland secretions. Underneath the hypodermis

is a homogeneous layer, the basement membrane, bm. Under this

is found the different muscle layers and the body parenchyma, figure 1.

The dorsal hypodermis, figure 3, is made up of columnar epithelial

cells somewhat longer than those on the ventral surface. No cilia were

found on these cells except near the edges of the animal. The rhabditi,

rb, were much larger and more numerous than on the ventral hypodermis.

They were found in groups of two or three. So thick were these that

the nuclei of the cells, in many cases, were pressed out of shape and often

concealed. The nuceli, nu, of the cells, when seen, appeared to have

122 B. R. WEIMER

the same structure as those in the ventral side. Beneath the cell layer

is found the basement membrane, bm. It is somewhat wider than on

the ventral surface but has the same appearance. Beneath this mem-

brane are the muscle layers and the parenchyma, mus. par. Imbedded

in the parenchyma are numerous rhabditi mother cells, rb. me, figure 1.

According to Woodworth, (’91) these rhabditi rapidly disintergrate

when extruded into the water to form a slime which, by entangling the

prey, aids the animal in procuring food. They may also be used for

protection. Whether or not these rhabditi play any part in the locomo-

tion of the animal, I was unable to determine.

In reviewing the literature on planaria there seems to be a difference

of opinion among investigators regarding the distribution of cilia on

the surface of the animal. Pearl (02) finds none on the dorsal surface

of Planaria maculata. Metschinikow (’66) and Kennel, (79), found

cilia covering the whole surface of Rhynchodesmus and Geodesmus

but Zacharias, (’88), states that the dorsal surface of a variety of

Goedesmus is bare. Vejdowsky, (’90), maintains the same for

Microplana, the cilia in the latter cases being confined to the ven-

tral surface or sole. Woodworth, (91), found the cilia on Phagocata

gracilis (collected near Cambridge, Mass.) to cover the entire surface

of the body. I was able, tho I used the same technique as Woodworth,

to find cilia only on the ventral surface and sides of the animal. How-

ever, these animals were collected in Falling Run, Morgantown, W. Va.

This difference in localities may account for the variation in ciliation.

Mosely, ’74, explains that this absence of cilia on the dorsal surface of

Bipalium may be due to the fact that the cilia on the dorsal surface of

land planarians are weaker thru comparative lack of function and are

more easily destroyed by reagents. However this is still an undecided

question.

SUMMARY

The results in tables 1 and 2 show that there is a preference shown for

a smooth surface rather than a rough one.

Tables 3 and 4, are interesting in that they substantiate the results

found by Olmstead, (’17), that unfed Planaria maculata are positively

geotropic, that is to say, have a tendency to keep the ventral side toward

the stimulus of gravity. Likewise these results show that Phagocata

gracilis is strongly negatively phototropic.

THIGMOTACTIC REACTIONS OF PHAGOCATA GRACILIS 123

Since no mucus was found filling the grooves and cracks in the glass,

it is clear that there must be no abnormal secretion of mucus and this

is further strengthened by the results found in tables 5, 6 and 7, that there

is an almost constant rate of locomotion on both a smooth and a rough

surface. The last three tables also show that there is a variation in

rate of locomotion which may be due to the different physiological states

of the animal.

A histological study of the hypodermis discloses the fact that no

cilia could be found on the dorsal side of the animal, which may be due

to a difference in the variety of the variety of the animals, since Wood-

worth, (’91), found cilia on the dorsal hypodermis of the same species,

collected in New England.

CONCLUSIONS

1. Phagocata gracilis is positively thigmotactic to a smooth surface.

2. This is not due to varying amounts of mucus secreted.

3. Phagocata gracilis is positively geotropic and strongly negatively

phototropic.

4. The rate of locomotion is the same for both a rough and am sooth

surface.

5. No cilia are found on the dorsal surface of Phagocata gracilis

collected at Morgantown, W. Va.

University of W. Va.

Morgantown, W. Va.

REFERENCES

OtmstEaD, J. M. D.

1917. Geotropism in Planaria maculata. Jour. Animal Behavior, Vol. VII,

pp. 81-86.

PEARL, R.

1902. The Movements and Reactions of Fresh Water Planarians. Quart.

Jour. Micr. Science, Vol. XLVI, pp. 508-714.

Wa ter, H. E.

1908. The Reactions of Planarians to Light. Jour. Exp. Zool., Vol. V, pp. 35-162.

WoopswortH, W. McM.

1891. Contributions to the Morphology of Turbellaria I. on the Structure of

Phagocata gracilis, Leidy. Harvard Mus. Comp. Zool., Vol. XVI, No.

1, pp. 142.

124 B. R. WEIMER

EXPLANATION OF PLATE

PLATE XI

Fig. 1. Transverse section of the animal anterior to the mouth, X 22.

Fig. 2. Portion of the ventral hypodermis, X 2000, approximately.

Fig. 3 Portion of dorsal hypodermis, < 2000, approximately.

LETTERING

bm. basement membrane

cil. cilia

c. mus. circular muscle

hyp. hypodermis

1. cil. limit of cilia

1. mus. longitudinal muscle

rb. mc.

mus. muscle

mus. par. muscle and parenchyma

nu. nucleus

par. parenchyma

ph. pharynx

rb. rhabite

rhabdite mother-cell

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

SOCIETY VOL. XXXVII

PLATE XI WEIMER

ADDITIONS TO OUR KNOWLEDGE OF UNIONICOLA

ACULEATA (KOENIKE)*

BY

ERNEST CARROLL FAUST

The material of Unionicola aculeata (Koenike) on which this paper

is based was secured from one of many specimens of Lampsilis luteola

Lamarck, from North Judson, Indiana. The host was determined by

Mr. Frank C. Baker, Custodian of the Natural History Museum of the

University of Illinois. The general organization of the mite conforms

to the type species described by Koenike (1890), but certain interesting

variations and certain structures yet inadequately described deserve

to be made a matter of record.

U. aculeata was originally described from Germany by Koenike, as

similar in many respects to U. crassipes (Miill.) and in others to U.

jiguralis (Koch). It differs from the former in the exact structure of the

palpus, in the number and distribution of the genital acetabula, and in

the structure of the chitinous ovipositor. Wolcott (1899) describes this

species from Michigan. The mite recorded by Soar (1899) as Ataw tav-

erneri has also proved to be U. aculeata. Piersig (1901) has made two

subspecies of U. aculeata, separating the American form, U. aculeata

sayi, from the European form, U. aculeata aculeata, on the basis of the

number of the tarsal claws, comparative lengths of the leg segments,

exact relations of the parts of the genital field, and a slight size difference.

The specimens collected by the writer conform to those described

by Wolcott in most respects. In size they belong to Piersig’s smaller

group, since the female measures about 680% long and the male

measures about 640 pu long.

Wolcott’s specimens have a terminal segment to the female palpus

which is somewhat attenuate distad, ending in four small claws. In the

writer’s material the palpus has a more distinctly thickened terminal

joint, with two finger-like claws and two thumb-like claws in the female

and with two finger-like claws and only one thumb-like claw in the male.

The penultimate joint of the palpus in Wolcott’s material has a needle

spine which is not present in the North Judson material. In the female

of this material the basal segment is in each case the thickest of the six.

*Contributions from the Zoological Laboratory ot the University of Illinois

under the direction of Henry B. Ward. No. 111.

126 E. C. FAUST

The joints are progressively narrower and longer from base to tip. In

the female there are three conspicuous spurs on the ventral side of the

basal joint, and two undivided sickle-shaped claws at the end of each

tarsus. Long needle spines are prominent on segments two to four.

Soar (1899) figures the claws of the first leg as cleft, a feature which

Wolcott (1899) describes for both U. aculeata and U. crassipes, while

those of the writer’s material are entire as in legs two to four.

Perhaps the most interesting feature of the entire body structure

of U. aculeata is the heteromorphic fourth leg of the male. On this

appendage both the joints and spines are curiously modified so that they

present a striking ornate appearance (fig. 5). The tarsus is a long atten-

uate flat:plate as in the female, with two undivided terminal claws and

two accessory spines. The main shaft of the tarsus is free from spines.

The tibia is short and thick with seven needle spines, two large heavy

spines and one small spine. The third segment has three large and three

small blunt spines, in two parallel lines at the outer edge of the joint.

The fourth and fifth joints are both shorter than joint three altho not

as short as the tibia. They are both supplied with several short bristles.

The basal joint is irregularly sculptured and bears four spines.

The genital field of the female resembles closely that described by

Wolcott, altho the cleft between the two plates is not distinguishable as

a separate structure and no posterior papillae are found in the writer’s

material. The external male genital organs (fig. 3) are very complicated,

consisting of an ornate sculpture of chitin, to which prominent muscle

bands are attached.

A thoro study of the new material and comparison with that described

by Koenike, Wolcott, and Soar, shows the wide range of variability

within the species, while at the same time it discredits Piersig’s separation

of the species into two subspecies. It seems much more desirable to

regard the species simply as highly variable rather than to maintain a

subdivision, since the lesser similarities and differences grade into each

other almost imperceptibly in various specimens.

Unionicola aculeata has been credited by some as a parasite of the

Unionidae and by others as free-swimming. Koenike (1915) has shown

that it is free-swimming during a considerable part of its life and seeks

the mussel at times of metamorphosis and propagation. This fixes our

knowledge of the periodicity of the parasitism, but leaves us in the dark

with regard to the degree of parasitism. The mites described in this

ADDITIONS TO KNOWLEDGE OF UNIONICOLA ACULEATA 127

paper were found embedded in the subdermal connective tissue of the

mantle and foot of the mussel (fig. 6). Their position was related to no

definite axis of the host. Around it was a thin tissue cyst. Inno case

were they found to have injured the host outside of the cyst. Thus,

altho an endoparasite, the evidence shows it to be only a temporary

lodger.

University of Illinois.

LITERATURE CITED

KoeENIKE, F. :

1890. Ein neuer Bivalven-Parasit. Zool. Anz., 13:138-140.

1915. Beitrag zur Kenntnis der Wassermilbe Unionicola aculeata (Koen.).

Arch. Hydrobiol. Planktonk., 10:308-319, 1 Taf.

Prersic, R.

1901. Hydrachnidae. Das Tierreich (Schulze). Lief. 13. 336 pp., 87 figs.

Soar, C. D.

1899. Atax Taverneri sp. nov.? Jour. Quekett Micr. Club, 7(2):219-221, 1 pl.

Wotcort, R. H.

1899. On the North American Species of the Genus Atax (Fabr.) Bruz. Trans.

Am. Micr. Soc., 20:193-259, 5 pl.

128 E. C. FAUST

EXPLANATION OF PLATE XII

Fig. 1. Dorsal view of female palpus, X 226; fig. 2, ventral view of first leg of

female, X 140; fig. 3, male genital organs, X 140; fig. 4, female, ventral view, X 100;

fig. 5, heteromorphic third leg of male, X 140; fig. 6, section thru outer portion of foot

of Lampsilis luteola, with encysted Unionicola aculeata, X 140.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

SOCIETY VOL. XXXVII

PLATE XII FAUST

DEPARTMENT OF NOTES AND REVIEWS

It is the purpose, in this department, to present from time to time brief original

notes, both of methods of work and of results, by members of the Society. All mem-

bers are invited to submit such items. In addition to these there will be given a few

brief abstracts of recent work of more general interest to students and teachers. There

will be no attempt to make these abstracts exhaustive. They will illustrate progress

without attempting to define it, and will thus give to the teacher current illustrations,

and to the isolated student suggestions of suitable fields of investigation —[Editor.]

METHODS FOR STUDYING LIVING TREMATODES

Studies on the living animal are of great importance in morphological

work on the Trematoda. Such studies have been entirely neglected by

most workers on the group. I believe that an increased use of living

material in the study of trematodes will make it possible to advance the

knowledge of the group more rapidly and to avoid many errors. Mater-

ial of living larvae and adult trematodes is easily obtained for class use,

and its use adds greatly to the students’ interest. Abundant material

of sporocysts, rediae and cercariae can usually be obtained by the exami-

nation of freshwater snails and material for the study of adults from the

intestines or lungs of frogs or snakes.

In miracidia or cercariae careful studies on locomotion reveal inter-

esting specific characters, which may give hints of the type of host in

which further development is carried on. The power of extention and

contraction is so great in most trematodes, especially in larval stages,

that a true conception of size and shape can be only gained from the

living animal. The pattern of the excretory system can only be made

out from living material. In fact it is almost impossible without care-

ful studies in the living condition to define sufficiently the structure of a

cercaria to insure specific determination. The amount of detail of

structure which can be quickly obtained from the study of living trema-

todes is often surprising. In one small distome about 2 mm. in length

it was possible from one living specimen not only to work out the con-

nections of the reproductive ducts and to gain some idea, from the direc-

tion of the beat of the cilia, of the functioning of the parts, but also to

make a camera lucida drawing under the oil immersion of the connections

of the female ducts.

Agamodistomes and adults for live study should be transferred to

slides in normal saline solution and covered with thin cover glasses.

130 AMERICAN MICROSCOPICAL SOCIETY

The water should be slowly removed from the preparation with a piece

of blotting paper until the pressure of the cover glass slightly flattens

the fluke without injuring any of its structures. Such a preparation,

when carefully sealed with vaseline, will often remain a whole day in

perfect condition for study and can be examined even with an oil immer-

sion lens. Sporocysts, rediae and cercariae are usually found in large

numbers in the digestive gland of the gasteropod host. The fully

developed cercariae should be mounted for study in the water from which

the snails are obtained, and the sporocysts, rediae and immature cer-

cariae in normal saline. By slowly removing the water from beneath

the cover glass with blotting paper, cercariae so small that they are

almost invisible to the naked eye can be slowed down and flattened so

that they can be studied under the highest powers of the microscope.

When dealing with small forms it is easier to make new mounts as the

one being studied becomes too dry, than it is to try to make a prepara-

tion more permanent.

At every stage of compression different structures are brought out.

Just before a cercaria goes to pieces in the process of drying the smaller

tubules of the excretory system are distended and the movements of the

flame-cells accentuated, so that they become clearly visible. By careful

observations and the use of a number of preparations the number and

position of the flame-cells and the relation of the tubules can be gradually

traced until the whole pattern of the excretory system is understood and

recorded. By this method I have been able to work out the excretory

systems of miracidia, rediae, agamodistomes and small adult trematodes.

The pattern of one system containing one hundred and twenty flame-

cells was successfully solved. I know of no other way by which such a

complicated excretory system could be worked out. The compound

binocular microscopes recently put on the market have proven very

helpful in the type of studies described above. This instrument is easy

on the eyes and gives depth to the object observed. Several intra vitam

stains have been tried. So far no method of intra vitam staining has

been found which gives a better picture than the unstained animal.

WILLIAM WALTER Cort.

University of California.

NOTES AND REVIEWS 131

A SUBSTITUTE FOR EUPARAL

In his original paper (La Cellule, 23, 427, 1906) Professor Gilson

omitted the method of preparing this medium because of its difficulties

and referred the matter to Griibler and Holborn. Under present cir-

cumstances it will perhaps not be a breach of etiquette to submit a method

which yields a medium with similar index and properties. According to

Gilson, Euparal is a solution of Sandarac in a mixture of Eucalyptol,

Paraldehyde, and Camsal.

Paraldehyde and eucalyptol can be purchased of any chemist’s supply

house. They should be dry and neutral. If purchased from the drug-

gist, who will probably substitute oil of eucalyptus, they should be redis-

tilled, reserving the fraction, 119° to 124° for paraldehyde, and 175-177°

for eucalyptol. The druggist’s stock is in both cases about one-third

something else.

Camsal is made by taking three parts of salol and two parts of cam-

phor, and warming gently until completely liquid. Thereafter the

mixture remains liquid, but should be kept well stoppered.

Sandarac as purchased is full of dust and ants. It may be purified

as follows: 30 grams of sandarac are placed in a 200 cc. flask and 150 cc.

of absolute alcohol added. Let stand with occasional shaking until

dissolved. This mixture is rather sensitive to water vapor from the air

and should be handled accordingly. Filter the solution thru a good

filter paper into a 300 cc. flask. This is best done by resting the short

funnel in the neck of the receiving flask and covering the whole with a

bell jar under which some anhydrous calcium chloride is placed. Filtra-

tion is much more rapid than Mayer’s albumin. The receiving flask is

now fitted with a two-hole stopper. Thru one hole passes a glass tube in

which a cotton filter has been placed; to catch dust, the rubber connections

must of course be clean. ‘This filter is connected to a calcium chloride

drying tower to remove water vapor. The other hole is connected thru

a receiving flask, if the alcohol is to be recovered, and thence to an

aspirator. Air is passed while the solution of gum is warmed gently to

50-60°. Do not bubble air thru the solution, that being unnecessary and

injurious. As the solution becomes thicker the temperature may be

slowly raised to 70° and finally to 80° to remove the last of the alcohol.

When the gum is moderately brittle on cooling the operation is ended.

To the gum in the same flask, add 20 cc. Eucalyptol, 10 cc. Paralde-

hyde, 10 cc. Camsal, cork and warm gently until a homogeneous solu-

132 AMERICAN MICROSCOPICAL SOCIETY

tion is obtained. This gives a medium with an index n= 1.483 to 1.486.

The index can be raised or lowered slightly by varying the proportions

used in making the solvent. Thus, the indices of the ingredients are

about: Eucalyptol 1.456; Paraldehyde 1.39, this varies with the pre-

paration used; Camsal 1.534; Sandarac 1.525. The essence d’euparal

is, of course, the solvent mixture used above. . The green tint mentioned

by Gilson as due to a certain copper salt is probably copper abietinate

which can be had of Merck or can be made of sufficient purity by any

student in the organic chemistry laboratory,

In my experience there is less difficulty in preparing this medium

than with some of the staining mixtures. It takes time but also little

attention. Slides mounted several months ago are in excellent condi-

tion and as near as one can judge the medium acts like Euparal. Sec-

tions can be mounted from 80% alcohol, either with or without passing

thru the essence.

E. S. SHEPHERD.

Geophysical Laboratory.

Washington, D.C.

CHROMOSOMES OF RANATRA SP?

During the summer of 1914 while working on the male germ cells of

another type, I prepared and sectioned some testes from a species of

Ranatra collected about Madison, Wis. The large number of chromo-

somes together with what seemed to be a very puzzling polymorphism

of spermatocytes induced me to defer a further investigation till a later

time. Recently the work upon this form has been resumed and has

progressed to a point where a preliminary and tentative statement may

be made.

The testes in the later nymph stages are especially valuable for sec-

tioning as they present in many cases the whole history of the germ cells

from the last spermatogonial divisions to mature spermatozoa. Sex

organs from adults collected in the spring, and up to mid-summer are

also generally favorable, but specimens taken in late summer and fall

show very few division stages.

My first material was composed of several testes from animals col-

lected in mid-summer and at that time believed to belong to but one

species. All of these were prepared together for study. Observations

NOTES AND REVIEWS 133

upon this mixed material show that there are at least two types of testes

as regards the chromosomes.

The first type has spermatogonia with 40 chromosomes of various

sizes; primary spermatocytes with 21, all of which divide equally; second-

ary spermatocytes with 21, two of which do not divide but pass directly

into different spermatids each of which then possess 20 chromosomes.

The two chromosomes which do not divide in the second division act

as a typical XY pair and always appear near the center of the chromosome

group in this division but their behavior is not sufficiently different to

enable them to be identified before this stage.

The second type has spermatogonia provided apparently with 8 or

10 more chromosomes than the first type. The primary and secondary

spermatocytes seem to have as a distinguishing mark a group of very

small chromosomes near the center of the larger group. Neither the

number nor the behavior of the chromosomes in the spermatids has been

determined although some interesting conditions are suggested by the

rather meager observations made to date.

Another interesting though not necessarily important fact is that

among the individuals collected in July few possessed testes of the

second type while a large percentage of those collected in September did

possess cells of that type.

In addition to the interest attached to spermatogenesis these forms

seem to offer an opportunity to determine possible correlation between

the chromosome differences and somatic variations as soon as the indivi-

dual origin of the two kinds of germ cells can be determined.

A. M. CHICKERING.

Beloit College.

NOTES ON COLLECTING AND MOUNTING ROTIFERS

C. F. Rousselet, the veteran English naturalist (J. Q. M. C., Nov.

1917) sums up methods which he has worked out for collecting,

handling, preserving and mounting rotifers.

For a collecting stick he recommends a walking stick with a telescopic

joint, with a ring net 6x5)4 inches and 6 inches long, made of bolting silk

No. 15 or 16. Silk lasts longer than mull and does not clog or shrink as

it does.

134 AMERICAN MICROSCOPICAL SOCIETY

The bottled materials collected are placed, on reaching home,

in aquaria with 7x7 inch parallel sides one and one-fourth inch in

depth from back to front. After a few hours the debris will have

settled, and if a strong light be placed at one face of the aquarium the

free-swimming rotifers will collect on the side toward the light, and can

be discovered with a lens and be picked up with a pipette. In solid

watch glasses these general collections can be examined quickly for new

species with the low power of the binocular and unfamiliar forms trans-

ferred to the live-box or the micro-glass trough for special study.

Narcotising the mass of rotifers in the watch glass is readily effected

by 1% cocaine. They may be killed and fixed by a drop of 4 to %%

osmic acid. They should be exposed to the osmic acid for a minute only

and then removed to formalin of 214% strength, changing it several

times until well washed.

The sorting out of different species is done under the binocular by

means of a bristle mounted in a suitable handle. They are then picked

up with a fine pipette and placed in an appropriate micro-cell, and finally

mounted in 214% formalin.

The ringing of the micro-cells may be done as follows: first a thin

ring of picture copal varnish; then several coats of Heath’s cement

(gold size-shellac-India rubber); finally finishing with three more coats

of gold size.

METHODS OF PRESERVING CERTAIN MARINE BIOLOGICAL SPECIMENS

F, Martin Duncan (J. R. M. S., Dec. 1917) brings together methods

which he has found most practical and successful in preserving marine

plant and animal life and in preparing it for microscopic examination.

Many of these methods are standard; but summarizing some of them may

be of value.

Anaesthetising

Place the smaller and specially sensitive medusze in just sufficient

sea-water for free expansion and swimming, and add two drops of 1%

solution of hydrochloride of cocaine, gently stirring with a glass rod.

Repeat at five minute intervals until the tentacles do not contract when

gently touched. Add 10-20 cc. of 4% formaldehyde solution, stirring

for several minutes. Store in 10% formaldehyde. Do not allow speci-

mens to remain in cocaine longer than absolutely necessary before adding

the formaldehyde, as the former softens the jelly of the medusz.

NOTES AND REVIEWS 135

~The author regards cocaine as, on the whole, the best anaesthetic for

the most of the smaller forms of marine life. Solutions of coacine must

be made anew since they do not keep well—becoming filled with fungoid

growths.

Hydroid zoophytes, simple and compound ascidians, holothurians,

anemones, and the like may be stupefied effectively with menthol.

This is slow in action and does not simulate to contraction. The animals

are submerged in clean sea-water and methol crystals are strewn over

the surface. Their solution is slow, and in twelve or twenty-four hours

depending on the size and sensitiveness of the animals and the amount

of water, the specimens will be narcotised in an extended position, and

may then be killed and fixed in any suitable fluid.

Fixing

The author prizes Bouin’s fluid (Picric acid, saturated aqueous solu-

tion, 75 parts; formalin 25 parts; glacial acetic acid, 5 parts) as the best

fixative for histological purposes. It has great power of penetration,

kills quickly, and fixes well. It allows after treatment of the most varied

sort. Next in desirability he considers saturated solution of corrosive

sublimate.

Weak osmic acid (a few drops of a 44% solution added to the water

in which the organisms are) is suggested for marine Protozoa. Radio-

laria are effectively killed and fixed in corrosive sublimate. Spherozoa

give good results in equal parts of sea water and 70% alcohol with a

trace of tincture of iodine added.

For Echinoderm larve an exposure of four minutes to a cold satura-

ted solution of corrosive sublimate is recommended. For whole mounts

dilute cochineal stain—as Mayer’s alcoholic cochineal formula.

Small sponges are placed, on collection, in 1% solution of osmic acid

and left there for five minutes, then transferred to strong alcohol and

changed twice. Stain sections in Mayer’s alcoholic cochineal.

Compound ascidians with contractile zooids may be handled to

advantage by placing in clean sea water, narcotizing with methol,

and then plunging for three to ten minutes in glacial acetic acid. Wash

in 50% alcohol and pass thru successive grades to a preserving strength.

Use no metal in the operation.

For small crustacea, both larve and adults, first treatment with 5%

formaldehyde in sea water is recommended. Transfer to 70% alcohol.

136 AMERICAN MICROSCOPICAL SOCIETY

For demonstration mounts it is necessary to guard against overstaining.

Weak alcoholic picro-carmine cleared in turpineol is advocated as a

means of staining.

THE SILVERMAN ILLUMINATOR FOR MICROSCOPES

This illuminator, invented by Professor Alexander Silverman of the

School of Chemistry, University of Pittsburg, is a small, circular tube

lamp which can be fitted quickly to any objective. It moves up and

down with the barrel and furnishes a diffused and uniform illumination

at the exact place where it isneeded. It is suitable both for low and high

power work, and may be used both for direct examination and for photo-

graphy of opaque objects.

Much structural detail is revealed by this device which the older

forms of illumination do not give. It isa low voltage tungsten lamp, and

may be supplied either in colorless glass or in daylight (blue) glass.

Its life is about 100 hours. There is no image of the source of illumina-

tion nor does the light strike the front of the lens except as reflected from

the object.

The intensity of the light reaching the eye is lower than in other

types of illumination, and yet because it is directed upon the spot ob-

served the observer sees more. There is no glare, no waste light, no

unduly contracted pupil, no unnecessary eye strain.

The lamps are manufactured by Ludwig Hommel & Co., Pitts-

burg, Pa.

TRANSACTIONS

OF THE

American

Microscopical Society

ORGANIZED 1878 INCORPORATED 1891

PUBLISHED QUARTERLY

BY THE SOCIETY

EDITED BY THE SECRETARY

T. W. GALLOWAY

BELOIT, WISCONSIN

VOLUME XXXVII

NUMBER THREE

Entered as Second-class Matter August 13, 1918, at the Post-office at Menasha,

Wisconsin, under act of March 3, 1879.

Che Collegiate Press

GrorcE Banta PuBLISHING CoMPANY

MENASHA, WISCONSIN

1918

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EX-OFFICIO MEMBERS OF THE EXECUTIVE COMMITTEE

Past Presidents Still Retaining Membership in the Society

Smion Henry GAGE, B.S., of Ithaca, N.Y.,

at Ithaca, N. Y., 1895 and 1906

A. CLirrorD Mercer, M.D., F.R.M.S., of Syracuse, N. Y.,

at Pittsburg, Pa., 1896

A. M. Briere, M.D., of Columbus, Ohio,

at New York City, 1900

C. H. E1cENMANN, Ph.D., of Bloomington, Ind.,

at Denver, Colo., 1901

E. A. Brrcer, LL.D., of Madison, Wis.,

at Winona Lake, Ind., 1903

Hnery B. Warp, A.M., Ph.D., of Urbana, IIl.,

at Sandusky, Ohio, 1905

HERBERT Oszorn, M.S., of Columbus, Ohio,

at Minneapolis, Minn., 1910

A. E. HERTZLER, M.D., of Kansas City, Mo.,

at Washington, D. C., 1911

F. D. HEALD, Ph.D., of Pullman, Wash.

at Cleveland, Ohio, 1912

CHARLES BROOKOVER, Ph.D., of Louisville, Ky.,

at Philadelphia, Pa., 1914

Caries A. Korom, Ph.D., of Berkeley, Calif.,

at Columbus, Ohio, 1915

M. F. Guyer, Ph.D., of Madison, Wis.,

at Pittsburg, Pa., 1917

The Society does not hold itself responsible for the opinions expressed

by members in its published Transactions unless endorsed by special vote.

TABLE OF CONTENTS

FOR VOLUME XXXVII, Number 3, 1918

Observations on Reproduction in Certain Parthenogenetic and Bisexual Nema-

todes Reared in Artificial Media, by Paul S. Welch and L. P. Wehrle........ 141

A New and Remarkable Diatom-eating Flagellate, Jenningsia Diatomophaga,

Nov. Gen., Nov. Spec., with Plate XIII; by Asa A. Schaeffer...................0000 177

Studies in American Stephanophialinae, with Plates XIV, XV; by Ernest Car-

POU SAGE: 4 tae iiadhy casio ctuantchaenehoecacioeena eect escke chav Alp asteree aera or 183

_ Notes and Reviews: Aquatic Microscopy for Beginners (Stokes)............:c:c:csessesese 199

Necrolory Dr Albert McGCalla 2 2 oe re Oe eee \ cians 201

TRANSACTIONS

OF

American Microscopical Society

(Published in Quarterly Instalments)

Vol. XXXVIT JULY, 1918 No. 3

OBSERVATIONS ON REPRODUCTION IN CERTAIN PAR-

THENOGENETIC AND BISEXUAL NEMATODES REARED

IN ARTIFICIAL MEDIA*

By Paut S. Wetca# and L. P. WEHRLE

CONTENTS PAGE

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*Contribution from the Entomological Laboratory, Kansas State Agricultural

College, No. 33.

142 WELCH AND WEHRLE

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INTRODUCTION

Nematodes present material for a wide variety of investigations.

Not only is the group very incompletely known, especially the free-living

species, but much also remains to be learned about their life processes.

One of the fertile, outstanding possibilities for investigation relates

to the remarkable features of reproduction presented by these animals.

Maupas (’00) and others have pointed out the striking diversity in the

methods of reproduction and the possibilities of this group in extending

the knowledge of sex determination, origin of parthenogenesis, origin

of bisexuality, and the nature of hermaphroditism. The development

of methods whereby these animals can be maintained in pure cultures

for indefinite periods adds to their value as material for critical study.

In this paper, the writers present results of observations on cultures

maintained continuously for more than three years. The data herein

presented are general in character and the results of study of certain

special problems will appear at a later date.

The writers wish to express their indebtedness to Dr. N. A. Cobb for

the identification of the specimens used in this work and to Mr. A. L.

Ford who, during the absence of the writers, maintained the cultures

through the summer months of 1917.

METHODS OF CULTURE

In connection with the work on which this paper is based, the writers

had occasion to use a number of methods for rearing nematodes in arti-

ficial media. Previous investigators devised means whereby certain

free-living and parasitic nematodes could be maintained for a number

of generations under laboratory conditions and some of their results

were used to advantage in the present work. In some cases, previously

known methods have been modified, while in others, new procedures

were developed. It is obvious that, in order to study these animals

REPRODUCTION IN CERTAIN NEMATODES 143

successfully, they must be reared under conditions other than those of

the natural environment and the fact that they can be cultured in arti-

ficial media for indefinite periods of time makes it possible not only to

observe continuously the details of activities and of life history but also

to carry on a large variety of experiments. The writers maintained

continuous cultures of Diplogaster erivora and Cephalobus dubius for

over three years. Cultures of the latter are still under observation

and are all descendants of one original stock. Since the published

methods of rearing the smaller nematodes appear in scattered sources,

the more important ones will be given brief notice here. In addition,

the methods employed by the writers will be discussed in detail.

METHODS OF OTHER INVESTIGATORS

Conte (00a, p. 374) cultured Rhabditis monohystera by employing

as nutritive media “la colle de pate trés epaisse les solutions de peptone

et les tranches de pomme de terre. Ces cultures etaient faites dans

des assiettes couvertes et, d’autre part sur porte-objets en isolant un

femelle fécondée dont j’etudiais ensuite la descendance.” He was

also able (’00b, p. 376) to rear Diplogaster longicauda “dans la colle de

pate.”

Metcalf (03, pp. 93-98) reared a nematode of uncertain identity

(Rhabditis brevispina ?), found in diseased corms, young stalks of Crocus

and cuttings of Petunia, Coleus, and Geranium on plates of asparagus

agar. Sterile agar was unsuitable, but a one per cent asparagus juice

agar, inoculated with a Fusarium and the bacteria which occurred with

the original stock of the nematodes and allowed to decay for about

two weeks, was satisfactory after it had been heated, filtered, and steri-

lized. In this medium, sterilized eggs developed rapidly and normally.

A certain fluidity of the medium was found to be desirable.

Potts (’10, pp. 443-446) readily cultured certain free-living nematodes

(Diplogaster maupasi, Rhabditis gurneyi, Rhabditis elegans, Rhabditis

duthiersi, Rhabditis sechellensis) in drops of nutrient media in watch-

glasses closed with a vaselined glass cover. Solutions of “brown” and

“white” peptone, used almost exclusively as the media, were allowed

to putrify until a cloudy growth of bacteria had formed throughout them.

In the presence of large numbers of bacteria, the nematodes thrived

. but, in sterile solutions, growth was suspended and eggs were deposited

144 WELCH AND WEHRLE

only at long intervals. Potts did not record the strength of the peptone

solutions—a matter of importance since, as will be shown later, an excess

of peptone has a deleterious effect on the worms. Two or three successive

generations were reared in a saturated solution of gelatin in water, and

mature individuals were secured from eggs in “solutions of amides like

tyrosin and leucin” but restriction of both growth and egg production

indicated that these media were inferior to peptone. Beef infusion was

also used to a very limited extent.

Oliver (’12) cultured an unknown nematode, found in the exudate

about the genitalia of dead guinea pigs, in a medium made by inoculating

a little of this exudate onto moist earth and slants of Musgrave’s amoeba

agar, kept at a temperature of about 75° F. Successful subcultures

were also made by using plain agar and ascites agar, plain agar and

amoeba agar giving the best results.

Johnson (’13, pp. 612-618) was able to culture Rhabditis pellio, a

parasite of the nephridia of the common earthworm (Lumbricus terres-

tris), by methods somewhat similar to those used by Potts, Conte, et al.

Filtered tap water or salt solution in watch-glasses to which were added

certain food materials constituted the basis of the cultures. The watch-

glasses were either covered individually by means of a vaselined glass

cover, or else several, without covers, were placed in a humid chamber

which gave a larger air space but minimized evaporation. The culture

medium was replenished from time to time. Experiments with Witte’s

peptone showed that a nearly saturated solution quickly proved fatal

to the nematodes and the same result accompanied weaker solutions

until a 0.15 per cent strength was reached. Although very dilute, this

solution proved satisfactory for one series of cultures, but afterwards it

was almost uniformly unsuccessful. A one per cent. hay infusion was

successful for only one series. Meat extract, decaying meat, a solution

of urea, and a hay infusion sterilized and inoculated with soil bacteria

were all virtually useless. Decaying earthworm had an advantage over

peptone in nutritive value, but presented the disadvantages of requiring

special treatment to prevent introduction of nematodes already present

in the worm; of becoming increasingly opaque as decay advanced, thus

rendering examination difficult; and of occasionally becoming foul

smelling and densely clouded, causing the death of all of the nematodes.

The best medium was prepared by removing the alimentary tract from

freshly killed Lumbricus terrestris and placing the body in a test tube

REPRODUCTION IN CERTAIN NEMATODES 145

which was then plugged with cotton-wool, heated in a steamer and kept

at a boiling-point for about two hours, decanted, filtered, and inoculated

with the bacteria which were associated with the worm in the soil. These

bacteria were secured by allowing a fresh Lumbricus terrestris to decay in

a small amount of water to which a little earth had been added. This

medium also possessed the disadvantage of occasional development of

bad odor and opaque scum, leading to the death of the entire culture.

Byars (714) made use of a synthetic medium, Pfeffer’s nutrient agar,

in connection with some studies of the plant nematode Heterodera radici-

cola and found it suitable for the preliminary development of the worms

up to their penetration into the tissues of experimental seedlings grown

in the same medium. Most of the individuals which remained outside

of the root of the seedling died within a few days, although a few

remained active for more thana month. The latter, however, did not

undergo their normal development into adults. It will be shown later

that this medium is much more successful for certain other species.

Merrill and Ford (’16, pp. 121, 126) found that Diplogaster labiata

flourished in water cultures to which had been added portions of the

macerated bodies of the beetle Saperda tridentata—the host of this nema-

tode. They state that “Different substances were tried with varying

success, but macerated beetles placed in water seemed to be the most

satisfactory”’ but these “different substances” were not specified. They

also reared Diplogaster erivora in similar preparations.

METHODS OF THE WRITERS

Equipment

The writers had occasion to employ a variety of culture methods

demanding a corresponding variety of equipment. Most of the cultures

were maintained in cells constructed as follows: Cylindrical glass mount-

ing-cells with ground edges were sealed to ordinary glass microscope

slides by means of pure vaseline and covered with a circular cover-glass,

the diameter of which was slightly greater than that of the glass mount-

ing-cell. The cover-glass was also sealed on with vaseline. For stock

cultures, the most suitable glass mounting-cell was found to be one having

a diameter of 20 mm. and a height of 10-15 mm., preferably 15 mm.

These tall cells made it possible to maintain a greater volume of the

liquid culture medium, thus providing for a larger stock and, more im-

146 WELCH AND WEHRLE

portant still, minimizing the danger of cultures drying out since it was:

discovered that they could not be sealed tightly but demanded at least

a small opening at the top. They offered the disadvantage of being

too high for examination with anything but very low powers of the com-

pound microscope. It was possible, however, to use the binocular

microscope, and since there was little occasion to use the stock cultures

for purposes other than maintenance, this disadvantage was of little

consequence.

Cultures maintained for experimental or detailed observational work

were kept in glass mounting-cells of another size, viz., diameter, 20 mm..,.

height, 6 mm. Such a cell surrounded a space large enough to enclose

a drop of the culture medium without involving the danger of the latter

coming into contact with the vaselined edges of the cell, a precaution

which had to be observed rigidly. That height also permitted examina-

tion with the ordinary low powers of the compound microscope without

removing the cell from the supporting slide.

Many of the glass mounting-cells used in this work have walls 1 mm.

thick. Cells having walls of 2 mm. thickness were found more advan-

tageous, especially for stock cultures, owing to the greater vaselined

area in contact with the slide, thus minimizing the danger of the watery

medium leaking out and permitting the culture to dry up. The thinner

cells were in more constant use owing to the difficulty of obtaining the

thicker ones, and, if care was taken to thoroughly vaseline the ground

edges and press them down onto the slide, they were quite satisfactory.

In special cases when greater space or capacity was desired, small

stender dishes were used. A limited use was made of a group of glass

mounting-cells sealed to the bottom of a Petri dish. For rapid observa-

tion of isolated individuals, Petri dishes alone were used for one series,

large drops of the nutrient medium retaining their identity when placed

on the dry, clean glass but not in contact with each other.

At times, culture slides of two kinds were used in place of the glass:

mounting-cells mentioned above. One form is the heavy plate glass,

76 x 26 mm. slide, 5 mm. thick and with a central, circular depression

4 mm. deep, and covered by sealing on a circular cover-glass with vase-

line. This form of receptacle was satisfactory in many respects but

was not usable for individual cultures because of its depth. Further-

more, it is more expensive than the glass mounting-cell. The other

culture slide is the ordinary 76 x 26 mm. form, having a central, shallow

REPRODUCTION IN CERTAIN NEMATODES 147

concavity with maximum depth of about 1 mm., closed by a vaselined

circular cover-glass. This type is useful for individual cultures.

Needles for transferring eggs, larve, and adults from one culture

to another were in constant use. These instruments were made by

setting into a small handle a fine needle, the point of which had been

bent into a small recurved hook. It was found convenient to have

several of these needles whose points were bent at different degrees.

Ordinary insect pins of sizes 0 and 00 were used to some extent but the

most useful form was the Japanned steel “Minuten Nadeln” commonly

used for pinning minute insects. These pins made especially useful

needles because of their very small diameter and extremely fine points,

thus facilitating their manipulation under the high power of the bino-

cular microscope and their use in transferring the minute nematodes.

Transferring brushes, which for part of the work were used instead

of needles, were made from small camel’s hair brushes by carefully cut-

ting out the brush until a very small central tuft of 3-5 hairs remained.

Such a brush was often used for transferring eggs.

Pipettes of the ordinary form were in common use for handling the

various fluids employed in the work. In order to avoid contamination

of the cultures, it was necessary to keep these pipettes properly labeled

and thoroughly clean. A special form of pipette for removing specimens

or for removing deteriorating culture fluids was made by drawing out

in a flame the open end of an ordinary straight pipette until the opening

was only about 0.3 mm. in diameter.

Media

In culturing nematodes, it is necessary to use some substance which

will serve directly or indirectly as food for the animals. The writers

have tried out a number of substances some of which have been distinctly

successful. Since the same substances were not in every case employed

for both of the species reared, the culture methods of each will be dis-

cussed separately.

Practically all of the culture media were more favorable under con-

ditions of dilution, in fact, some of the most successful ones demanded

extensive dilution, otherwise they were inimical to the worms. Dis-

tilled water was uniformly used, thus eliminating the danger of contami-

nating the cultures from that source, as would be the case with tap-water.

Preliminary experiments with both Cephalobus dubius and Diplogaster

148 WELCH AND WEHRLE

erivora showed that all stages of the life history lived for.some time,

several days in many cases, in distilled water alone, and when a small

amount of nutritive substance was added, the medium became suitable

for continuous culture. Apparently, the usual toxicity of ordinary

distilled water had no deleterious effect on the worms.

Diplogaster erivora.—Since the original stock of. this species was

found in the eggs of grasshoppers, the yolk of these eggs was at first

used as a medium and, as might be expected, gave good results. Eggs

from a number of species of grasshoppers appeared to be of equal value.

Owing to the occasional difficulty or inconvenience in getting grasshopper

eggs, the eggs of certain Coccinelide, of the Colorado potato beetle, of

certain unidentified insects, and the ovaries of grasshoppers were success-

fully substituted. Furthermore, the softer tissues of young grasshopper

nymphs, army worm pupz, variegated cutworm pupe, Hessian-fly

pupz, and pupz of a number of other insects were found to constitute

good media when prepared in the proper way. It is very probable that

a wide variety of insect eggs and tissues would serve this purpose equally

well.

The procedure in the use of the above mentioned substances depended

upon the kind of culture desired. Most of the stock cultures, as well as

the majority of the life history cultures, were maintained in the glass

mounting-cells already described. When grasshopper eggs were used,

they were first carefully cleaned externally and stripped of their shells,

since fragments of the latter when present tend to render the examination

of the culture more difficult. The contents were placed in a small

amount of distilled water and thoroughly triturated in order (1) to dis-

tribute more uniformly the yolk throughout the culture and render it more

readily available to the nematode, and (2) to facilitate the examination

of the material for evidence of previous nematode infestation, thus aiding

in the maintenance of pure cultures. This precaution is necessary since

small nematodes often occur on the surface and apparently within some

of these insect eggs and pupx. The writers found them in connection

with Hessian-fly puparia, both on the surface and apparently in the

pup, although in the latter case there is the possibility that they might

have been included by manipulation. However, Marchal has reported

Osborn, ’98, p. 41) nematodes in the puparia of this insect. Insect

tissues mentioned in a foregoing paragraph were sometimes used in a

REPRODUCTION IN CERTAIN NEMATODES 149

similar way, removing, of course, all fragments of the integument. The

proper amount of the diluted nutriment was placed in the glass mounting-

cell which already contained the appropriatc amount of distilled water.

The method of adding the food materials thus prepared was deter-

mined by the purpose of the culture. For stock cultures, two or three

drops from a pipette were added every 3-5 days to the 1-1.2 c.c. of dis-

tilled water in the mounting-cell. A larger amount of food could be

added without serious results to the organisms but it was not needed for

nutritional purposes, and it had the disadvantage, at least in certain

cases, of tending to increase the opacity of the culture.

When there was any occasion for using large quantities of the material

for a medium, good results were secured by putting sterilized soil in a

covered stender dish, moistening it liberally with distilled water, and

transferring to it a pupa or other food object which was then perforated

and inoculated with the nematodes from a stock culture. These cul-

tures lasted for considerable periods of time, developing nematodes in

quantities.

While the above-described methods were satisfactory, it was incon-

venient in the maintenance of a long series covering different seasons of

the year to keep a constant supply of the food materials at hand and to

have a continuous stock of any one kind for experimental purposes. For

that reason, a search was made for some food which is approximately

constant in composition and easily available at any time. Among

other things, the yolk of hen’s egg was tried and found successful. Ex-

periments with different amounts of this substance showed that, in a cul-

ture containing 1-1.3 c.c. of distilled water, the nematodes thrived when

the quantity represented by three or four dippings of the points of ordin-

ary forceps into the yolk was added. Almost any larger amount may

be used but increased opacity and the occasional undue putrefaction

make it undesirable. Amounts of food represented by one or two dippings

of the forceps were, in general, found to be distinctly inferior in results

and evidently represent too poor a culture. A single hen’s egg supplied

food material for a number of days. A small opening, approximately

1 cm. in diameter, was made through the shell and the adjacent albumen

allowed to escape. Sterile forceps were used to lift out the desired

quantities of the yolk and then the opening in the shell was sealed over

with a gummed label or piece of gummed cloth and the egg kept in a cool

150 WELCH AND WEHRLE

place. When needed again, the gummed covering was removed, the

desired quantity of yolk secured, and the opening again sealed. The

amount of yolk adhering to the points of forceps is variable but the

method was found to be both rapid and practical in the operation of

ordinary cultures.

Liebig’s extract of beef, diluted to different degrees with distilled

water, was tried as a nutrient medium and found to be usable so far as

maintaining the animals was concerned but its partial opacity impaired

its usefulness.

Since Byars (’14, p. 323) found Pfeffer’s synthetic agar useful in

culturing certain nematodes parasitic on plants, it occurred to the writers

to experiment with it as a possible medium for rearing Diplogaster erivora.

The medium used was as follows: calcium nitrate, 4 grams; potassium

nitrate, 1 gram; magnesium sulphate, 1 gram; potassium dihydrogen

phosphate, 1 gram; potassium chloride, 0.5 gram; ferric chloride, trace;

distilled water, 6 liters; and agar, 12 grams. The formula used by Byars

calls for powdered agar but the writers used the shredded agar cut up

into small bits. This fluid was used directly from the stock solution

without change and, while it appears from all the trials that it is some-

what inferior to certain other media used, nematodes were maintained in

this solution for long periods of time. According to the experience of the

writers, cultures in this medium, when properly cared for, are fairly

satisfactory, but since there was some evidence of inferiority, parallel

series of tests were carried on for 20 days, comparing the relative merits

of Pfeffer’s solution and hen’s egg as media. Using the rate of develop-

ment and reproduction as indices of the value of the media, four of the

five series showed evidence of the superiority of the hen’s egg, one of the

five showing a slight advantage for the Pfeffer’s solution.

Maceration cultures used by the writers have already been described

and it seems very probable that, barring the danger of contamination,

they might be effective and that a number of different animals could

be used as sources of food supply. Mr. A. L. Ford reared Diplogaster

erivora for one year on the macerating bodies of adult Saperda tridentata

and Hydrophilus triangularis. He also found it possible to use macerated

beef but the cultures proved somewhat unsatisfactory because of the

offensive odor. This nematode was found by Merrill and Ford (’16)

parasitizing termites and Mr. Ford has informed the senior writer that

he tried the macerating bodies of these insects as a medium but found it

REPRODUCTION IN CERTAIN NEMATODES 151

unusable since a mould almost invariably appeared in the cultures, filling

them with threads and obscuring the entire preparation. The writers

also tried macerated termites with similar lack of success from the same

cause.

Cephalobus dubius——Maupas (00, p. 611, 613) found some difficulty

in culturing the parthenogenetic nematodes, including this species, stat-

ing that “elles se prétent mal 4 des cultures en grand.”” However, the

writers have been able to culture Cephalobus dubius in countless numbers

at any time. Since studies of this species were not begun until cultures

of Diplogaster erivora had been in progress for about two years, the

methods which had proved most satisfactory were tried for Cephalobus

dubius and were likewise found to be successful. Several series of cul-

tures were maintained in Pfeffer’s solution but many of the life history

studies were made using the hen’s egg preparation. Both media were

used as described in the preceding section, and both proved suitable for

prolonged cultures, indications pointing to the hen’s egg being slightly

preferable. Grasshopper eggs, Colorado potato beetle eggs, and pupz

of Hessian-fly also proved suitable but were not always readily available.

As has been mentioned, other workers made some use of peptone

as a nutrient medium for the culture of nematodes. After a number

of trials with different strengths of peptone, a solution was found which

has proved thoroughly satisfactory and, at the present writing, cultures

of Cephalobus dubius have been running in this medium for months, with-

out any indication of weakness. The strength of solution used by Potts

is not given in his paper. Johnson discovered that strong solutions

are inimical to the nematodes and that dilution to as low as 0.15 per

cent. was necessary, this strength proving satisfactory for one series

of Rhabditis pellio. The writers have had a similar experience, finding

that strong solutions kill Cephalobus. dubius in a short time but that

very weak ones are quite efficient. In the work on which this paper is

based, 0.8-4.6 per cent. solutions were used. In fact, it was found un-

necessary to make up solutions of definite strength, except for special

Studies, since the stock cultures could be kept in flourishing condition

by occasionally (every four to six days) adding to the water in the mount-

ing-cell containing the nematodes the smal] amount of peptone which

would adhere to the point of a common dissecting needle. This method

of measure was, of course, subject to variation but within its limits the

152 WELCH AND WEHRLE

varying quantity seemed to produce no appreciable effect on the cultures.

and indicated a range of over five per cent. within which the solution

is suitable. The exact maximum and minimum dilutions for this species

were not determined. It should be mentioned in this connection that

even longer intervals between “feeding” were possible if necessity de-

manded. The writers had one peptone culture made up in the above-

described way which existed for over four weeks without renewal of the

food, the nematodes thriving and reproducing during the whole period.

As stated on a preceding page, Potts found that it was necessary

for peptone solutions to putrefy until a cloudy growth of bacteria had

developed and that it was “only in the presence of great numbers of

bacteria, or the substances formed by them, that the nematodes thrive

so well.”? In cultures of Cephalobus dubius maintained by the writers,

it was not necessary to develop any putrefaction of the peptone solution

but the dry peptone was added directly to the water of the rearing cell.

When new cultures were necessary, distilled water was placed in the

cell, a small amount of peptone on the end of a dissecting needle was

transferred to it, and the nematodes then introduced. This procedure

was followed a great many times and without any appreciable diminution

in the activities of the animals. Furthermore, cultures made up in this

way have been kept for a month without developing cloudiness and the

nematodes grew and reproduced rapidly. Since no attempt was made to

keep these cultures sterile, bacteria did develop in them to some extent

and it may be that in those cultures which remained perfectly clear a

limited amount of bacteria was present. It should be mentioned in

this connection, however, that a distinct cloudiness did appear in some

of the cyltures after standing several days, developing gradually into

a distinct brown color. This occurred rather commonly in summer and

was possibly due, in part at least, to the accumulation of bacteria. But,

with this particular nematode, the development of the brown color

was accompanied by a general deterioration of the culture and necessi-

tated either a removal and renewal of the greater part of the culture medi-

um or a transferrence of the nematodes to a new solution. It would

then seem that Cephalobus dubius requires only a very low development

of bacteria in the culture medium, if it is required at all, and that an

undue development of the cloudiness is usually detrimental.

REPRODUCTION IN CERTAIN NEMATODES 153

General Procedure

Certain general methods of procedure were found to be preferable,

and, in some cases, necessary to the maintenance of the cultures. In

stock cultures reared in glass mounting-cells, the optimum amount of

medium was found to be about two-thirds to three-fourths the capacity of

the cell. Most of the cells were kept approximately half filled. Smaller

amounts could be used but involved the danger of the cell drying up be-

tween observations.

Early in the work, it was noticed that when a stock culture was sealed

over completely with a vaselined cover-glass unfavorable conditions

became established and the culture would often die out. Experiments

were performed to determine the effect of the presence or absence of

an air-space above the medium and it was definitely demonstrated

that such a space must be provided, hence the above-mentioned proced-

ure of never filling the cells more than three-fourths full. Furthermore,

it was found that even with an air-space provided, unfavorable condi-

tions would develop in a few days if the cell was sealed air tight. This

situation was avoided by slipping the cover-glass back so that a small

crescent-shaped opening between the edge of the cover-glass and the

walls of the mounting-cell was produced. This method was entirely

satisfactory although it involved a certain loss of the water by evapora-

tion, thus necessitating an occasional restoration to the original level

in the culture-cell. The exact reason for this demand for ventilation is

not known. Johnson (’13, p. 612), who also discovered the necessity

for providing against completely closed cultures, thought that it was neces-

sary to permit the escape of gaseous decomposition products originating

in the culture medium. It is true that in cultures made from such

materials as yolk of hen’s egg, insect eggs, and ovaries of grasshoppers,

decomposition products were formed since they tend to quickly develop

offensive odors. On the other hand, Martin (’13, pp. 94-97; 143) has

pointed out the indispensability of free oxygen to the development of

the embryos of nematodes and it may be that the demand for oxygen

is the explanation. Since many of the culture media used were under-

going decomposition and since this process draws upon the available

oxygen, it is possible that the oxygen supply in one of the sealed culture-

cells, having either a small air-space or no air-space at all, would soon

be reduced to an unfavorable extent.

154 WELCH AND WEHRLE

Occasionally, even the partially closed stock cells, especially those

containing peptone, developed not only a cloudy appearance but also

a brown color which gradually increased in intensity. The specific

cause of this color is not known but it was evidently a result or an accom-

paniment of the general activity of organisms in the cell and almost in-

variably led to the death of the nematodes if the conditions were allowed

to continue. Thus it was necessary either to transfer nematodes to

an entirely new culture cell or else remove the old medium and replace

it with new. To accomplish the latter, the point of a finely drawn

pipette was thrust just below the surface of the medium in the stagnant

cell and the liquid very slowly removed. In this way, the eggs, young,

and adult nematodes which are always at the bottom were but little dis-

turbed and few of them lost in the process. Then the old medium was

replaced with distilled water and a small amount of peptone added.

If necessary, a number of the nematodes can be transferred to new cul-

ture-cells by means of the transferring needles already described. In

stocking a new culture, only a few nematodes need be transferred.

In studies of the reproduction, development, growth, and activities

of these nematodes, it was necessary to isolate individuals or eggs in

order to follow definitely and accurately the sequence of events. To

accomplish this end, cultures of a different type were used. The culture-

cell was made up in the same way as those for th: stock cultures except

that it was necessary to use a cell not over 5 mm. high. A single drop

of distilled water from a pipette was centrally placed in the bottom of a

clean, dry cell. This drop, if carefully placed, retained its integrity.

If the drop spread and came into contact with the edges of the cell, it

was discarded. To this drop of water was added a tiny bit of the food

material (the quantity which will just cling to the extreme point of a

dissecting needle) and the nematode or the egg transferred to it. Since

these single-drop cultures have a large air-space compared with the

bulk of the fluid and since they must be examined frequently in connec-

tion with the recording of data, these cells were kept tightly sealed and

in that way the danger of drying up was avoided. During the micro-

scopical examination, the whole cell was removed from the slide, thus

leaving the culture drop intact. It was necessary to guard against

undue evaporation during the exposure of the drop and all losses had to

be replaced. Transference of the nematodes, young or adult, was easily

accomplished by means of the needles already described. Eggs were

REPRODUCTION IN CERTAIN NEMATODES 155

~ transferred by the same means and likewise by the drawn out pipette

already described. The latter method was advantageous in transferring

a number of eggs at a time since they could be massed together in the

culture-cell and then drawn up in the pipette with a relatively small

amount of liquid. Furthermore, eggs were transferred by means of the

brush described in an earlier paragraph but this method was inferior

to the ones discussed above.

Starting Cultures from New Stock

Since Cephalobus dubius has been found but once and the entire

series of cultures is from the original stock, the writers have had little

experience in transferring this species from the conditions of nature to

those of the culture-cell. However, Diplogaster erivora has been brought

in from outside conditions and established in cultures a number of times.

New stock used by the writers usually came from grasshopper eggs and

was secured by breaking up the eggs in culture-cells, diluting the yolk

with distilled water, covering the cell in such a way as to leave an air-

space and a ventilating opening, and putting it aside under temperature

conditions of about 68-77° F. Often, after a few days, this nematode

would appear in the culture whence it could be transferred to a more

dilute medium of the same kind and then later to a different medium,

if desired. Mention has already been made of the fact that Merrill and

Ford (716) found this nematode parasitizing termites. Mr. Ford has

informed the senior writer that he could almost invariably secure a

culture by the following method: A number of termites are killed and

placed on the surface of water saturated soil in a small container. After

some hours, nematodes usually emerge from the heads of the termites

and continue their activities on the moist surfaces of the dead insects.

In a few days, the disintegrating bodies are often swarming with the

worms. ‘Then a number of them are removed to a culture-cell containing

the desired medium. Of those first transferred, the majority may die

but usually a few will survive the change and reproduce, starting a new

stock. Evidently, in a parasitic form, the transition from the body of

the termite to the conditions of a culture-cell is a severe one but the

immediate progeny of those which persist flourish in the new medium and

thereafter maintenance is simple. This mortality of the parasitic nema-

todes, when removed to cultures, confirms, in part, the experience of

Johnson (’13, p. 613) who frequently had great difficulty in starting cul-

156 WELCH AND WEHRLE

tures of Rhabditis pellio, although, when once started, the difficulties of

maintenance were considerably reduced.

Temperature Conditions of Maintenance

While no definite experiments have as yet been made to determine

the exact maximum and minimum temperatures for these nematodes,

certain observations are worthy of mention here. Temperature records

for rooms where some of the cultures were kept showed that both species

withstand successfully a considerable variation of temperature. In one

room, the daily maximum-minimum records sometimes showed a dif-

ference of as much as 33° F. Such variation usually occurred during

the winter months when the room temperature occasionally dropped from

74° to 42° F. Under these conditions, daily variation of 15-25° F. were

common and a minimum temperature of 40° F. for a limited time (4-6

hours) apparently had no serious effect on the cultures. In this same

room during the summer months, the variation was considerably less

but both maximum and minimum temperatures were much higher, e.g.,

92° and 84°F. It was found that when the room temperature rose much

above 80° F., cultures began to show signs of weakness.

In cultures of Cephalobus dubius, the nematodes began to die under

conditions of 90° F. These cultures were removed to an underground

concrete cave where an almost constant temperature of 80° F. was

maintained and evidence of strengthening was apparent. During the

following thirty days (July), when the temperature in the cave gradually

approached 87° F., the cultures showed increasing signs of weakness and

ultimately maintenance became difficult. In the following month, when

the temperature began to fall, signs of strengthening were evident as soon

as 80° F. was approached. When 78° F. was reached, the cultures were

soon flourishing again. In other respects, the conditions of rearing were

the same throughout this period. Evidently, the maximum temperature

for this species is near 90° F. and the optimum below 80° F. At the

present time, stock cultures are thriving under laboratory conditions

of 65-75° F.

Preliminary studies with reference to the influence of temperature

upon Diplogaster erivora were conducted by means of an air conditioning

machine having two large breeding chambers in which constant tem-

peratures (within very narrow limits) of 80° and 90° F., respectively,

were maintained. These nematodes could not be reared in the 90° F.

REPRODUCTION IN CERTAIN NEMATODES 157

chamber. Cultures submitted to that temperature soon showed decrease

of activity, suspended reproduction, and early death. Fresh cultures

reared in the 80° F. chamber and transferred to the 90° F. chamber

suffered the same fate. Nematodes reared in the 80° F. chamber also

exhibited some decrease in activity but continued to live and multiply,

producing four generations in forty-three days. Cultures, kept under

greenhouse conditions at a mean temperature of about 75° F., increased

more rapidly than did those within the 80° F. chamber and the rate of

development was greater, five generations being produced in forty-three

days. Still other cultures were maintained in the laboratory under a

variety of temperature conditions, and, according to the experience of

the writers, 65-75° F. is favorable for rearing Diplogaster @rivora.

PARTHENOGENESIS IN CEPHALOBUS DUBIUS MAUPAS

DISTRIBUTION

Cephalobus dubius is evidently a cosmopolitan species. Maupas

(00, pp. 555-556) states that it is very common in Algeria and that he

found it in a sample of red earth collected in the environs of Tananarivo,

Madagascar. In these localities, the nematode lives in rather poor earth

and is said to withstand long desiccation and to revive when the moist

conditions are re-established. Dr. N. A. Cobb states in a letter that it

occurs in various parts of the United States and is found in moist situa-

tions, feeding upon animal matter. He also states that on several occa-

sions it has been found on the surface of various insect eggs, and that the

young withstand desiccation.

SOURCE OF MATERIAL

The original stock for all of the writers’ cultures was secured accident-

ally in connection with the study of another nematode. In a prelimi-

nary experiment to determine whether Diplogaster erivora, a species

found feeding on the contents of grasshopper eggs, has the ability to

penetrate insect eggs or whether it depends upon some other agency

to provide the means of entrance, ordinary loam, sterilized by steam,

was placed in a jar. Grasshopper eggs, cleaned of all soil particles but

not subjected to special treatment, were introduced into this soil and

specimens of Diplogaster erivora in water added to the adjacent soil.

When examined later, the eggs were found broken and deteriorating.

158 WELCH AND WEHRLE

Under magnification, nematodes were seen in the egg contents and were

transferred to culture-cells. It soon became evident that they were

unlike Diplogaster erivora, and when submitted to Dr. N. A. Cobb for

identification they proved to be Cephalobus dubius Maupas. The original

source is thus in doubt, but since the soil in the jar had been sterilized,

it seems very probable that this nematode was carried into the culture

on the surface of the grasshopper eggs, either as an egg or in some sub-

sequent stage of development. Since there is evidence that at least

the immature individuals of this species can successfully withstand

desiccation, it is possible that they might have occurred on the surface

of the insect eggs in that state, resuming activity when introduced into

the moist conditions of the experiment.

PARTHENOGENESIS

Maupas (’00), in his extensive study of reproduction in nematodes,

found seven species in cultures of which he saw no trace of males. These

seven species, distributed among six different genera, included Cephalobus

dubius. He was inclined to admit the possible occasional occurrence

of males but states that if they do appear they must be very rare.

Since the beginning of this work, the writers have watched carefully

in the large number of successive generations for any appearance of males

but in vain. Mature individuals, isolated in culture-cells, always de-

posited eggs. Similarly, isolated eggs or young that attained maturity

always produced individuals which deposited eggs. Maupas expressed

uncertainty as to the constant absence of males because of the fact that,

in his studies, the greater part of the apparently parthenogenetic nema-

todes “se prétent mal 4 des cultures en grand” and that with one excep-

tion he was unable to examine very large numbers. That uncertainty

does not seem so significant since the writers were able, with their

methods of culture, to get this nematode in almost any quantity and

a very large number was examined for the possible appearance of males.

Since the nematodes were, in part, maintained under certain differences

and variations of food and temperature, it would seem that if males

ever do appear in this species they must be extremely rare and apparently

must develop under conditions of culture different in some unknown re-

spect from those of the writers. Cultures of this species are still under

observation in order that this matter, among others, may be more thor-

oughly tested. If it is later discovered that males do occasionally occur,

REPRODUCTION IN CERTAIN NEMATODES 159

it will be interesting to determine whether they have all of the activities

and functions of the sex or whether they resemble the imperfect males

which occasionally occur in several of the hermaphroditic nematodes.

As Lankester (’17, p. 504) points out, parthenogenesis signifies “an

exceptional and historically super-induced modification of the normal

process of sexual reproduction or gamogenesis in which the female gamete

or egg-cell does not unite with a male gamete or sperm-cell to form a

‘zygote,’ but proceeds to develop independently.” It is a well known

fact that protandric hermaphroditism (“Syngonism’’) is common among

the free-living nematodes and Cobb (15, p. 95; ’16, pp. 198-199) suggests

the desirability of re-examining the supposedly parthenogenetic forms

to determine whether some of them are not actually “‘syngonic.” This

suggestion was made on the basis of a study of a series of syngonic free-

living nematodes in which the spermatozoa present were smaller and

smaller until they reached the optical limits of present instruments and

he finds himself unable to assert the non-existence of spermatozoa in

certain nematodes merely because he has not succeeded in finding them.

This suggests that since both the parthenogenetic and the hermaphrodi-

tic nematodes have the unmodified form of the female, the former may,

at least in some cases, possibly be instances of masked hermaphroditism.

The writers have not thus far made any microscopical examination

of the gonads to determine the above-mentioned point and no comment

can be made upon it except that the complete absence of males in con-

tinuous cultures maintained for over three years seems to indicate the

parthenogenetic method of reproduction. If the animal were herma-

phroditic, it would necessitate that every egg produced be fertilized and

that fertilized eggs invariably produced hermaphrodites in order for the

results of the writers’ cultures to have been possible. Furthermore,

as Maupas and others have pointed out, the hermaphroditic forms show

an unbalanced relation between the number of spermatozoa and ova

produced and the unfertilized ova do not develop but disintegrate after

they are laid. In the case of Cephalobus dubius, all of the eggs laid were

capable of development. Also, in the majority, if not in all hermaphrodi-

tic nematodes, males appear even though they may in some species be

rare and imperfect in their sexual functions. At present, there seem

to be no grounds for considering Cephalobus dubius other than a par-

thenogenetic form.

160 WELCH AND WEHRLE

Ovi position

In order to obtain data on the reproductive capacity of the females,

oviposition records were made as follows: Eggs, isolated from vigorous

stock cultures, were placed individually in a single drop of distilled water

in a glass mounting-cell. Frequent observations were made and the

time of hatching carefully noted. A trace of yolk of hen’s egg was then

added and daily observations continued until the death of each individual.

When oviposition began, the number of eggs appearing in the cell each

day was recorded and the female transferred to a new culture, thus avoid-

ing the labor of removing the eggs from the cell.

Cultures of thirty-six different individuals, representing a number of

successive generations, were carried through the life cycle to cessation

of oviposition and subsequent death and showed considerable variation

in the length of the egg-laying period. These cultures were all main-

tained under the same conditions with respect to food but the tempera-

ture was that of the laboratory and subject to an average daily variation

of about 18° F., the extremes being approximately as follows: minimum,

40-58° F.; maximum, 66-76° F. Temperature was the only factor which

varied to any extent and the precise influence of this factor was not de-

termined. Under these conditions, the duration of the egg-laying period

was found to have a variation of 6—44 days, the average being about 16 days.

This variation seems surprisingly large and leads to the suspicion that those

nematodes with a short egg-laying period are individuals which, for some

reason, had their normal existence shortened. But, since no account

was taken of those individual life histories which were ended at a time

when the egg production rate was high and only those which showed

an acceleration followed by a subsequent retardation were considered,

it does not appear that all such instances can be so interpreted. It

should be stated that the maximum oviposition period of 44 days given

in this set of records is much higher than any of the others, the next

lowest being 31 days.

Egg-laying, when once initiated, continues uninterruptedly until its

decline at the end of the life of the individual. None of the records

showed any evidence of definite periods of cessation of egg-laying followed

by active resumption of oviposition. Only two instances of 24-48 hour

periods with no oviposition appears in the records and these occur at

the end of the life of the nematode. The eggs are laid singly and at a

REPRODUCTION IN CERTAIN NEMATODES 161

varying rate depending upon the age of the parent. The most charac-

teristic feature of the records on all individuals presenting any evidence

at all of a normal life cycle is the rather gradual increase in the daily

rate of egg-laying during the first part of the oviposition period and the

more or less gradual diminution in the last part. In every case, the maxi-

mum daily number of eggs deposited appears well within the oviposition

period, ordinarily near the middle. Usually the initial and concluding

daily rates are very slow, the latter often ceasing completely before the

death of the nematode. In a few individuals only was the initial daily

record above 10. The increase and decrease of the daily rate were not

regular for any of the individuals studied. The maximum production

of 27 eggs in one day occurs in the record of nematode No. 36. <A few

other records are close rivals. According to Maupas (’00, p. 560), “ Par

une température de 20° c., le maximum d’ceufs pondus, dans les vingt-

quatre heures, est de 12 4 13.”’ It is evident that the daily rate of egg

production is, at least at times, much higher than Maupas’ record would

indicate.

The total number of eggs per individual varied from 33 to 285, the

average being 139. In general, those nematodes which lived longest

and thereby had the greatest oviposition period produced the largest

number of eggs but, in the writers’ records, no constant relation exists

between the duration of the oviposition period and the total number

of eggs. For instance, nematode No. 55 produced 101 eggs in 9 days but

nematode No. 79 deposited only 77 eggs in 13 days; nematode No. 24

produced 270 eggs in 44 days but nematode No. 85 produced 285 eggs in

30 days. Maupas (’00, pp. 561-562) reported a maximum of 415 eggs

deposited over an egg-laying period of about 4 months.

In addition to the above, another set of oviposition records were made

from cultures kept in an underground, concrete cave, where, during the

period involved, the daily temperature variation was within 5° F., usually

within 4°F. The average maximum temperature was 77.8° F., the varia-

tion being 75-79° F., and the average minimum temperature was 73° F.,

the variation being 71-76° F. While the temperature was not com-

pletely controlled, the fluctuation was small and the results are of interest

when compared with those already described. The remaining conditions

of the cultures were like those of the other series with the exception that

peptone was used instead of the yolk of hen’s egg. Six individuals were

162 WELCH AND WEHRLE

successfully carried through their complete life cycle and the egg-laying

period varied from 15 to 39 days. The total number of eggs deposited

by each individual varied from 78 to 234. This series of cultures also

presented the characteristic increase and diminution of the daily rate

of oviposition which has already been described. The lack of a definite

relation between the length of the egg-laying period and the total number

of eggs deposited was also apparent although the general tendency for

the longer periods to be accompanied by the larger number of eggs was

evident. Owing to the difference in the numbers of individuals studied

in the two series, a more detailed comparison is not possible.

After oviposition, the development of the embryo goes on rapidly

and within a few hours the egg, under magnification, shows signs of

internal activity. Records on the development of 88 uggs from deposi-

tion to hatching showed 3.3 days as the average egg period, the variation

being 2.5-4 days. This is in close agreement with Maupas’ statement

(00, p. 561) “les ceufs mettent trois jours 4 parcourir leur embryogénie

jusqu’a éclosion.”

Immature Period

Length of Larval Stage——In hen’s egg cultures kept under the con-

ditions of the laboratory, the larval period of 48 individuals of different

progenies showed a variation of 8-17 days, although the usual variation

was 8-14, 17 occurring but once and 14 being the next lowest number. —

The average larval period, based upon the above-mentioned records, is

10.1 days. In this paper, the larval period is regarded as extending

from the moment of hatching to the deposition of the first egg, and not

to the cessation of growth as will be discussed later. The appearance of

the genital pore is an indication of maturity or closely approaching

maturity but it was thought best to use the first oviposition as the final

limit of the immature stage. Maupas (’00, p. 561) reported the larval

period to be 10-11 days, at a temperature of 68° F. He also used the

appearance of the first egg as evidence of the completion of larval

existence.

In peptone cultures maintained under the more uniform temperature

conditions of the underground cave described previously, the larval

period of 10 individuals had a variation of 8-14 days, the average being

10.8 days. It will be noted that this agrees closely with the cultures

reared under laboratory conditions and it appears that limited differences

of temperature and the comparative culture values of peptone and yolk

REPRODUCTION IN CERTAIN NEMATODES 163

of hen’s egg had no striking effect on the time interval demanded for the

attainment of sexual maturity.

Rate of Growth—Careful, daily measurements of length of 8 indi-

viduals reared in peptone cultures under the cave conditions were made

for about 22 days, a period of time well beyond maturity and the cessa-

tion of growth. It was noted that the period of growth and the Jarval

period are not coterminal, but that growth usually continue. 3-6 days

after the production of the first egg. During this time, the increase in

body-length has a variation of 0.019-0.076 mm., the average being

0.0527 mm.—about 10 per cent. of the average body-length of the com-

pletely formed individual.

At hatching, the larva is only 0.140-0.179 mm. long, the average of

16 specimens being 0.162 mm., and, from that time to the attainment of

the full-grown condition, growth is continuous and approximately uni-

form. The growth curves of different individuals are very similar. The

average daily increase in length was found to be 0.0258 mm., the varia-

tion being 0.019-0.03 mm. There is no evidence of a definite maximum

daily rate of growth anywhere in the period and apparently growth,

when completed, ceases almost as abruptly as it begins. The average

growth period was approximately 16.3 days, with a variation of 14-18

days.

Length of Life

The complete life showed a variation of 20-61 days. Only 1 nematode

lived 61 days, but 8 of 29 individuals lived 40-50 days, and 11 lived 30-40

days. The variation indicated above appears to be rather large and

may have been due to some peculiar combination of factors in the dif-

ferent cultures, although all were given the same kind of food in approxi-

mately the same amount and kept under the conditions of the laboratory.

Maupas (00, pp. 561-562) records one individual which lived for approxi-

mately five months. The egg-laying period has already been discussed

and it thus appears that if the first eggs of the parent be considered, a

new generation can be produced in the cultures every 11-19 days, or about

two generations per month.

REPRODUCTION IN DIPLOGASTER ZZRIVORA COBB

SOURCE OF MATERIAL

This nematode was first discovered in 1914 infesting the eggs of

grasshoppers after they had been deposited in the ground. Specimens.

164 WELCH AND WEHRLE

were sent to Dr. N. A. Cobb who reported them as representing a new

species. Later, Merrill and Ford (’16) found nematodes infesting the

termite, Leucotermes lucifugus, which, when submitted to Cobb, proved

to be the same species as the one found in grasshopper eggs and he

described it under the name Diplogaster erivora. Merrill and Ford made

a special study of this nematode in relation to the termite host and pub-

lished, in addition to Cobb’s original description of the species, certain

data on the reproduction and habits of this worm as observed in cultures.

The present account includes the results of a more prolonged study of

certain features of the reproduction, based upon continuous culture

studies of more than three years.

OVIPARITY

Merrill and Ford (’16) have already pointed out the fact that males

and females are continually present and that the males are functional.

Cobb, in the same paper, described the morphological features of the two

sexes. The writers have also found this species reproducing exclusively

by the bisexual method. No evidence of hermaphroditism or partheno-

genesis appeared, although evidence of such phenomena was sought con-

tinually

Copulation

Copulation was easily studied in the cultures and the observations

of the writers essentially confirm the brief account of Merrill and Ford

(16, pp. 125-126). Increased activity on the part of the male before

mating was noticed. A female will copulate with several different males

in a short time and the same is true of the behavior of the male towards

different females. As will be shown later, observations indicate that a

female must mate two or more times in order to produce fertile eggs

throughout the adult life.

Fertile Eggs

The normal, fertile egg of Diplogaster erivora is oblong, the average

dimensions from forty-five measurements being 0.064 mm. and 0.035

mm., the variation being within 0.007 mm. ‘The outer covering consists

of a tough, membranous coat. Eggs are always deposited singly.

Infertile Eggs

An egg, which has seemingly never been fertilized and from which a

nematode never develops, is designated as an infertile egg in these

REPRODUCTION IN CERTAIN NEMATODES 165

studies. Such eggs are larger than the fertile ones, their dimensions being

about 0.084 mm. and 0.043 mm. They are slightly variable in shape

but are usually suboval. The envelope consists of a very thin membrane

and encloses the finely granular, light colored contents. The general

appearance of such eggs is sufficiently different from that of the fertile

eggs to make it comparatively easy, with a little practice, to recognize

them at sight in the cultures.

Relation of Copulation to Egg Production

An interesting relation between copulation and the production of both

fertile and infertile eggs exists in this species. Fertile eggs were never

deposited by a female previous to the initial copulation. A female, upon

being mated, will, for a time, lay fertile eggs after which infertile ones

will be deposited until another mating occurs. Upon being mated the

second time, the female will again produce fertile eggs. For example,

female No. 12 was mated at maturity, after which the male was removed

from the cell. Twelve fertile eggs were deposited by this individual

during the next 48 hours. During the following 60 hours, 8 egg- were

extruded, all of which were infertile. After 6 days, this female was

mated a second time and as a result, within 3 hours after copulation,

fertile eggs were again deposited. Fifteen fertile eggs were laid during

the following 3 days and 2 fertile eggs were found in the body of the

female at death. Essentially, the same results were obtained in several

different individuals maintained under similar conditions. On the other

hand, female No. 88 was kept constantly exposed to males throughout

her mature life and fertile eggs were uninterruptedly deposited until her

decease. After the first mating of this female, eggs soon appeared and

the number rapidly increased until well towards the end of the 2nd day

at which time the maximum deposition of 19 eggs occurred. From this

time on, there was a gradual reduction in the number of eggs produced

until cessation occurred at the end of the 6th day, death ensuing about

24 hours later.

Occasionally, a female would completely exhaust her ability to deposit

fertile eggs before any infertile ones were produced but, in the majority

of cases, the approaching cessation of fertile egg production was indicated

not only by the reduction in the number but also in the appearance of a

mixture of fertile and infertile eggs leading to the final disappearance of

the former. After a second or subsequent mating, the same phenomenon

166 WELCH AND WEHRLE

usually occurred except in the reverse order, although, in some cases,

there was an abrupt cessation of the infertile egg production after re-

mating the female. Commonly, the maximum‘ egg production follows

the first mating, subsequent ones being followed by the deposition of a

smaller number of eggs over a shorter length of time, but this is not a

constant feature since a few exceptions appear in the records, as for

example, female No. 81 was mated 3 times within a period of 11 days

and the maximum production followed the third mating.

It thus appears that in order for the female of Diplogaster erivora to

attain her complete reproductive capacity, she must be mated at iatervals

throughout the egg producing period. One mating is insufficient but

the reasons for this insufficiency are not definitely known at present. In

all of the matings, the period of production of fertile eggs never exceeded

3 days and it may be that this represents the extent of the life of the

spermatozoa after they have been transferred to the female. There is

no reason for believing that the fertilization is other than /ysterogamic

(Lankester, ’17, p. 505) and possibly fertile eggs cease to appear when

all the spermatozoa of a single mating have been exhausted, but, if this

be the case, there must be considerable variation in the number of sper-

matozoa transferred to the female at copulation since, according to the

writers’ records, the number of fertile eggs resulting from a single mating

varied widely, as for example, initial matings resulted in 7-113 fertile

eggs. Possibly the length of copulation, which is known to vary in this

species, determines, in part at least, the number of male cells transferred.

Maupas (00, pp. 586-587; 601-602) pointed out that there is a striking

imperfection in the protandric hermaphroditic nematodes in which fertile

eggs will be produced until the supply of spermatozoa is exhausted and

then the same nematode will continue to produce infertile eggs in num-

bers 2 or 3 times greater than the fertile ones. The evidence seems to

support the contention that this condition is the result of the failure of

the ovo-testis to develop enough male cells. The infertile eggs invariably

deteriorate. He also found that the occasional males of these herma-

phroditic species sometimes fertilized the hermaphroditic individual

after it had exhausted its own supply of spermatozoa. These cases

may be comparable, if not homologous, to the condition in Diplogaster

erivora. Certainly, the disadvantage to the species is similar. Since

all of the infertile eggs of Diplogaster erivora deteriorate, the reproductive

capacity of a single female is limited greatly unless males are ever present

to fecundate her.

REPRODUCTION IN CERTAIN NEMATODES 167

VIVIPARITY

At times, another form of reproduction occurs in the life history of

this nematode in which living young appear within the body of the moth-

er. This phenomenon existed many times in the cultures and was care-

fully studied. The first indication of this change in the normal repro-

ductive procedure is the appearance of one or more very tiny nematodes

within the body of the parent and inside of the uterus. These young

move about actively and increase in size at a rapid rate. In time, they

break their way through the wall of the uterus into the body-cavity of

the mother and begin to actively attack her internal organs, soon causing

her death. The young continue to feed upon the body contents of the

dead parent until, in many cases, the whole interior is hollowed out,

leaving nothing but the transparent cuticula within which the developing

young wriggle about. From this empty parental cuticula, the young

escape to the exterior and take up an independent existence in the sur-

rounding nutrient medium. Approximately one-third to two-fifths of

the complete growth may be attained within the parent. Merrill and

Ford (716, p. 126) observed this phenomenon in the parasitic strain

of this species which they studied and they state that “Usually they were

unable to escape, although instances were observed where they escaped

through the genital pore of the mother.” They also figure a dead female

containing 14 young, all of about the same degree of development. It

might be inferred from the above quotation that few of these young are

able to complete their development, but the long observations on which

this paper is based show that, in the strain from the grasshopper eggs,

not only is the phenomenon fairly common but that the large majority

of the young so developed escapes through the genital opening of the

mother or through some rupture of her body-wall. Not only do such

young complete their growth but they are perfect individuals and capable

of reproduction. The number of young developing within the parent

is variable. The writers have records of as many as 20 appearing at about

the same time and also some evidence that even more may be so produced.

On the other hand, the number may be as low as 3 or 4. These larve

develop into both males and females and, since living young were never

observed in females which had not at some time been exposed to males,

it appears that they arise from fertilized ova and not from parthenogene-

tic ones.

168 WELCH AND WEHRLE

Reproduction of this viviparous sort has been observed before in

nematodes. Maupas (’00) found it to be a common occurrence in

Rhabditis elegans, Rhabditis caussaneli, and Diplogaster robustus in which

the eggs are not deposited as rapidly as they arrive in the uterus but tend

to accumulate there, the delay causing them to be deposited ultimately

in an advanced stage of development. Some of the young hatch in the

uterus and are expelled along with the unhatched eggs but when the sup-

ply of spermatozoa is exhausted and the infertile eggs pass into the uterus,

the young hatch, feed upon the infertile eggs accumulating there, grow,

rupture the wall of the uterus, scatter in the general cavity of the mother,

disorganize and devour the internal parts, and ultimately escape to

the exterior. Pérez (Conte, ’00b, p. 375) observed viviparity in Rhabditis

teres, Conte (’00a; ’00b) recorded it in Rhabditis monohystera and Diplo-

gaster longicauda, and Southern (’09, pp. 93-94) described the occasional

appearance of young within the body of the mother in Rhabditis brassice.

Merrill and Ford (’16, p. 120) apparently found a similar occurrence in

Diplogaster labiata in which “Occasionally a young nematode hatched

within the body of a dead female,” but no statement is made as to the

ultimate fate of such individuals.

The factors initiating and influencing the appearance of this vivi-

parity are not definitely understood. Conte (00a) found that Rhabditis

monohystera “vivipare sur colle de pate, est ovipare sur peptone” and that

the conditions of development are influenced by the nature of the nutri-

tive medium. In another paper, Conte (’00b) points out that Maupas

(99) attributes the appearance of this viviparity to two causes, inanition

and senility. Conte, however, found evidence that putrefaction in

the medium was a cause, at least in the case of Rhabditis monohystera.

“Tout en admettant avec lui que, dans certaines espéces, l’inanition et

la sénilité aménent le parasitisme embryonnaire, je crois que ce phéno-

méne peut étre provoqué par d’autres causes et notamment, ches Rhab-

ditis monohystera, pax la putréfaction du milieu. D’une fagon générale,

je pense qu’il est en relation avec un état morbide de la mére.” In

connection with his studies of the production of eggs or young in these

nematodes, he finds it possible to distinguish the following stages which

are closely related to the character of the nutritition:

Absolute oviparity—deposition of unsegmented eggs.

Relative oviparity—deposition of eggs undergoing segmentation.

' REPRODUCTION IN CERTAIN NEMATODES 169

Ovo-viviparity—deposition of gegs containing active embryos.

Viviparity—deposition of young which hatched in the uterus of the

mother.

Embryonic parasitism—consumption of morbid mother by her off-

spring.

There is certainly a tendency in Diplogaster erivora for this form of

reproduction to appear towards the end of the reproductive period and

while the parent may live and show body movements for a brief period

after young appear within her body, her reduced vitality is apparent

and is an accompanying feature, if not the causative one, of this phenom-

enon. However, the writers have evidence that age may not always

be an accompanying factor, in fact, there is circumstantial evidence that

any set of conditions which interferes seriously with the well-being of

the female may lead to the appearance of living young within her body,

even early in the reproductive period. It is therefore the opinion of the

writers that this viviparity is the result of reduced vitality of the mother

rendering her incapable of discharging the eggs. It also appears that

eggs formed early in the reproductive period may hatch within the body

as well as those produced at the end, and the writers have thus far dis-

covered no positive evidence of any inherent predisposition of the last

formed eggs for internal hatching.

There is no question that this appearance of young within the body

of the mother constitutes a form of reproduction and that the resulting

offspring are just as capable of continuing the species as those arising

from hatching outside of the body. In reality, these two forms of repro-

duction are only superficially distinct and the use of the terms oviparous,

ovo-viviparous, and viviparous is mainly one of convenience rather than

one of exact distinction. Lankester (’17, p. 505) holds that “really all

animals are viviparous, for the birth-product is a living thing whether

it is a naked egg-cell or more or less advanced in development. The

enclosure of the birth-product is a shell or case, which has given rise to

the term ‘oviparous’ is not of any value as indicating the real degree of

development of the young at birth, for in some cases unfertilized egg-

cells, in others mere discs of developing embryonic cells (as in birds, etc.),

and in yet other cases well-shaped young ranging from the early larva

of Some invertebrates up to the completely formed miniature of the adult,

as In some of the shell-bearing snails, may be enclosed within an egg shell

when ‘laid’ by the mother. There is accordingly no general importance

170 WELCH AND WEHRLE

to be attached to the distinction between ‘viviparous’ and ‘oviparous’

animals.” In this paper, these terms have been retained for the sake

of convenience.

PROPORTION OF SEXES

In the cultures studied by the writers, the total egg production of

females varied widely, the average being about 55 eggs per female. The

maximum observed was 196. In an attempt to determine the propor-

tion of the sexes and the number of eggs and young per female, eggs

selected at random were isolated, each in a separate culture. Females

resulting from these eggs were mated at regular intervals throughout

their lives. A complete daily record was kept of all eggs laid by these

females and the sex of the resulting young determined by rearing them

to maturity. Twenty-two females were studied in this way and, of the

437 resulting offspring, 182 developed into males and 291 became females.

Since it was not possible to eliminate a certain mortality among the eggs

and larvze, the numbers given above can be regarded only as a general

indication. They do show, however, that, in contrast to some of the

bisexual species, the males are very common although the females are in

the majority. These results agree, in general, with those of Merrill and

Ford (’16, p. 126).

Records from the progeny of 22 females show that in the vast majority

of cases both males and females appear in each progeny and also that in

most of the progenies, females were numerically dominant. Since all

the evidence indicates that the species is exclusively bisexual in its mode

of reproduction, a sufficient number of males is demanded to maintain

the generative processes, but since it was observed that a single male

may and often does copulate with a considerable number of females, it

seems probable that the smaller number of males indicates no important

disadvantage in the multiplication of the species. In fact, the numerical

dominance of the females coupled with fewer but sexually active males

may possibly facilitate the production of a larger number of offspring,

even if the locomotor ability is poorly developed.

Cobb (18, p. 477), in discussing the comparative rarity of males

in free-living nematodes, states that ‘“There is reason to think that in

some of the species the males are short-lived, and that this is the reason

they are so rarely seen. The males are often so much smaller than the

females that they are easily overlooked, or mistaken for young, so that in

REPRODUCTION IN CERTAIN NEMATODES 171

such cases the rarity of the males may easily be overestimated.” Since

the length of life of the males and females of Diplogaster erivora is vir-

tually the same and since the length of the female exceeds the length of

the male by only about one-fifth, it would seem that these features have

not affected the observations on this species.

RATE OF GROWTH

The young nematode, upon emerging from the egg, is active and moves

about in much the same way as the adult. Its average length at emer-

gence is about 0.238 mm. and the average diameter about 0.015 mm. A

number of specimens were carefully studied in individual cultures for

the rate of growth, all being reared under the same conditions and all

living 12-23 days after hatching. Careful measurements were made at

the time of emergence from the egg and at regular intervals of 24 hours

thereafter throughout the life of each individual. Data were secured

from 4 males and 5 females which were carried through their entire

existence, each manifesting all of the activities of a normal individual

and living for some time after growth had ceased. From the sedata,

growth curves were constructed for the increase in length and diameter.

These curves showed a striking similarity, not only in the different indi-

viduals of the same sex, but also in the individuals of the different sexes.

Furthermore, the curves for the increase in length and the increase in

diameter showed very close correspondence in every case. Growth

begins immediately at hatching and continues uninterruptedly for a

period, after which it ceases permanently. Composite graphs con-

structed on the basis of the individual growth curves showed that growth

in both length and diameter ceases, on the average, on the 8th-9th day

after hatching. In all cases except one, the variation from this average

was very slight, the exception showing no growth after the 4th day. The

average length of the males at the end of the growth period was 0.864 mm.

and the diameter 0.052 mm., while the length of the females was 1.105

mm. and the diameter 0.068 mm. The length of life of the adult follow-

ing cessation of growth varied with the individual from 6 to 15 days

inclusive.

LENGTH OF LIFE

The length of the life of this nematode is, no doubt, subject to varia-

tion under different conditions. One series of eggs kept under average

172 WELCH AND WEHRLE

temperature conditions of about 75° F., variation within 7°, required

an average of 17.9 hours from the time of oviposition to hatching,

the variation being 17-20 hours. A series of 29 individuals showed an

average larval period of 3.75 days, the variation being 1.3-8 days.

Twenty-three individuals of the same series had an average adult life

of 13 days, the variation being 5-21 days. The average life of the

individual outside of the egg was found to be about 17.1 days. Including

the egg stage, the average life was approximately 18 days. Assuming

copulation on reaching maturity, a generation can be secured in about

4.5-5 days. But very little difference was observed in the length of life

of males and females.

SUMMARY

METHODS

1. Some of the free-living and semi-parasitic nematodes can be reared

generation after generation in artificial media and their study thus

facilitated. Two species, Cephalobus dubius Maupas and Diplogaster

erivora Cobb, were cultured continuously for over three years.

2. Cylindrical glass mounting-cells sealed with pure vaseline to

ordinary microscope slides and covered with vaselined cover-glasses

were found to be the most suitable containers for cultures. Methods

of transference are described.

3. Of the numerous substances used as media, a very dilute solution

of peptone, diluted yolk of hen’s egg, and Pfeffer’s synthetic agar were

most extensively used. The eggs, ovaries, and body tissues of a number

of insects were found to be favorable when properly prepared.

4. Unfavorable cultural conditions developed in stock cultures which

were tightly sealed. Provision for ventilation was necessitated.

5. In starting cultures of Diplogaster erivora from new stock taken

from nature, mortality in the first generation was high but usually a few

survived in the new medium and subsequent maintenance then became

simple.

6. Temperatures above 80° F. are unfavorable, 90° F. and above

proving fatal. These nematodes withstand a considerable fluctuation

of temperature, e.g., 32° F. in 24 hours. Temperatures as low as 40° F.

can be withstood, at least for a limited time. Optimum temperature

conditions seem to be near 65-75° F.

REPRODUCTION IN CERTAIN NEMATODES 173

CEPHALOBUS DUBIUS

1. Observations on long continued cultures of Cephalobus dubius

involving many individuals and generations revealed no traces of males

and reproduction seems undoubtedly parthenogenetic. If males ever

appear, they must be extremely rare and develop under conditions of

culture different in some unknown respect from those employed by the

writers.

2. The egg-laying period has a variation of 6-44 days, average about

16 days. Oviposition, once initiated, continues uninterruptedly. The

daily rate increases somewhat gradually to the middle of the period and

then declines. As many as 27 eggs per day may be deposited. The

total number of eggs per individual showed a variation of 33-285, average

139. Apparently, all eggs were capable of normal development.

3. Under cultural conditions, larve emerge from eggs 2.5-4 days

after oviposition and reach maturity in 8-14 days.

4. The growth period and the larval period are not coterminal, growth

usually ceasing 3-6 days after sexual maturity, during which time an

increase in body-size of about 10 per cent. occurs. Larval growth is

continuous and approximately uniform. The average daily increase

in body-length is about 0.026 mm. Growth curves of different individ-

uals are very similar.

5. The length of life has a variation of 20-61 days. Computing from

the first eggs of a parent, a new generation can be secured in cultures

every 11-19 days.

DIPLOGASTER ZRIVORA

1. Diplogaster erivora is bisexual and males are completely functional.

One female may copulate with several males and males may behave simi-

larly towards different females.

2. Both fertile and infertile eggs are deposited. Fertile eggs follow

mating for a time, after which infertile eggs are laid until a second mating,

after which the same sequence usually occurs. Constant exposure to

males may completely prevent deposition of infertile eggs. Approaching

cessation of fertile egg production is often indicated by the appearance

of a mixture of fertile and infertile eggs. Usually, maximum oviposition

follows the first mating.

3. All of the evidence indicates that the female must be mated at

intervals throughout the egg producing period in order to fulfil her com-

174 WELCH AND WEHRLE

plete reproductive capacity. The insufficiency of one mating is not

definitely understood but it seems probable that infertile eggs appear

upon the exhaustion of the spermatozoa received from a single mating.

A similar imperfection in the reproductive ability appears in the pro-

tandric hermaphroditic nematodes and is explained by Maupas and others

as due to exhaustion of the limited supply of spermatozoa.

4. Viviparity occurs from time to time. Young appear, first within

the uterus, then later within the body-cavity of the mother, feeding upon

her internal organs, untimately causing her death. In many cases, the

interior is completely consumed, leaving nothing but the transparent

parental cuticula from which the young escape to take up independent

existence in the surrounding medium. Young so produced evidently

originate from fertilized eggs and develop into both males and females

capable of normal reproduction.

5. This form of viviparity has been observed by other workers and

certain factors have been proposed as being responsible for the appearance

of this phenomenon, namely, inanition and senility (Maupas), and putre-

faction of the medium (Conte). The writers have incomplete evidence

that it is the result of any factor or group of factors which reduce the

vitality of the mother. Definite evidence was secured to show that,

at least in Diplogasier erivora, age is not always an accompanying feature

but viviparity may appear at any time during the egg-laying period.

6. Females are numerically dominant, both in mass populations and

in single progenies. Males, however, are common and very rarely

absent, even in a single generation. Although fertilization is evidently

necessary for any reproduction, it is probable that the smaller number

of males is compensated for by their constant presence and their ability

to copulate with several females.

7. Growth curves based upon daily measurements of length and

diameter are strikingly similar for all individuals, irrespective of sex.

Composite graphs showed the cessation of growth occurred on the 8th-

9th day. The length of life following cessation of growth has a variation

of 6-15 days..

8. The average length of life under cultural conditions is about 18

days. A new generation can be secured every 4.5-5 days. No marked

difference in the length of life of males and females was observed.

REPRODUCTION IN CERTAIN NEMATODES 175

LITERATURE CITED

Byars, L. P.

1914. Preliminary Notes on the Cultivation of the Plant Parasitic Nematode,

Heterodera radicicola.

Phytopathology, 4:323-327.

Cobb, N. A.

1915. Proceedings of the Helminthological Society of Washington.

Journ. Parasit., 2:93-95.

1916. Proceedings of the Helminthological Society of Washington.

Journ. Parasit., 2:195-200.

1918. Free-living Nematodes. Fresh-water Biology, by Ward, H. B., and

Whipple, G. G., pp. 459-505.

Conte, M. A.

1900a. De J’influence du milieu nutritif sur le développement des Nématodes

libres.

C. R. Soc. Biol., 52:374-375.

1900b. Sur les conditions de ponte des Nématodes.

C. R. Soc. Biol., 52:375-376.

Johnson, G. E.

1913. On the Nematodes of the Common Earthworm.

Quart. Journ. Micr. Sci., 58:605-652.

Lankester, E. R.

1917. The Terminology of Parthenogenesis.

Nature, 99:504-505.

Martin, A.

1913. Recherches sur les conditions du développement embryonnaire des Néma-

todes parasites.

Ann. Sci. Nat., Zool., (9), 18:1-151.

Maupas, E.

1899. La mue et l’enkystement ches les Nématodes.

Arch. Zool. exp., (3), 7:563-628. 3 pls.

1900. Modes et formes de reproduction des Nématodes.

Arch. Zool. exp., (3), 8:463-624. 11 pls.

Merrill, J. H., and Ford, A. L.

1916. Life History and Habits of Two New Nematodes Parasitic on Insects.

Journ. Agr. Research, 6:115-127.

Metcalf, H.

1903. Cultural Studies of a Nematode Associated with Plant Decay.

Trans. Am. Micr. Soc., 24:89-102.

Oliver, W. W.

1912. The Cultivation of an Ectoparasitic Nematode of a Guinea Pig on Bac-

teriologic Media.

Science, (n.s.), 36:800-801.

176 WELCH AND WEHRLE

Osborn, H.

1898. The Hessian Fly in the United States.

Division of Entomology, U. S. Dept. Agr., Bull. 16,n. ser. S8pp. 2 pls.

Potts, F. A.

1910. Notes on the Free-living Nematodes.

Quart. Journ. Micr. Sci., 55:433-484.

Southern, R.

1909. On the Anatomy and Life-history of Rhabditis brassice, n. sp.

Journ. Econ. Biol., 4:91-95. 1 pl.

A NEW DIATOM-EATING FLAGELLATE 177

A NEW AND REMARKABLE DIATOM-EATING FLAGELLATE,

JENNINGSIA DIATOMOPHAGA NOV. GEN., NOV. SPEC.

AsA. A. SCHAEFFER

Diagnosis. Shape, cylindrical, 180 microns long by 40 microns in diameter;

very metabolic. Flagellum, large, 150 microns long. Cuticula with spiral striations

1 to 2 microns apart; numerous movable club shaped appendages about one and one-

half microns long on the striations. Large central nucleus, 35 microns in diameter.

Large contractile vacuole near the anterior end. Several rod-like structures in the

pharynx immediately internal to a large mouth at the anterior end. Numerous bodies

in the form of rings or strongly biconcave discs from 1 to 3 microns in diameter, in the

endoplasm; large clear spheres up to 6 microns in diameter sometimes present. Loco-

motion, creeping. Food, exclusively diatoms. Reproduction, asexually, by longi-

tudinal fission. Habitat, marshes, among algae and diatoms.

This remarkable flagellate has come under my observation on three

different occasions: February 27 and March 31, 1916, and March 5, 1918.

In the three instances, material was collected from the Lonsdale marshes

near Knoxville, and allowed to stand in vessels in the laboratory for

several months. The organism may be considered rare, for frequent

examinations of material from these marshes during the past five or six

years has revealed its presence only three times.

This flagellate does not occur in large numbers, not more than a few

hundred being found at any one time. And their length of life in the

culture is as short as their number is small. In none of the three cases

mentioned did they remain in the culture for more than a week. Its

rarity, and the difficulty with which it may be cultured are unfortunate

from the point of view of the biologist, for its predatory animal nature

stands out in striking contrast to the plant-like character of some of its

immediate relatives.

I propose for this organism the generic name Jenningsia, in honor

of my friend Professor H. S. Jennings, and the specific name diatom-

ophaga.

Jenningsia is one of the largest members of the flagellates. Although

the average length is about 180 microns, several individuals were found

which measured 260 microns, exclusive of the flagellum, which in these

individuals measured 170 microns, and in the smaller individuals, 150

microns. The shape of the body is cylindrical, with slightly tapering

anterior end and very blunt but also slightly tapering posterior end. The

178 A. A. SCHAEFFER

flagellum is very stout and during locomotion is directed straight ahead,

only the apical part being usually in active motion (figs. 1, 2).

In the general character of its movements this organism resembles

peranema. Locomotion is accomplished only by creeping, the organism

being incapable of swimming. Violent metabolic movements accompany

locomotion especially when an obstacle is encountered in its path. Only

rarely is the organism stretched out straight. When the direction of

locomotion is changed or when a strong stimulus is received from another

organism the shape assumed during locomotion is entirely lost in a very

violent twisting and kneading movement, which soon, however, gives way

again to the cylindrical shape characteristic of locomotion. A very slight

stimulus is sufficient to cause very marked metabolic movements.

The exact manner in which locomotion is brought about could not be:

ascertained satisfactorily, but it was observed that the tip of the flagellum

was habitually bent in the form of a small loop which was used somewhat

in the manner of a paddle so as to pull the organism along. During

locomotion, it is worthy of note, the organism rolls over frequently

though not regularly, differing in this respect from the peranemas.

Externally, Jenningsia is radially symmetrical like the euglenas.

The body is covered with a thin cuticle which is marked with spiral

striations that take their origin in the mouth. Small slender spindle-

shaped structures about one and one-half microns in length, which may

readily swing about in the water, are attached to the striations at irregular

intervals. These cuticular spindles are most numerous near the extreme

anterior end.

Internally there are found: a large central oval nucleus about 35

microns in diameter with a central denser body about 15 microns in dia-

meter; a complicated contractile vacuole system (similar to that found

in euglenas) near the anterior end, which functions several times a minute;

a complicated pharynx provided with stiff rod-like elements; and num-

erous strongly biconcave discs or rings of clear bluish green material,

ranging from one to three microns in diameter. One or more large

vacuoles are also occasionally found in the posterior half of the organism.

Of the structures just enumerated the pharynx is of especial interest

although it is very difficult to understand its detailed operation while

feeding. In the moving organism one can see two rod-like elements

lying with their anterior ends in a clear space free from protoplasm and

A NEW DIATOM-EATING FLAGELLATE 179

protected by a strong arched structure on the anterior side, the arch

representing the lips of the closed mouth. When the animal is com-

pressed under the cover glass the mouth is forced open and three large

rods are now seen which are forced partly outwards (fig. 3). In this

process some of the small rod-like elements disappear, which may indicate

that these apparent rods are only folds of cuticle occasioned by the

closure of the mouth and the retraction of the pharynx. Although I

have seen the animal devour diatoms on several occasions I have been

quite unable to determine exactly how the rods of the pharynx operate

during this process.

Another element of interest are the numerous rings or discs scattered

throughout the endoplasm (figs. 1, 4). They are of all sizes as stated

above and are of a clear but pale bluish green color. They are unaffected

by the ordinary stains or iodine but dissolve without visible change in

solutions of H2SO,. In their general appearance, number, and behavior

towards reagents they are similar to the crystals in amebas and may

possibly be formed in a similar manner. They are not active under the

polariscope. It is probable that these rings or discs are some by-product

of metabolism.

In one culture of few but large individuals, numerous spherical bodies

were observed which stained deeply with haematein. No food was

observed in any of these individuals.

The most interesting thing about this organism from a general point

of view is its mode of nutrition. Itis completely holozoic. As indicated

by its proposed specific name, its food is living diatoms of all sizes up to

100 microns in length. I have examined in all several hundred of these

organisms and nearly all of them had from one to five diatoms, mostly

of medium size, in them. No other food objects have been observed.

The feeding process is very difficult to observe in detail, owing to the

violent metabolic movements, the activity of the complicated pharynx,

and the speed with which the food is devoured. The organism moves

along with the tip of the flagellum moving about until it comes into

contact with a diatom. If hungry, the flagellum is brought into contact

with the diatom as much as possible while the animal continues with its

forward movement. When the anterior end of the organism comes

nearly into contact with the diatom, the posterior end rears up and vio-

lent metabolic movements set in. The anterior end is brought over the

180 A. A. SCHAEFFER

diatom and is seen to spread out. The basal part of the flagellum is

also seen to move about as if it had a part in the actual swallowing of the

food particle. Presently the diatom is seen inside the flagellate, which

moves away within a few seconds, the whole process of feeding taking

place within about 20 seconds. Although I saw a number of instances

of feeding and took particular pains to see whether the pharyngeal rods

were actually protruded, or were used merely in distending the mouth, I

was unable to determine their exact function. I incline to think how-

ever, that the rods were not protruded beyond the mouth opening.

On several occasions I tested their reactions to carmine particles without

obtaining positive responses, although one specimen so tested later

devoured a diatom. All the evidence therefore indicates that Jenningsia

is a predacious animal that feeds exclusively on living diatoms.

I observed but one instance of reproduction—asexual—which was

accomplished by longitudinal splitting, beginning at the anterior end and

extending backwards. One of the daughters inherits the old flagellum,

the other grows a new one during division.

Jenningsia affords a very good example of the ease with which fun-

damental instincts and habits may be changed in phylogeny, for it is an

animal descended from plants. The exclusively predacious instincts of

this animal contrast strongly with the true holophytic mode of nutrition

of some of the closely related family of euglenas. In so far as the actual

process of feeding is concerned we have exhibited within the span of the

single order Euglenineae all the general methods of nutrition known

among nucleated organisms: true holophytic nutrition by means of

chlorophyll among euglenas; saprophytic, the ingestion of decomposing

nitrogenous foods either liquid (astasias) or solid (peranemas); holozoic,

ingestion of carbohydrate or protein materials—pieces of animal or

vegetal tissues (peranema); holozoic, ingestion of small masses of bacteria

or pieces of animal and vegetal tissues and on living protophyta (dinema);

holozoic, predacious, injestion only of living, moving organisms that,

in a real sense, have to be captured (Jenningsia). No better illustration

than this could be found of the combined plant and animal characters

of the Flagellata.

The change from a holophytic to a holozoic type of nutrition in the

Euglenineae has been made possible by the development of the pharynx.

In the euglenas the pharynx is a simple tube-like depression at the

anterior end whose chief function seems to be that of an efferent drainage

A NEW DIATOM-EATING FLAGELLATE 181

canal for the contractile vacuole. But it also serves as a means of taking

into the body small solid particles as has been shown by placing carmine

in the culture fluid. The taking in of solid matter is however not an

essential function for the euglena, but it is of interest in that it fore-

shadows saprophytic and holozoic instincts and modes of nutrition in the

so-called colorless euglenas—the astasias—and in the peranemas.

The astasias have gone a step further than the euglenas. They have

developed a larger pharynx and, we may suppose, one better adapted to

taking in liquidfood. Atthe same time they have also lost the chloro-

phyll from their bodies.

The peranemas have likewise gone a step further than the astasias in

the development of the pharynx. In these organisms the pharynx is

provided with special rods which make it possible to open and close the

pharynx, and also to act somewhat like a suction apparatus by means of

which solid matter may be eaten with despatch. Most of these organ-

isms are small, so that they are restricted in their food to bacteria or small

pieces of disintegrating organisms. In Jenningsia we have however as

a most important development, a large size, so that it is possible for it

to feed on such large actively moving organisms as diatoms in a truly

predatory manner.

_ In company therefore with a progressively developing tendency in

these organisms from holophytic to holozoic nutrition in a physiological

sense, we have also, on the morphological side, a progressive develop-

ment of the pharynx and of the size of the organism which makes possible

the capturing and eating of relatively large masses of solid food and

actively moving organisms.

University of Tenn.,

Knoxville, Tenn.

182 A. A, SCHAEFFER

EXPLANATION OF PLATE

Fig. 1. Jenningsia diatomophaga in locomotion. Body length, 180 microns,

flagellum length, 150 microns, cv, contractile vacuole; d, ingested diatoms; e, excretion

bodies; m; mouth; n, nucleus; p, pharynx; r, pharyngeal rods; v, vacuole.

Fig. 2. Photomicrograph of living J. diatomophaga in locomotion. The dark

masses represent ingested diatoms. The basal part of the flagellum is seen at the

anterior end.

Fig. 3. Sketch of anterior end compressed under cover glass to force open the

mouth, m. s, cuticular striations originating in the mouth and running spirally around

the organism; r, the pharyngeal rods.

Fig. 4. Enlarged view of “excretion” discs. a, top view; b, cross section.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

SOCIETY VOL. XXXVII

Fic. 4

PLATE XIII SCHAEFFER

STUDIES ON AMERICAN STEPHANOPHIALINAE*

With Two Plates

ERNEST CARROLL FAUST

Since the time of O. F. Miiller helminthologists have recognized

certain distomes which are characterized by a coronet of papillae around

the oral opening. These were first classed as Armata, which group in-

cluded also all echinostome species. The more modern studies of Looss

and Odhner have shown that certain of these species are related to the

Allocreadiidae. With Bunodera luciopercae (nodulosa) as the type Looss

(1902:453) proposed the subfamily Bunoderinae for the then known

forms, Crepidostomum metoecus, C. farionis (laureatum), Distomum petalo-

sum and Bunodera luciopercae. However, the enormous extent of the

uterus crowding the entire posterior part of the body in the case of

Bunodera and the scanty coiling of the uterus entirely anterior to the

anterior testis in the case of the other species constitutes so marked

a difference that the latter species were necessarily removed from the

Bunoderinae. Odhner (1905, 1910) placed them in the Allocreadiinae.

Nicoll (1909) showed that they were more closely related to the Allo-

creadiinae than to the Bunoderinae, but that certain differences possessed

by the entire group justified the creation of a new subfamily, Stephano-

phialinae. The writer’s study of the American representatives of this

group stands in support of Nicoll’s thesis, and contributes certain data

which mark out this subfamily more definitely.

The following description supplements Nicoll’s original diagnosis.

The integument is usually aspinose, but in the case of a few representa-

tives of Stephaniphiala farionis (fig. 3) spines are found around the oral

sucker. The circumoral papillae are always six in number, altho there

is a tendency for the dorsal papillae of Crepidostomum spp. to bifurcate.

The yolk glands in this subfamily extend from the region of the pharynx

to the posterior end of the body. Moreover, the excretory bladder

extends to the anterior border of the anterior testis, whereas in the Buno-

derinae it is a small pouch posterior to the testes. In the Allocreadiinae

as at present constituted there is no common type of excretory bladder.

In the genus Allocreadium it terminates behind the anterior testis.

*Contributions from the Zoological Laboratory of the University of Illinois

No. 115.

184 E. C. FAUST

There are recognizable in the subfamily Stephanophialinae three

genera, Stephanophiala, Crepidostomum and Acrolichanus. Stephano-

phiala contains the cosmopolitan species, S. farionis (lawreatum), and

a new species, S. vitelloba. Crepidostomum contains the type, C. metoe-

cus, C. cornutum and a new species C. illinoiense. Acrolichanus is mono-

typic, being represented by A. petalosa. The type species of the Buno-

derinae, B. luciopercae, has been found in the North American perch,

Perca flavescens, by Stafford (1904:489) and also by Lander.

The writer is indebted to Professor Henry B. Ward for valuable data

and material, and to Professor Edwin Linton, Professor Henry L. Osborn,

Dr. Arthur R. Cooper and the Bureau of Animal Industry of Washington

for the loan of material.

Stephano phiala Nicoll 1909

This genus was created by Nicoll in 1909 to separate Crepidostomum

farionis of Miller (laureatum Zeder) from C. metoecus. The separation

was made on the basis of 1) disposition and number of the oral papillae,

2) position of the genital pore, 3) size of the cirrus pouch, and 4) extent

of the uterus with size and number of the ova. These are the characters

which require that Crepidostomum farionis be placed in a separate genus

from C. metoecus, but Nicoll’s definition of Stephanophiala is obviously

obscure and demands more precision on the one hand and more flexibility

on the other.

Redesignation of Stephanophiala. Minute to medium-sized Allo-

cread species with six subequal rounded papillae encircling the oral sucker

laterally and dorsally. Prepharynx and esophagus short as in the entire

subfamily; pharynx about one-half size of the oral sucker. Forking of

the gut a little distance in front of acetabulum. Testes median tandem,

larger or smaller than acetabulum. Ovary lateral or median. Uterus

composed of a few coils between anterior testis and acetabulum, with

the vagina leading directly to the genital aperture. Cirrus pouch a mus-

cular S-shaped sac which may lie entirely dorsal to the acetabulum or

may extend a short distance behind, but never extending as far caudad

as in Crepidostomum. Genital pore always anterior to the forking of

the gut. Vitellaria marginal, somewhat dorsal, from the pharynx to.

posterior end but usually encroaching on the middle field in the region

of the testes and farther caudad. Eggs few to many, averaging about

75 in length by 45y in trans-section.

STUDIES ON AMERICAN STEPHANOPHIALINAE 185

In this genus are included Stephanophiala farionis (O.F.M.) and

new species, S. vitelloba, from the Bitter Root Valley, Montana.

Stephano phiala farionis (O. F. Miiller)

This species was first described by O. F. Miiller in 1788. It has

passed thru a vicissitude of generic names including Distoma, Distoma

(Crossodera), Crossodera, Lobostome, and Crepidostomum. It has

more frequently been known as /aureatum of Zeder than as farionis, altho

the former specific designation was not applied until some twelve years

after Miiller’s name fartonis was proposed. In the Old World the species

has been described from Central Europe, Scandinavia, and England.

In the Western Hemisphere it has been reported from Canada by Stafford

(1904) and Cooper (1915) and from Yellowstone National Park by

Linton (1893). Several Salmonidae and Percidae have been described

as host of this species.

The writer’s material for the study of this species in North America

consists of parasites of Salmo mykiss lewisii, (Gir.) collected by Linton

from Yellowstone National Park and described by him (1893) and, in

addition, material collected by Dr. B. H. Ransom, from Park Co.,

Montana, in 1904, and lent by the Bureau of Animal Industry, Washing-

ton, D.C. The study of the New World material shows it to belong to

the species S. farionis, but it shows likewise that the species is widely

variable in several characters.

Detailed descriptions of the worm have been given by Olsson (1878:

24), Blanchard (1891:481) and Nicoll (1909:425) and need not be con-

sidered here. However, a summary of the constancy and variability

of certain characters may well have a place in this paper. In length the

mature worm varies from 1.2 mm. (Ransom material) to 6 mm. (maxi-

mum record of Olsson). The ventral sucker is always larger than the

oral sucker, altho no constant ratio has been found. The fluke has a

short prepharynx, a small pharynx and an esophagus shorter than the

pharynx. The gut forks a little distance anterior to the acetabulum.

The excretory bladder extends to the anterior border of the anterior

testis. The testes are median tandem, somewhat larger than the aceta-

bulum. Anterior to them is the ovary, which may be lateral or median

in position. Most variable of all is the size and extent of the cirrus pouch.

Its posterior limit in some individuals may be near the center of the

acetabulum as Nicoll has found in his material, but in other specimens

186 E. C. FAUST

it extends behind the posterior margin of the acetabulum. In all cases,

however, its internal structure is the same, and the anterior end always

extends anterior to the genital pore. The amount of coiling of the

uterus is variable. The eggs vary in number from few to about one hun-

dred. ‘They vary in size from 62 to 82y in length and 40 to 59y in cross

section.

The papillose Allocread species have been uniformly described as

aspinose. A small per cent of specimens of the Ransom collection have

spines on the integument in the region dorsal and lateral to the oral

sucker (fig. 3). They are so definite in character as to leave no room

for uncertainty. The small number of individuals of this collection so

armed makes it inadvisable to consider them as a distinct variety.

The recognition of the American specimens, especially those of Linton

and Ransom as Stephanophiala farionis shows the wide distribution of

this species. The name S. transmarina proposed by Nicoll (1909) for

the American specimens of this species consequently becomes untenable

and is to be considered as a synonym.

Stephanophiala vitelloba nov. spec.

Stephanophiala vitelloba was taken from the gall bladder of a single

specimen of Coregonus williamsoni Gir. among several dozen examined

from the Bitter Root River, at Fort Missoula, Montana, Feb. 18, 1916.

Ten specimens were found at the head of the gall duct.

The worm is quite cylindrical, but tapers in an ovoid fashion at the

posterior end. At the anterior end there is a slight bending ventrad,

so that the oral sucker is directed anteroventrad. Topping the dorsal

portion of the oral sucker are six subequal blunt papillae, about 30 yu

wide and 20 » in depth. About one-third of the distance caudad on the

ventral side the acetabulum bulges out as a truncate cone. The fluke

is 1.11 to 1.4 mm. long by 0.277 mm. wide at the middle of the body.

The oral sucker measures 0.148 mm. in trans-section and the acetabulum,

0.176 to 0.185 mm. The integument is free from spines but has small

wartose prominences.

The oral atrium leads thru a very short prepharynx into a barrel-

shaped pharynx, 74 pw in diameter and about 100 u in length. At the

posterior end of the pharynx is a short esophagus, behind which the

digestive tube bifurcates, each cecum clasping the anterior face of the

acetabulum and then running caudad to a position near the posterior

STUDIES ON AMERICAN STEPHANOPHIALINAE 187

end of the worm. The wall of each cecum is composed of about eight

to ten cells in cross section, with rounded walls lining the lumen of the

pouch (fig. 11, ce). The cecum is oval in cross section, about 35 yw in

frontal diameter and 64 u along the sagittal plane. The lumen is scarcely

as large as the cells of the cecum wall. Each cell is filled with fine

granular material and has an oval nucleus 6 yw in section. The general

appearance of the cell is vesicular, altho no vacuoles have been found.

The excretory pore is terminal. From it, extending cephalad, near

the dorsal face is a long sacculate bladder. It is slightly pouched at the

posterior end but soon becomes distinctly tubular. It continues for-

ward as a single tube to the region of the ootype where it bifurcates,

sending forward two lateral vessels. Beyond that point the system has

not been traced. The bladder has no granular inclusions.

The male genital organs consist of two testes, efferent ducts, muscular

cirrus and seminal vesicle. The testes are rounded glands lying one

behind the other on the ventral side of the body at the beginning of the

posterior half of the body. They measure about 100 uw in diameter.

Each testis (fig. 11) contains a great number of spherical cells, arranged

almost exactly in concentric rows. The cells are about 6 yw in section.

Toward the center of each gland are found various stages of maturation

up to spermatids and spermatozoa. The maturing cells occur in large

aggregates, so that they form large clumps of densely staining bodies

in the gonocoel. From the outer margin of each testis at the anterior

end a vas deferens arises. The two efferent ducts proceed separately to

the anterior end of the ovary in which region they merge into the pos-_

terior end of the cirrus sac. The cirrus is long and muscular. It bends

on itself near the middle of the acetabulum and proceeds again forward

in the region of the left cecum, ending at the genital pore, anterior to the

forking of the ceca. The portion which constitutes the seminal vesicle

is located at the S-bend in the sac. Prostate glands occur in the anterior

half of the cirrus pouch.

The female genitalia consist of ovary, oviduct, seminal receptacle,

Laurer’s canal, vitellaria and vitelline ducts, shell gland and uterus.

The ovary lies just anterior to the anterior testis, overlapping it in part.

It is spherical, lying slightly to the side of the mid-plane (figs. 5, 12, 0)

and measures 110 to 120 win diameter. A coil of the uterus lies below it.

The immature ovarian cells are similar to those of the testes, but some-

what smaller. The short oviduct proceeds ventrad (fig. 8) where it

188 E. C. FAUST

opens into the ootype. On the left of the ootype is a pyriform recep-

taculum seminalis and branching from its neck is a tubular sac, Laurer’s

canal, directed dorsad. It ends blindly near the dorsal wall. On the

right hand side is a clump of cells, eight or ten in number, with deeply

staining cytoplasm and small spherical nuclei. These constitute the shell

glands of authors. Extending lateral right and left from the ootype are

the vitelline ducts. Their plane practically divides the animal into equal

halves. The vitellaria are the most characteristic features of the fluke.

Lateral to the ceca they extend from the extreme anterior to the extreme

posterior ends. At the posterior end they bend forward inside the ceca,

extending almost to the ootype. In section they appear as distinct

chorda, the outer series large and at times divided into branches, while

the smaller inner glands constitute a single stem. Each chordum in

section is composed of aggregates of vesicular cells of irregular polygonal

outline and large spherical nuclei which display great numbers of mitotic

figures. The entire cell is rich in chromatic materials.

From the ootype the uterus coils forward, opening to the right of the

cirrus sac. It contains from four to eight eggs. The eggs are oval, with

a lemon colored shell and operculum at one end. They measure about

77 » in length by 42 uw in trans-section. The egg is filled with granular

cytoplasm and has, in addition, from twelve to twenty-four ovate vitel-

line cells.

Crepidostomum Braun 1900

This genus was created by Braun (1900) to include the species Dis-

tomum farionis (laureatum) and D. metoecus, designating papillose Allo-

creadine species, which unlike Bunodera, have the uterus confined anter-

ior to the anterior testis. The discovery of other species related to these

shows that these Old World species are to be regarded as types of distinct

genera. The present study shows the close relationship of Crepidostomum

metoecus, C. cornutum, and C. illinoiense. On the basis of a study of

these three species a redesignation of the genus follows.

Redesignation of Crepidostomum. Minute to inframedium aspinose

Allocreadine species with six oral papillae, of which the ventral pair con-

sist of laterally extending muscular processes which taper to an acute

point. Dorsal papillae showing a tendency toward bifurcation. Short

prepharynx and esophagus; pharynx minute. Gut forking occurs a

little or a considerable distance in front of acetabulum, depending on

STUDIES ON AMERICAN STEPHANOPHIALINAE 189

expansion or contraction of prepharynx and esophagus. Excretory

bladder extending to anterior border of anterior testis. Testes median

tandem. Ovary lateral or median, just anterior to testes. Vitellaria

from pharynx to posterior end. Vitelline follicles sparsely scattered

anterior to ootype. Cirrus sac large, muscular, often convoluted, ex-

tending from ootype to front of acetabulum. Genital pore ventral or

posterior to forking of gut. Uterus consisting of several coils anterior

to anterior testis. Eggs one to several, varying in size from 55 by 40.9 p

for C. metoecus to 70 by 41 w for C. cornutum. Found in various fishes in

North America and in Vespertilio lasiopterus in Europe. Young of

American species have been found in Cambarus spp.

Crepidostomum cornutum (Osborn) 1903

This species, originally designated as Bunodera cornuta, was de-

scribed from material from Micropterus dolomieu, Ambloplites rupestris,

and Ameiurus nebulosus, taken from Chautauqua Lake, New York.

It has since been found in Canada by Stafford (1904:490) in the distomu-

lum stage in Cambarus sp. and by Cooper (1915:193) as adults in Microp-

terus dolomieu, Ambloplites rupestris, and Amieurus lacustris and in

the distomulum stage in Cambarus spp.

The material on which the original description is based (Osborn 1903)

bears evidence of being not one species but three species, namely, a

Crepidostomum species which must be regarded as the cornutum type,

Acrolichanus petalosa (Lander), and a Bunoderan species, probably

luciopercae. Specimens from Professor Osborn which the writer has

been enabled to examine consist of Crepidostomum cornutum and Acro-

lichanus petalosa. The fact that the cornutum individuals have a muscu-

lar cirrus sac and a short uterus coil never encroaching on the territory

of the testes or farther caudad precludes any possibility of regarding

them as Bunoderans. While figures 1 to 6 of Osborn’s paper are ac-

ceptable as representatives of C. cornutum, fig. 7 is distinctly a Bunoderan.

Thus it becomes necessary to redescribe C. cornutum and limit the type

more definitely.

The size of C. cornutum is variable. Its length ranges from 0.9 to

3.0 mm. and its width from 0.2 to0.9 mm. While it is slightly narrower

at the posterior end than in the region of the acetabulum, the tapering

is so gradual as to be almost inconspicuous. The oral sucker is commonly

as wide as the body or even wider, its diameter varying from 0.33 to

190 E. C. FAUST

0.46 mm. The acetabulum is considerably smaller, from 0.15 to 0.31

mm. in trans-section. It lies in the anterior quarter of the body.

Of the six papillae crowning the oral sucker the ventral pair are

lateral extentsions of the sucker itself, giving the appearance on the

whole of turned flanges (fig. 14). The four remaining papillae are equal

in size and inconspicuous in detail.

The orifice leads thru a short prepharynx into a small oval pharynx

about 50 in cross section. Behind the pharynx is a short esophagus

of equal length. The digestive tract usually bifurcates some little dis-

tance in front of the acetabulum. Long tubular ceca extend to near the

caudal extremity of the body.

The excretory system as far as it has been made out consists of a long

bladder arising from a caudal excretory pore and ending near the anterior

border of the anterior testis. The tubules have not been studied.

Turning to the genital organs, large testes, capable of considerable

elongation or widening, lie tandem in the posterior half of the body.

Osborn (1903:69-71) has described slender vasa deferentia which run

from the testes along the dorsal side of the fluke, merging into a single

duct at the base of the cirrus sac (fig. 6). From ventral aspect the ovary

is seen sometimes on the right, sometimes on the left. It is oval and

considerably larger than the receptaculum seminalis. The ootype is

located in about the center of the body. Its relation to the female

genital organs is shown in Osborn’s figure 6. The vitellaria are situated

in rather definite chorda, closely surrounding the ceca on the sides.

Anterior to the ootype they are composed of minute follicles. They

usually extend from the pharynx to the posterior end of the body. They

do not encroach on the median field as do the follicles in C. illinoiense.

The uterus in the mature worm consists of several coils anterior to

the anterior testis, with a terminal portion directed forward over the

acetabulum toward the genital pore. The genital pore is ventral or

slightly posterior to the bifurcation of the gut. Osborn (1903:72) has

found its anterior end to be muscular. The eggs of C. cornutum are

not nunerous; they range up to about twenty. They measure 65 to

71 uw in length by 414 in cross diameter. The cirrus sac is a long coiled

muscular organ arising in the region of the ootype. At times its con-

volutions separate the ovary from the receptaculum seminalis. The

STUDIES ON AMERICAN STEPHANOPHIALINAE 191

writer has found both the vesicle and the ductus ejaculatorius to be ex-

tensively muscular. As is commonly found in the Stephanophialinae

the ductus is surrounded by prostate glands.

Crepidostomum illinoiense nov. spec.

This minute fluke was taken from the intestine of the crappie, Pomo-

xis sparaides (Lac.), at Havana, Illinois, July 11, 1910, by Dr. H. J.

Van Cleave for Professor Henry B. Ward, to whom the writer is indebted

for the material. A large number of specimens were secured from the

intestine of the host.

The worm is elongate in outline, with a greatest width of 0.15 to

0.18 mm. in the region of the acetabulum, posterior to which it gradually

tapers to a distinctly conical end. The acetabulum measures from 76

to 88u in trans-section, while the oral sucker is almost twice as large.

Crowning the oral sucker is a cluster of six papillae, two ventral, two

dorsolateral and two distinctly dorsal (fig. 17). The ventral papillae

emerge from the sides of the posterior margin of the oral sucker, but are

always intimately connected with an anterior folding of the sucker. Each

papilla extends laterad about i5ythen is flexed dorsad some 5 to 7y,

terminating in a distinct point. The dorsolateral papillae are triangular

with rounded corners. They extend forward and dorsad. The dorsal

papillae lie directly above the orifice. They are separated from one

another by an inconspicuous sinus. Each dorsal papilla is bifurcate,

altho the notch is not deep. Altho there is no fundamental muscular

connection between the dorsal and the dorso-lateral papillae, folds of the

integument stretch across the intervening notch, giving the appearance

superficially of a sympapillose condition. The body is unarmed.

The mouth opens thru a short prepharynx into a spherical pharynx

of 26 diameter. Behind this organ is an esophagus of equal length.

The bifurcation of the digestive tract occurs just a little anterior to the

acetabulum. ‘The ceca are long attenuate tubes reaching to the extreme

posterior limits of the worm.

The excretory system (fig. 19) was found only in sections. The pore

is caudal in position. For a short distance forward the bladder is muscu-

lar, but as it bends dorsad its lining is entirely parenchymatous. The

tube gradually becomes more and more attenuate in the vicinity of the

testes and ends just dorsal to the anterior wall of the anterior testis.

192 E. C. FAUST

The ovary is a medium-sized reniform body lying just behind the

acetabulum. In ventral view it is covered on the right side by the cirrus

pouch and on the left by the uterus. In a median line just behind the

ovary lies the ootype, and on the right, posterior to the ootype, is a large

pyriform receptaculum seminalis. The testes lie tandem in the third

quarter of the body. The anterior one is sometimes compressed longi-

tudinally so that it reaches laterad almost to the ceca. The cirrus pouch

is an extremely long muscular organ, originating some distance posterior

to the ootype and extending forward over the acetabulum to terminate

at the genital pore, ventral to the forking of the gut. It is capable of

considerable eversion. The vitelline glands at the margins of the body

extend from pharynx to posterior end of the body. Anterior to the

acetabulum they are few and small. In the region of the testes they

encroach on the median organs and fill the entire dorsal portion of the

worm posterior to the testes. The uterus is an uncoiled tube which runs

directly to the genital pore. The eggs occur singly or at most in pairs.

The mature egg measures 63 by 40 yw. The shell of the immature egg

is colorless, while the yolk follicles are yellow; the shell of the mature

egg is a dark golden yellow, concealing the color of the yolk material.

Acrolichanus Ward 1918

The name Acrolichanus was substituted by Ward (1918:396) to re-

place Acrodactyla of Stafford 1904, preoccupied. The genus at the

present time includes a single species, first described by Looss (1902:454)

as Distomum petalosum Lander. The type material was secured from

the Lake sturgeon, Acipenser rubicundus Le S., from the vicinity of Ann

Arbor, Michigan. The species has also been found by Stafford (1904)

and Cooper (1915:194, 195) in the same host in Canadian waters. It is

highly probable that Distomum auriculatum Wedl (?) of Linton (1898:

491) and Bunodera lintoni Pratt (Linton 1901:435) are synonyms of

Acrolichanus petalosa. The type D. auriculatum Wed), described from

Europe as a parasite of Acipenser ruthendus (Wedl 1857:242, 243) is

so inadequately described that it seems unwise to give it a systematic

position.

Redesignation of Acrolichanus. Inframedium Stephaliphialine spe-

cies with six oral papillae, of which the ventral pair is draped over the

anterior end of the oral sucker. Excretory bladder dilated with con--

spicuous constriction at posterior end. ‘Testes median tandem or slightly

STUDIES ON AMERICAN STEPHANOPHIALINAE 193

oblique. Ovary close behind acetabulum; vitellaria composed of sparse-

ly scattered follicles. Cirrus sac ending in a large conspicuous sphincter.

Acrolichanus petalosa (Lander) 1902

Acrolichanus petalosa has been referred to frequently in the literature

but has never been adequately described. The writer has had access

to material and records of this species from the following sources: 1)

original drawings of Lander’s type material, 2) drawings made by Pro-

fessor Henry B. Ward of material secured from Cambarus sp., at Ann

Arbor, Mich., 1893 (Ward 1894:180), 3) material on which Cooper’s

record (1915) is based, and 4) specimens of Osborn’s material from

Ambloplites rupestris.

A. petalosa is inframedium in size, averaging from 1.5 to 2.5 mm. in

length and 0.32 to 0.54 mm. in width. The acetabulum lies at the

posterior limit of the anterior third of the body. It measures 0.16 to

0.32 mm. in cross section. The oral sucker measures from 0.27 to 0.45

mm. The body is aspinose. The disposition of the oral papillae as

seen from the front and below is strikingly similar to a notched geranium

leaf. The six lobes are practically equal; the ventrals are directed caudad

and their folds continue mesad so that they meet in an acute notch just

under the oral sucker (fig. 20). A very short prepharynx leads into

a pharynx which is at times oval but is more often campanulate.

A short esophagus bends dorsad from the pharynx (fig. 21), so

that the ceca frequently appear to airse from the base of the phar-

ynx. The ceca originate some little distance anterior to the acetabulum

and extend as large pouches to the subcaudal region of the worm. The

cells of the ceca are small, from ten to fourteen being found in a cross sec-

tion.

The excretory bladder arises posteriorly from an inconspicuous caudal

pore. For a short distance it consists of a small tube, but soon enlarges

into an irregular pouch lined with a thin layer of cells (fig. 26). The

pouch ends at the anterior margin of the anterior testis, from which region

a pair of lateral tubes can be traced forward for a short distance in pre-

served specimens. The writer is indebted to Professor Henry B. Ward

for Lander’s data regarding the details of the tubule system. From

the sides of the anterior extremity of the bladder a pair of single tubules

arises. Each tubule soon bifurcates, one branch coursing forward and

the other caudad. Capillaries ending in flame cells arise along each

194 E. C. FAUST

lateral tubule. As many as six flame cells have been found anterior

to the forking of the system, while eight have been found posterior to

this separation. The anterior limit of the system is in the region of the

pharynx. .

The ovary, a medium-sized ovoid gland, is usually located lateral to

the median line, immediately behind the acetabulum. The receptacu-

lum seminalis is very minute. The testes are large ovate organs, lying

in the third or fourth quarter of the body. The ootype is median, just

behind the ovary. Laurer’s canal which is on the left (fig. 25) meets

the duct from the receptaculum seminalis and both open into the ootype

from above (fig. 27). The sparsely scattered vitellaria extend from the

pharynx to near the posterior end of the body. Their transverse ducts

empty into the ootype from a ventrolateral direction.

The uterus emerges from the posterior margin of the ootype. After

coiling once it runs forwards to the right of the acetabulum to the genital

pore. The eggs are few to several in number, averaging up to twenty-

five in some cases. Each egg measures 70 to 72 in length by 40 to

50 w in cross section. The operculum is small but distinct (fig. 23). As

in the eggs of the other Stephanophialinae, many yolk cells are found.

The small efferent ducts from the testes converge in the plane of the

ovary. The muscular cirrus sac begins at this junction. It is daucine

in shape, except for a lateral bulge in the portion dorsal to the acetabulum.

This bulge is due to the convolution of the seminal vesicle which occupies

the middle region of the cirrus sac. The ductus ejaculatorius is con-

spicuously muscular. Both the seminal vesicle and the ductus are sup-

plied with glands. The opening of the ductus lies posterior to the forking

of the gut. Itis provided with a powerful sphincter (fig. 27).

The structure of the oral papillae is of a deep-seated character. It is

made up of an outermost basement membrane, within which are several

enveloping muscle strands, the nuclei of which can be made out plainly

(fig. 28). Within the center of the papilla is a core of longitudinal

muscle strands. Both of these series are distinct from those making up

the oral sucker. .

The main features of the worm are made out in very young distomula

(fig. 29). These include the campanulate pharynx, distinct genital

cells, representing testes, ovary and anterior sphincter of cirrus sac,

sparsely scattered vitellaria, and the constriction of the excretory bladder

at the posterior end. Eyespots are also present in young worms.

STUDIES ON AMERICAN STEPHANOPHIALINAE 195.

KEY TO THE SUB-FAMILY STEPHANOPHIALINAE

1 (4) Cirrus pouch small to inframedium, mostly lying over the acetabulum; genital

pore anterior to the forking of the gut; papillae subequal ........-.-

Stephanophiala, 2.2 ai aos sna, epee 2

2 (3) Oral sucker smaller than acetabulum; testes larger than acetabulum; eggs

few to many, varying in size from 62 to 85y by 40 to 59y; integument occa-

Slonally SpImOSe #4. cc 1.25 <i eee ante tac eae = S. farionis (O. F. M.)

3 (2) Oral sucker smaller than acetabulum; testes smaller than acetabulum; eggs

EW Ril ph Wy AD Be carla aipeg seted heme eer es 98 or 8 S. vitelloba Faust

4 (1) Cirrus pouch large, extending some distance behind the posterior limit of the

acetabulum; genital pore posterior or ventral to the forking of the gut; ven-

tral papillae large, differentiated ......----- 2s ee eee eee 5

5(10) Ventral papillae extending laterad, tapering to acute points; cirrus pouch

well developed, muscular, tapering to a small ductus ejaculatorius . .

Crepidostomum ...... 6

6 (7) Dorsal papillae entire, not tending to bifurcate; pharynx very small... .

.... C. cornutum (Osborn)

7 (6) Dorsal papillae tending to bifurcate; pharynx about one-half diameter of oral

SHIR 2569 OR het CE Ae lus ea Ore int Chee ea rae Bthe nas rome teeing” Sc 8

8 (9) Inframedium in size, testes very large......... C. metoecus Braun

9 (8) Minute, testes relatively small .............. C. illinoiense Faust

10(5) Ventral papillae extending as folds over the anterior portion of the oral cavity;

ductus ejaculatorius with a powerfully muscular end . . . Acrolichanus

Sin tle ispeciesmarcatnt ears ova ce ot aace a ete versie rr en retepses A. petalosa (Lander)

Biology of the Stephano phialinae

While no faunistic-biological reconnoissance of the Stephanophialinae

will be attempted in this paper on account of the incompleteness of

many of the records, the data are nevertheless sufficiently adequate to

indicate some of the important biological relations of the group.

With the exception of Crepidostomum metoecus Braun all of the de-

scribed species are parasites of fresh-water fishes. Stephanophiala

farionis (O.F.M.), the species most widely distributed geographically,

has also the greatest host distribution. European investigators have

reported it from Trutta fario (L.), T. trutta (L.), Epitomynis salvelinus

(L.), Thymallus thymallus (L.), and Coregonus oxyrhinchus (L.), while

American students have found it in Salvelinus fontinalis Mitch., Perca

flavescens (Mitch.), Eupomotis gibbosus (L.), Boleosoma nigrum (Raf.),

Etheostoma iowae J. and M., Stizostedion vitreum (Mitch.), and Salmo

mykiss lewisit (Gir.). In addition Stafford (1904:490) has reported this.

species from Necturus maculatus. Crepidostomum cornutum (Osborn),

196 E. C. FAUST

while limited to the Eastern part of the United States and Canada, has

been found in Micropterus dolomieu Lac., Ambloplites rupestris (Raf.),

Ameiurus lacustris (Walb.), and A. nebulosus (Le S.). Acrolichanus

petalosa (Lander) has been found in various localities in the Great Lakes

and St. Lawrence Basin, in each case from the same species of host,

Acipenser rubicundus Le S. The species described as new in this paper,

Stephanophiala vitelloba and Crepidostomum illinoiense, have each been

found in but a single host in a single locality.

The normal seat of these parasites is the anterior part of the small

intestine, altho they have been recorded from the posterior end of the

intestine, the pyloric ceca, the gall bladder, and the stomach. It is also

a matter of record that the related species Bunodera luciopercae (O.F.M.)

has wandered out of its host after the death of the latter.

Sufficient data are not at hand to determine annual cycles of infection

in the host.

The life history of no one of the species of the Stephanophialinae

has been worked out. That there is an intermediate host is a certainty,

since immature specimens of Crepidostomum cornutum and Acrolichanus

petalosa have been found in abundance in the crawfish, and cysts of

immature Stephanophiala farionis have been taken from Hexagenia sp.

But the type of cercaria is not known. However, since the cercaria of

the near relative, Allocreadium isoporum, is a rhopalocercous type and

the precocious Allocread species, Cercaria macrostoma Faust, is a cysto-

cercous type, it is highly probable that the Stephanophialine species

have cercariae that are provided with heavy tails. It is evident that

the cercariae have pigmented eyes, since representative species of all

three genera of this group show the remains of these eyes in their imma-

ture stages. Moreover, since the miracidium of Bunodera luciopercae

has pigment eye-spots, it is possible that the miracidia of the Stephano-

phialinae are also pigmented.

A tentative life history scheme for the various species of Stephano-

phialinae may be outlined as follows:

STUDIES ON AMERICAN STEPHANOPHIALINAE 197

miracidium- > sporocyst-> cercaria- > distomulum- >adult distome

or with heavy

redia tail and

eye-spots

See = veer.

in snail in insect in fresh water

or fishes (exception-

crustacean ally in other ver-

_ tebrates)

IMPORTANT REFERENCES

Blanchard, R.

1891. Notices Helminthologiques. Mem. Soc. zool., France, 4:420-489; 38 figs.

Braun, M.

1900. Trematoden der Chiroptera. Ann. d. K. K. Naturhist. Hof-mus., 15;

217-236; 1 Taf.

Cooper, A. R.

1915. Trematodes from Marine and Fresh-Water Fishes. Trans. R. Soc.

Canada, (3)9:181-205; 3 pl.

Linton, E.

1893. On Fish Entozoa from Yellowstone National Park. Rept. U. S. Com. of

Fish and Fisheries, 1889-1891 :545-564; 5 pl.

1898. Trematode Parasites of Fishes. Proc. Nat. Mus., 20:507-548; 15 pl.

1901. Parasites of Fishes of the Woods Hole Region. Bull. U. S. Fish Com.

for 1899 :405-492; 34 pl.

Looss, A.

1902. Ueber neue und bekannte Trematoden aus Seeschildkréten. Nebst

Eréterungen zur Systematik und Nomenclatur. Zool. Jahrb., Syst., 16:

411-894; 12 Taf.

Miiller, O. F.

1788. Zoologica danica. 4 vol.in 2. Havniae. 225 pp., 160 pl.

Nicoll, Wm.

1909. Studies on the Structure and Classification of Digenetic Trematodes.

Quar. Jour. Micr. Sci., n.s., 53:391-487; 2 pl.

Odhner, T.

1905. Die Trematoden des Arktischen Gebietes. Fauna Arctica, 4:291-372;

Subat.

1910. Nordostafrikanische Trematoden. Fascioliden. Results Swedish Zool.

Exp., 1901. Stockholm. 170 pp.; 6 Taf.

Olsson, P.

1876. Bidrag till Skandinaviens Helminthfauna. Stockholm. 35 pp.; 4 Taf.

Osborn, H. L.

1903. Bunodera cornuta sp. nov.: a New Parasite from the Crayfish and Cer-

tain Fishes of Lake Chautauqua, N. Y. Biol. Bull., 5:63-73; 7 figs.

198 E. C. FAUST

Stafford, J.

1904. Trematodes from Canadian Fishes. 1. Zool. Anz., 27:481-495.

Ward, H. B.

1894. On the Parasites of the Lake Fish. Proc. Am. Micr. Soc., 15:173-182, 1 pl.

1918. Parasitic Flatworms, in Ward and Whipple’s Fresh-Water Biology, 365-

453, 113 textfigs.

Wedl, C.

1857. Anatomische Beobachtungen iiber Trematoden. Sitz. K. Akad. Wiss.,

Wien. Math.-naturwiss., 26:241-278, 4 Taf. .

EXPLANATION OF FIGURES

(Code ages ree hes: cirrus pouch Sy ee ee shell gland

"Cotes rue eam cecum |B Le Nera anterior and posterior testes

56 ca Pea vitelline duct EUS Sart, ener ene uterus

CEN neers egg Veet pax eet bation vitellaria

Pe ware genital pore Vl, tna anaes vas deferens

IL CS 2. eres Laurer’s canal VS nase seminal vesicle

Oi das eae ovary Se Rien excretory bladder

Die pharynx SLE eo ootype

Bea eee receptaculum seminalis

DESCRIPTION OF FIGURES

PLATE XIV

Stephanophiala farionis. 1.—Ventral view, X 54; 2.—lateral view of oral sucker

and papillae, X 105; 3.—detail of portion of integument lateral to acetabulum, showing

spines, X 180; 4.—egg, X 180.

Stephanophiala vitelloba. 5.—Dorsal view, X 54; 6.—dorsal view of head, show-

ing papillae, X 105; 7.—egg, X 240; 8.—detail of sex organs in region of ootype, X

180; 9.—cross section of worm just anterior to acetabulum, X 180; 10.—cross section

thru ovary, X 180; 11.—cross section thru posterior testis, X 180; 12.—cross section

thru region of ootype, X 180; 13.—detail of lateral papilla, X 180.

PLATE XV

Crepidostomum cornutum. 14.—Ventral view, X 34; 15.—detail of anterior end,

X 24.

Crepidostomum illinoiensis. 16.—Ventral view, X 105; 17.—detail of anterior

end, ventral view, X 180; 18.—egg, X 330; 19.—diagram of lateral aspect, X 75.

Acrolichanus petalosa. 20.—Ventral view, X 34; 21.—median sagittal section,

X 34; 22.—detail of anterior end, X 24; 23.—egg, X 330; 24.—cross section thru region

of genital pore, X 75; 25.—cross section thru region of ootype, X 75; 26.—cross sec-

tion thru anterior testis, X 75; 27.—detail of genital organs, X 50; 28.—section thru

lateral papilla, X 105; 29.—ventral view of young fluke, X 34.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

SOCIETY VOL. XXXVII

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TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

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DEPARTMENT OF NOTES AND REVIEWS

It is the purpose, in this department, to present from time to time brief original

notes, both of methods of work and of results, by members of the Society. All mem-

bers are invited to submit such items. In addition to these there will be given a few

brief abstracts of recent work of more general interest to students and teachers. There

will be no attempt to make these abstracts exhaustive. They will illustrate progress

without attempting to define it, and will thus give to the teacher current illustrations,

and to the isolated student suggestions of suitable fields of investigation.—[Editor.]

AQUATIC MICROSCOPY

To those of us who remember the first thrills of work with the micro-

scope it is not easy to understand why there are not more amateur stu-

dents of nature who cultivate a life interest in the microscope. Very

much of the early enthusiasm of this Society was given to it by those

who were using the microscope in amateur exploration of the marvels of

nature. The American schools and colleges make much use of the

microscope, but those who are most interested in these things tend to

go on into the study of some more or less technical aspect of the subject.

It would seem however, if our teaching were as effective and inspiring

as it should be, that more of our students would thruout life continue

an amateur interest in this charming field.

Perhaps, as we grow older in America and achieve more leisure and

more general interest in culture, we may again increase the number of

those who untechnically use the microscope for the mere love of its

interesting revelations. In England there are many such, and numerous

simple handbooks have been issued which helpfully guide the beginner.

Dr. Stokes has been a long-time exponent and writer for such students

in this country. His little book, now in its fourth edition, has done its

part to keep alive amateur interest. The author undertakes in the

simplest and most concrete way possible to answer the questions of the

embryo naturalist, such as:—‘How can I best find and collect the

microscopic plants and animals? How can I tell them apart and deter-

mine what they are and how they are named? What outfit will I need,

and how can I prepare my material for profitable study?”

These questions are answered in the following chapters: I, The

Microscope and its Parts; II, Common Aquatic Plants Useful to the

200 AMERICAN MICROSCOPICAL SOCIETY

Microscopist; III, Desmids, Diatoms, and Fresh Water Algz; IV, Rhizo-

pods; V, Infusoria; VI, Hydra; VII, Aquatic Worms (and Insect Larve);

VIII, Rotifera; IX, Freshwater Polyzoa; X, Entomostraca and Phyllo-

poda; XI, Water Mites and Water Bear.

The especial virtues of the book are in its untechnical language, its

simple descriptions, its concrete keys and synopses, and its outline draw-

ings. The author states that, with the exception of a few western forms,

every type of organism mentioned in the book was taken by him from a

single pond in central New Jersey. Aside therefore from its value as a

guide for the study of nature, it has a value as a contribution to intensive

study of a limited locality. The same features which make it useful to

the independent beginner will make the book helpful to the students of

elementary Biology in high school and college, and stimulate to more

effective field work.

Aquatic Microscopy FOR BEGINNERS, by Alfred G. Stokes. Fourth edition, revised and enlarged;

324 pages, illustrated. John Wiley & Sons, New York and London. Price $2.25 net, postpaid.

AMERICAN MICROSCOPICAL SOCIETY 201

Albert McCalla, M.A., Ph.D., F.R.M.S., died suddenly of heart

failure at 9:15 p.m., on Thursday, June 6th, 1918, at his late residence,

2316 Calumet Avenue, Chicago. He was 72 years of age and his demise

came after more than a year of ill health. He was the son of Thomas

McCalla, one of the first bankers of Chicago, and Marianne Davisson,

and was the brother of the late Mary Ella McCalla. Of Scotch descent,

he was of South Carolina and Virginia lineage.

Mr. McCalla was much interested in scientific research and received

a number of degrees. He was possessed of unusual expert ability with

the microscope and was the inventor of an attachment widely used in

days gone by.

He graduated from the old Chicago High School and was the winner

of the Foster Medal; graduated from Monmouth College; was one of the

founders of Beta Theta Pi at the old Chicago University. He was a

member of the following organizations:

Fellow Royal Microscopical Society, London.

Past-President American Microscopical Society.

Past-President Llinois Microscopical Society.

Member American Association for the Advancement of Science.

Member First Presbyterian Church, Chicago.

Member Sons of the Revolution.

Member Beta Theta Pi Fraternity.

He taught at Parsons College and later at Lake Forest College.

He is survived by his wife, Eleanor Hamill McCalla, daughter of the

late Honorable and Mrs. Smith Hamill, and four children, Helen Wayne

McCalla, Thomas Clarendon McCalla, Major Lee A. McCalla, U.S. A.,

and Paul Hamill McCalla.

TRANSACTIONS

OF THE

American

Microscopical Society

ORGANIZED 1878 INCORPORATED 1891

PUBLISHED QUARTERLY

BY THE SOCIETY

EDITED BY THE SECRETARY

T. W. GALLOWAY

BELOIT, WISCONSIN

VOLUME XXXVII

NuMBER FouR

Entered as Second-class Matter August 13, 1918, at the Post-office at Menasha,

Wisconsin, under act of March 3, 1879. Acceptance for mailing at the

special rate of postage provided for in Section 1103, of the

Act of October 3, 1917, authorized Oct. 21, 1918

The Collegiate Press

GrorcE BANTA PUBLISHING COMPANY

MENASHA, WISCONSIN

1918

ee ee

TABLE OF CONTENTS

FOR VOLUME XXXVII, Number 4, 1918

A New Species of Rynchelmis in North America, with Plate XVI by F. Smith

EAT el Wig BG Ml DT (ol SEES ee Me Nee SE Re ee Done Ss ea oe ED Ek Lr

Development of the Wolffian Body in Sus Scrofa Domesticus, with Plate XVII

FOP RER by bdward! |. Angles Ay Meo TD). s..cstcscss Saco see ne

Variation in the Horizontal Distribution of Plankton in Devils Lake, North

WOR Oba ab yy irik! Gx MODENE § 2ct.05- sesh orig et eee ee ee

Notes and Reviews: Genetics in Relation to Agriculture (McGraw-Hill); Nitrate

Cellulose as a Substitute for Celloidin, by Chas. H. Miller

OFFICERS

President?) Ts) is GRTRRIN ee ae el ate ie eee ee eee ee Pittsburg, Pa.

First Vice-President? 1. Mi WRELPLEV 2 ios eee ee ee ee St. Louis, Mo.

second Vice President: ‘C..O. HMsTemEw ON i es sale ee a Los Angeles, Cal.

weer etary: Lai Wa GALLO WAN ates oles ue estes coer ere ee Beloit, Wis.

Treasurer: TH. J. VAN CLBAVE 43.20.65. 3.0)don ene ee Urbana, Ill

Custodian: MAGNUS (PFLAUM. Jo.ss.0icseot eee Se Meadville, Pa.

EX-OFFICIO MEMBERS OF THE EXECUTIVE COMMITTEE

Past Presidents Still Retaining Membership in the Society

Smon Henry GAGE, B.S., of Ithaca, N.Y., :

at Ithaca, N. Y., 1895 and 1906

A. CLIFFORD MERcER, M.D., F.R.M.S., of Syracuse, N. Y.,

at Pittsburg, Pa., 1896

A. M. Bere, M.D., of Columbus, Ohio,

at New York City, 1900

C. H. E1GENMANN, Ph.D., of Bloomington, Ind.,

at Denver, Colo., 1901

E. A. BrrcE, LL.D., of Madison, Wis.,

at Winona Lake, Ind., 1903

Hnery B. Warp, A.M., Ph.D., of Urbana, IIl.,

at Sandusky, Ohio, 1905

HERBERT OsBorNn, M.S., of Columbus, Ohio,

at Minneapolis, Minn., 1910

A. E. Hertzter, M.D., of Kansas City, Mo.,

at Washington, D. C., 1911

F. D. HEALD, Ph.D., of Pullman, Wash.

at Cleveland, Ohio, 1912

CHARLES BROOKOVER, Ph.D., of Louisville, Ky.,

at Philadelphia, Pa., 1914

Cartes A. Korom, Ph.D., of Berkeley, Calif.,

at Columbus, Ohio, 1915

M. F. Guyver, Ph.D., of Madison, Wis.,

at Pittsburg, Pa., 1917

The Society does not hold itself responsible for the opinions expressed

by members in its published Transactions unless endorsed by special vote.

TRANSACTION

OF ;

American Microscopical Society

(Published in Quarterly Instalments)

Vol. XXXVII DECEMBER, 1918 No. 4

A NEW SPECIES OF RHYNCHELMIS IN NORTH AMERICA*

F. Smit AND L. B. DIcKEY

The worms described in this paper are part of a series of Oligochaeta

obtained by Miss Bessie R. Green from the vicinity of Flathead Lake in

Montana during the summer of 1914, while at the Biological Station

maintained by the University of Montana. The Rhynchelmis speci-

mens were collected in July by A. G. Vestal and M. J. Elrod, for whom

the species is named, from a creek near the Station, and included several

mature specimens and a number of immature ones.

But two species and a variety of Rhynchelmis have previously been

known. R. limosella Hoffmeister is a common European species, and

the Asiatic species R. brachycephala and its variety bythia have been

somewhat recently made known by Michaelsen (1901 and 1905). We

now describe a distinct but somewhat closely related species from North

America.

A modification of the definition of the genus in a few characters is

necessary, and a still closer relationship between Rhynchelmis and the

North American genera Sutroa and Eclipidrilus becomes apparent.

RHYNCHELMIS HOFFMEISTER

Setae simple. Spermiducal pores paired on 10.f Oviducal pores

paired in intersegmental groove 11/12. Spermathecal pores paired on

8. Longitudinal muscle layer completely separated into eight longitu-

dinal bands. Transverse blood vessels, two pairs, in each of most

somites. Spermaries and spermiducal funnels paired in 10, or two pairs

in 9 and 10; sperm ducts, one or two pair, opening into a pair of long atria.

Ovaries paired, in 11. Spermathecae paired, in 8, without diverticula

opening into the spermathecal ducts, ampullae communicating with the

alimentary tract.

* Contributions from the Zoological Laboratory of the University of Illinois, No.

106.

} Arabic numerals are used to designate the somites, counted from the anterior end.

208 SMITH AND DICKEY

RHYNCHELMIS ELRODI SP. NOV.

Length, 47-65 mm. Somites, 133-177. Proboscis long and slender.

Setae closely paired. Clitellum on 9-17. Spermiducal, oviducal, and

spermathecal pores nearly in seta line ab. Longitudinal muscle bands

not spirally rolled at edges. Ventral vessel forked in 7, and connected

with dorsal vessel in 1. First nephridia in 13. Spermaries paired, in

10. Spermiducal] funnels, one pair; sperm ducts, one pair, imbedded in

the walls of the atria. Albumen glands lacking. Spermathecae, one

pair in 8; communicating by ducts with the alimentary tract.

From the mucky banks of a creek near the Biological Station at Flat-

head Lake in western Montana.

Holotype and paratypes in the collection of the senior author (Cat.

No. 1058).

The more important facts of structure were gained from the study of

a series of sagittal sections of the 33 anterior somites of one specimen, and

of two series of transverse sections from the anterior 18 somites of each of

two other specimens, of which one is the type.

EXTERNAL CHARACTERS

Alcoholic specimens, apparently sexually mature, are 47-65 mm. in

length, and 0.9-1.25 mm. in diameter in the region of the clitellum, where

the diameter is greatest. In the anterior half of the worm the body is

nearly circular in cross section, unlike other described species of Rhyn-

chelmis, and elsewhere.it is not decidedly quadrilateral. In one appar-

ently complete specimen, the number of somites is but 133, while in

another it is 177. The number of somites in other specimens varies be-

tween these extremes and approximates 150. The anterior part of the

prostomium is prolonged into a slender tentacle-like proboscis. The

setae are closely paired and the distances between the pairs are approxi-

mately indicated by the formula; aa*:bc:dd=3:5:5. The setae are sig-

moid, slightly more curved at the distal end, slender, and simple. The

average length is about 0.27 mm., and the diameter at the nodulus is

about 0.01 mm. The nodulus is at about one-third of the length of the

seta from the distal end.

The clitellum is developed on 9-17 and encroaches slightly on the

adjacent somites. It is most strongly thickened on 10-16, and is devel-

* Letters are used to designate the setae of either side of a somite, beginning with

a for the most ventral one and proceeding in order to d for the most dorsal one.

NEW SPECIES OF RYNCHELMIS IN NORTH AMERICA 209

oped ventrally as well as dorsally. The spermiducal pores are paired on

10, slightly anterior to 10/11, and nearly in line with the ventral setae,

The oviducal pores are small, in 11/12, and in line with the ventral setae.

The spermathecal pores are paired on 8, posterior to the ventral setae.

INTERNAL CHARACTERS

The brain lies dorsad of the mouth, in the first somite, and is similar

in form to that of R. limosella, as figured by Vejdovsky (1876). The

ventral nerve cord is closely adherent to the body wall throughout its

length. The layer of longitudinal muscle fibers is in eight distinct bands,

as in other species of the genus, but the edges of these bands are not

rolled as in R. limosella (Vejdovsky, 1884, pl. 16, figs. 1 and 2), and in R.

brachycephala and its variety, as described by Michaelsen (1905:62-63).

The alimentary tract is simple in character, like that of the other species.

The ventral vessel forks in 7 and the two anterior branches unite near

the brain with the dorsal vessel. A pair of transverse vessels in the pos-

terior part of each of somites 2-6, connect the dorsal vessel with the

branches of the ventral; and similar transverse vessels in 7-12, connect

the dorsal and ventral vessels. In one specimen there is a similar vessel

on one side of 13.. The paired posterior transverse vessels of somites

posterior to 12 are connected with the dorsal vessel only. They have a

few caecal branches and often extend only part way down the sides of the

body. There is a pair of transverse vessels in the anterior part of each

of most somites posterior to 7. The first pair are somewhat shorter and

more simple, but those of somites posterior to 8 extend to the ventral

side and have several caecal branches. In the somites that have been

examined, posterior to 12, each of these vessels is connected with the

ventro-lateral wall of the intestine by a branch which extends obliquely

dorsad and mesad from that part of the vessel lying in the ventro-lateral

part of the body cavity. Ventro-intestinal vessels connect the ventral

vessel with the ventro-median wall of the intestine (fig. 1). In somites

10 or 11 to 18 or 19 inclusive, these vessels, usually three in number, enter

peculiar glandular bodies which are closely associated with the ventro-

median wall of the intestine and correspond to the blutdriisen described

by Michaelsen (1901:178) in R. brachycephala. These blood glands

(fig. 1) are more intimately united with the wall of the intestine in R.

elrodi than are those of the other species.

210 SMITH AND DICKEY

In the specimens examined, the most anterior nephridia are in 13 or

14, and they are more or less irregularly distributed posteriorly. There

are sometimes a pair in a somite, sometimes a single one, and often none

at all. Just posterior to the septum which supports the nephridial fun-

nel, there is an enlargement similar to that found in a considerable num-

ber of other species of lumbriculids. The nephridiopores are in the line

of the ventral seta bundles and a short distance anterior to them.

There is but one pair of spermaries and they project freely into 10

from their attachment to the posterior face of 9/10. A pair of sperm

sacs extend posteriorly on either side of the alimentary tract, from their

openings in septum 10/11, at least as far as to somite 30, in some speci-

mens. ‘The spermiducal organs are similar in their main features to

those of other species of the genus; but there is no trace of more than one

pair of sperm ducts or spermiducal funnels, and those present belong to

somite 10. The funnels are on 10/11, below and laterad of the openings

of the sperm sacs, and the dorsal edges of the funnels extend into the sacs,

along their ventral wall for a short distance. In tracing each sperm duct

from the funnel towards the external pore, we havea relatively slender duct

which extends posteriorly through several somites in the cavity of the cor-

responding sperm sac, to a position at which it enters the posterior end of

a much larger and tubular atrium which extends anteriorly into 10 and

then, bending ventrally, joins the body wall, posterior to the ventral

setae, and opens to the exterior at the spermiducal pore. There is a

general correspondence between the main features of the spermiducal

organs, as outlined above, and those of the other species of the genus;

but a more detailed study yields distinct differences, as will appear later.

From the funnel the sperm duct first extends ventrad along the septum

and then anteriad to the atrium which it follows closely to the place of

their union. The duct and atrium are merely in contact in somite 10,

but in the anterior part of the sperm sac the duct becomes more strongly

flattened against the atrial wall, and about opposite 11/12, in the type

specimen, it enters the tissue of the atrial wall (fig. 2, sd) and follows it

to a point near the posterior end of the atrium, where duct and atrium

merge and their cavities become continuous. ‘This intimate relation of

duct and atrium is more like the condition found in certain species of

Eclipidrilus than it is like that of the other species of Rhynchelmis.

In the type specimen the atrium extends posteriorly to 15, and in the

other sectioned specimens not so far. Numerous small glandular masses

NEW SPECIES OF RYNCHELMIS IN NORTH AMERICA 211

or prostate glands which are much like those of other species of the genus,

are attached to the outer surface of the atrium (fig. 2, pr). The ectal

ends of the atria are apparently protrusible and may function as penial

organs. In somite 9, in other species of Rhynchelmis, there are organs,

either one or a pair, which are known by various names: albumen glands,

Kopulationsdriisen, etc. There are no recognizable traces of such

organs in R. elrodi.

There is but one pair of ovaries and these are in 11 and are attached

to the septum 10/11. The paired ovisacs extend posteriorly from 11/12

and closely invest the corresponding sperm sacs except where ova prevent.

They extend through several somites posteziad of the sperm sacs. Paired

oviducal funnels are on the anterior face of the septum 11/12, and the

very short oviducts open to the exterior in the segmental groove 11/12

in line with the ventral setae. Paired spermathecae in 8, correspond

closely with those of the other species of the genus. They open to the

exterior posteriad of the ventral seta bundles of 8; the ducts are without

diverticula; and the ampullae open through narrowed duct-like portions

into the alimentary tract. In one specimen the spermathecae have no

connection with the alimentary tract and the diameter of the lumen is

much less than normal. This is probably due to degeneration, since the

spermaries are small and apparently at a stage of inactivity and yet the

sperm sacs are well distended with sperm cells.

SYSTEMATIC RELATIONSHIPS

The new species has important characters that ally it closely with the

Eurasian species of Rhynchelmis, and others in which it is nearly related

to Sutroa (Beddard, 1892; Eisen, 1888, 1891) and Eclipidrilus. The

simple pointed setae, and much elongated atria are characters shared by

all of them. In having an intervening somite between the spermathecal

and atrial somites; in the communication between the spermathecae and

the alimentary tract; and in the lack of differentiation of each atrium into

a “sperm reservoir’ or “storage chamber” and a penial organ with

narrowed connecting duct; it resembles the species of Rhynchelmis and

Sutroa and differs from those of Eclipidrilus (Michaelsen, 1901:150;

Smith, 1900:473). It is nearer to Rhynchelmis than to Sutroa, in having

the spermathecae paired and without diverticula; but resembles the latter

rather than the previously known species of the former, in having no

atrial remnants (albumen glands) in somite 9. To the writers the rela-

EM SMITH AND DICKEY

tionships to Rhynchelmis seem more significant and they include it in

that genus. One important difference between Rhynchelmis and Sutroa

disappears when we find simple, paired spermathecae, and absence of

atrial remnants in 9, characterizing the same species. It is interesting

to note that the possibility of the existence of such a species of Rhyn-

chelmis as R. elrodi has already been forecast by Michaelsen (1908:163).

“Tch bin in meinen Betrachtungen dieser Reduktionsverhiltnisse

dann noch einen Schritt weiter gegangen. Von Rhynchelmis brachy-

cephala ausgehend, sagte ich mir, dass es kein morphologisch sehr bedeut-

samer Vorgang sei, wenn nun die rudimentiren, Samentrichterlosen

Samenleiter des vorderen Paares und die verlassenen, ihrer Hauptfunk-

tion enthobenen Atrien des vorderen Paares ganz schwinden. Es wiirde

dann ein Zustand des minnlichen Geschlechtsapparates eintreten, der

mit dem urspriinglich einfachpaarigen Apparat durchaus iiberein-

stimmte.”’

In R. limosella (fig. 3) there are two pair of spermaries and spermiducal

funnels in 9 and 10, and two pair of sperm ducts joining the paired atria

of 10. In 9 there are paired organs resembling atria but without the

atrial function since no sperm ducts are connected with them. They are

the “albumen glands” and presumably represent an additional pair of

atria which in ancestors were joined by the sperm ducts connected with

the spermiducal funnels of 9. In R. brachycephala (fig. 4) and its variety

bythia, the spermaries and spermiducal funnels of 9 have disappeared

and there is a partial disappearance of the related pair of sperm ducts,

while the atrial organs of 9 are still represented. In R. elrodi (fig. 5)

there is a complete disappearance of the reproductive organs of 9, and

we have simply the single pairs of spermaries, spermiducal funnels, sperm

ducts, and atria in 10. We also have a single pair of ovaries and of ovi-

ducts which are in 11. The location of the single pair of spermathecae

in R. elrodi, two somites anterior to the one containing the male organs,

which would otherwise seem rather peculiar, is easily understood on the

assumption that this species has been derived from ancestors similar to

R. limosella._ In accordance with the views of Michaelsen, these in turn

were presumably derived from Lamprodrilus-like ancestors in which

each pair of sperm ducts had its own pair of atria independent of others.

NEW SPECIES OF RYNCHELMIS IN NORTH AMERICA 213

LITERATURE CITED

BEDDARD, F. E.

1892. A Contribution to the Anatomy of Sutroa. Trans. Roy. Soc. Edinburgh,

37 195-202.

EISEN, GUSTAV.

1888. On the Anatomy of Sutroa rostrata, a New Annelid of the Family of Lum-

briculina. Mem. California Acad. Sci., 2:1-8.

1891. Anatomical Notes on Sutroa alpestris, a New Lumbriculide Oligochete

from Sierra Nevada, California. Zoe, 2:322-334.

MICHAELSEN, W.

1901. Oligochaeten der Zoologischen Museen zu St. Petersburg und Kiew.

Bull. Acad. Imp. Sci. St. Petersburg, (5), 15:137-215.

1905. Die Oligochaeten des Baikal-Sees. Wiss. Ergebn. Zool. Exped. Baikal-

See, unter Leit. v. A. Korotneff. 1 Lief., pp. 1-68.

1908. Pendulations-Theorie und Oligochiten, zugleich eine Erérterung der

Grundziige des Oligochiten-Systems. Mitt. Nat. Mus. Hamburg, 25:

153-175.

SmiTH, F.

1900. Notes on Species of North American Oligochaeta. IV. Bull. Ill. State

Lab. Nat. Hist., 5:459-478.

VEJDOVSKY, FRANZ.

1876. Anatomische Studien an Rhynchelmis Limosella Hoffm. (Euaxes filiros-

tris Grube). Zeit. f. wiss. Zool., 27:332-361.

1884. System und Morphologie der Oligochaeten. 166 pp., Prag.

214 SMITH AND DICKEY

EXPLANATION OF PLATE XVI

Fig. 1. Rkynchelmis elrodi. Transverse section through the posterior part of

somite 17: int, intestine; bg. blood gland; vi, ventro-intestinal vessel; vv, ventral

vessel; ss, sperm sacs.

Fig. 2. The same. Transverse section through the atrium near the place of en-

trance of the sperm duct: at, atrium; sd, sperm duct; pr, prostate glands; s, developing

sperm cells. Semi-diagrammatic.

Fig. 3. Rhynchelmis limosella. Diagram showing relations of the reproductive

organs of one side: sy, spermary; sd, sperm duct; at, atrium; al, albumen gland; oy,

ovary; od, oviduct; st, spermatheca.

Fig. 4. Rhynchelmis brachycephala. Similar diagram: adapted from figure of

Michaelsen (1901:179).

Fig. 5. Rhynchelmis elrodi. Similar diagram.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL

SOCIETY VOL. XXXVII

PLATE XVI SMITH AND DICKEY

DEVELOPMENT OF THE WOLFFIAN BODY 215

DEVELOPMENT OF THE WOLFFIAN BODY IN

SUS SCROFA DOMESTICUS

Epwarp J. ANGLE, A.M.M.D.

The results embodied in this paper are from studies undertaken some

years ago. At that time it was realized that the investigation was far

from complete and publication was delayed with the hope of further

study—a hope never realized—of the source of origin of the several por-

tions of the urinary tubules. In the light of recent research the publica-

tion of the paper at this late date is largely that the illustrations may

prove of some permanent value.

HISTORICAL

The Wolffian bodies (Corps de Wolff, Urniere, Mesonephros, Primi-

tive Kidney) were discovered by Casper Fr. Wolff in the year 1759, who

regarded them as representing the embryonic period of the true kidney

(Metanephros). They received their present name from H. Rathke in

1825; this only applied to the Wolffian bodies of birds, as Rathke termed

the same organs in mammals Okensche KGrper. In 1824 Jacobson intro-

duced the name of primordialniere and discovered that in birds these

bodies secreted uric acid, which was conducted away by the allantois.

The first mention of the mesonephros in man was made by J. Frey

Meckel (1809) in his work on comparative anatomy. Meckel describes

in fairly accurate language the mesonephros of an embryo 1 mm. long;

but evidently was in doubt as to what the organ was, as he later asks the

question—“are these structures the common source from which lungs,

liver, kidneys, adrenals and sexual organs later have their origin?”

The older writers (Wrisberg, Dzondi, Oken, Emmert and Hochstetter)

had many fanciful theories regarding the role of the Wolffian body, re-

garding it either as the beginning of the kidney or as the horn of the

utereus.

Rathke (25) led the way to a true conception by discovering the origin

of the true kidney in an embryo chick of six days and showed that the

Wolffian body was related to the kidneys as the gills are to the lungs.

The glomerulus of the mesonephros was discovered by J. Miiller (30).

The honor is due Miiller for having first accurately and correctly followed

the developmental changes of this organ in a large series of embryos.

216 ANGLE

The smallest of Miiller’s embryos hada length of 20mm. In this embryo

he describes the adrenals, which are quite large and covered by the true

kidney, the kidney and ureter, the Wolffian bodies with their conducting