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Cover :

Color varieties of the Chinese Giant Salamander ( Andrias davidianus) from aquaculture farming operations in China. Photo Sumio Okada.

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Amphibian & Reptile Conservation 5(4): 1-1 6.

Survey techniques for giant salamanders and other

aquatic Caudata

'ROBERT K. BROWNE , 2 HONG LI, "DALE MCGINNITY, 4 SUMIO OKADA , 5 WANG ZHENGHUAN,

"CATHERINE M. BODINOF, 7 KELLY J. IRWIN, “AMY MCMILLAN, AND "JEFFREY T. BRIGGLER

1 Center for Research and Conservation, Royal Zoological Society of Antwerp, Antwerp, BELGIUM Polytechnic Institute of New York University,

New York, New York 11201, USA 3 Nashville Zoo, Nashville, Tennessee 37189, USA 4 Laboratory of Biolog}’, Department of Regional Environment,

Tottori University, Tottori 680-8551, JAPAN 5 School of Life Sciences, East China Normal University, 200062, Shanghai, CHINA 6 University of Mis-

souri, Department of Fisheries and Wildlife, Columbia, Missouri 65211, USA ''Arkansas Game and Fish Commission, Benton, Arkansas 72015, USA

8 Buffalo State College, Buffalo, New York 14222, USA 9 Missouri Department of Conservation, Jefferson City, Missouri 65109, USA

Abstract.— The order Caudata (salamanders and newts) comprise ~13% of the ~6,800 described am-

phibian species. Amphibians are the most threatened (~30% of species) of all vertebrates, and the

Caudata are the most threatened (~45% of species) amphibian order. The fully aquatic Caudata family,

the Cryptobranchidae (suborder Cryptobranchoidea), includes the the world's largest amphibians, the

threatened giant salamanders. Cryptobranchids present particular survey challenges because of their

large demographic variation in body size (from three cm larvae to 1.5 m adults) and the wide variation

in their habitats and microhabitats. Consequently, a number of survey techniques (in combination) may

be required to reveal their population and demography, habitat requirements, reproduction, environ-

mental threats, and genetic subpopulations. Survey techniques are constrained by logistical consider-

ations including habitat accessibility, seasonal influences, available funds, personnel, and equipment.

Particularly with threatened species, survey techniques must minimize environmental disturbance and

possible negative effects on the health of targeted populations and individuals. We review and compare

the types and application of survey techniques for Cryptobranchids and other aquatic Caudata from a

conservation and animal welfare perspective.

Key words. Survey techniques, giant salamander, amphibian, Caudata, Cryptobranchid, conservation

Citation: Browne RK, Hong L, McGinnity D, Okada S, Zhenghuan W, Bodinof CM, Irwin KJ, McMillan A, Briggler JT. 2011 . Survey techniques for giant

salamanders and other aquatic Caudata. Amphibian & Reptile Conservation 5(4):1-16(e34).

Introduction

Amphibians are suffering from one of the greatest rates of

decline and extinction of any vertebrate class. One of the

most unique, iconic, and threatened amphibian clades in

the Caudata are the fully aquatic Cryptobranchids (fam-

ily Cryptobranchidae; suborder Cryptobranchoidea). All

three Cryptobranchids are fully aquatic and include the

world’s largest amphibians: the Critically Endangered,

Chinese giant salamander ( Andrias davidianus), the Near

Threatened, Japanese giant salamander (A. japonicus ),

and the North American giant salamander ( Cryptobran -

chus alleganiensis), commonly known as the Hellbender

(CNAH 2011).

The conservation potential of Cryptobranchids ex-

tends beyond their immediate conservation needs. As

iconic species, Cryptobranchids offer an ideal opportu-

nity to develop public awareness and government and

institutional support for water catchment management.

In Japan, A. japonicus has become a national symbol,

attracting publicity including parades with large floats,

education and environmental awareness campaigns, and

village conservation programs. Similarly, in the People’s

Republic of China, the release of A. davidianus from

farm stock has received widespread government support

and formal public recognition, and this species is becom-

ing a symbol for watershed conservation. There is also an

increasing momentum toward establishing C. allegani-

ensis as an icon for watershed conservation in the USA

(Browne et al. 2012a, b).

However, in addition to public and government

support, the conservation of Cryptobranchids and oth-

er aquatic Caudata relies upon scientific knowledge of

their conservation genetics, population demography

and size, habitat and microhabitat variables, reproduc-

Correspondence. Emails: jxindakittylhong@gniaiI.com; 3 dmcginnity@nashville.org; 4 shichibu@mocha.ocn.ne.jp; 5 zhwang@

bio. earn. edu. cn; 6 bodinofc@missouri. edu; 7 kirwin@agfc. state. ar. us; % mcmillam@buff (dost ate. edu; 9 Jeff. Briggler@mdc.mo.gov;

'robert.browne@gmail.com (Corresponding author).

amphibian-reptile-conservation.org

December 2011 | Volume 5 | Number 4 | e34

Browne et al.

Figure 1. Andrias davidianus is the largest and most threatened Cryptobranchid, and can reach 200 cm in total length and 59 kg in

weight. Image Robert Browne.

tion and life stage survival, and environmental threats.

The most appropriate survey techniques to achieve this

knowledge will depend on survey objectives in concert

with logistical constraints including the type of habitat

surveyed (Dodd 2009). The choice of survey techniques

must consider interacting factors, including the species’

autecology, targeted life stages, and season, as well as

water depth, velocity, and clarity (Dodd 2009). Survey

techniques must minimize environmental disturbance

and possible negative effects on the health of the targeted

individuals and populations through the spread of patho-

gens and trauma to individuals.

The conservation needs of Cryptobranchids vary

widely between the three species. Andrias davidianus

was until recently considered almost extinct in nature.

However, recent evidence shows that there are a num-

ber of relict populations distributed throughout China.

The few remaining populations (in lowland areas) are

fairly genetically homogenous, probably due to anthro-

pogenic transport and the building of canals over China’s

~6,000 year history of civilization. Nevertheless, there

are genetically distinct populations remaining (Tao et al.

2005), and ongoing molecular studies may reveal even

finer population structure (R. Murphy, pers. comm.) and

further Evolutionarily Significant Units (Crandall et al.

2000 ).

Andrias davidianus has a considerable aquaculture

potential, and more than 1000 licensed aquaculture fa-

cilities are in production in China with up to 106 indi-

viduals in stock. In concert with aquaculture, there are an

increasing number of restocking programs using aqua-

culture brood stock. However, aquaculture brood stock

is subject to genetic drift, a process that reduces genetic

diversity over generations. Additionally, the source of the

aquaculture brood stock is often unknown, and examples

such as the unmanaged release and escape of aquaculture

stock of Pacific salmon ( Oncorhynchus spp.) have result-

ed in a loss of genetic variation or out breeding in wild

populations (Reisenbichler and Rubin 1999). Therefore,

surveys are needed at all potential release sites to reveal

the presence of relictual populations to avoid compro-

mising the long-term conservation of A. davidianus and

other Cryptobranchids. Their population genetics must

also be assessed to enable the provision of genetically

competent individuals for release (Reisenbichler and Ru-

bin 1999)

Consequently, the major conservation needs of A.

davidianus, besides watershed restoration, limiting wild

harvest, and pathogen management, are assessing the

presence of relictual populations and their conservation

genetics, and then matching the genetics of released stock

with those found in nature. When these requirements are

satisfied, the survey focus must include selecting suit-

able release sites, then release of juveniles or adults, and

ongoing assessment of the survival and reproduction of

released individuals. Because there are few remaining A.

davidianus in nature, it will be difficult for surveys to

associate habitat variables with carrying capacity (Zhang

et al. 2002). However, surveys can identify remaining

populations, provide genetic samples, and assess the suc-

cess of restocking programs (Wang et al. 2004).

The conservation of A. japonicus relies on the main-

tenance of the populations that generally still remain in

suitable habitats (Tochimoto et al. 2008). Although A.ja-

ponicus was harvested in the past, strict protection is now

in place to prevent this species from exploitation. How-

ever, threats include habitat modification and other an-

thropogenic changes, including pollutants, and the intro-

duction of A. davidianus in some systems. Consequently,

the conservation needs of A. japonicus include surveying

Figure 2. Genetic drift and selection for color traits in A. da-

vidianus have resulted in orange, piebald, and albino strains.

Image Robert Browne.

amphibian-reptile-conservation.org

December 2011 | Volume 5 | Number 4 | e34

Survey techniques for giant salamanders

Figure 3. Andrias japonicus is the second largest Cryptobran-

chid and reaches 150 cm in total length and 44 kg in weight.

Image Sumio Okada.

population densities and demography, habitat variables

including channelization and watershed characteristics,

assessing the effects of obstacle removal to migration,

such as dams, and the provision of artificial habitats on

survival and recruitment (Browne et al. 2012a, b).

The conservation needs of C. alleganiensis include

identifying the most enigmatic threat to any Cryptobran-

chid and perhaps any amphibian species. Cryptobran-

chus alleganiensis has generally been declining over

most of its range (Wheeler et al. 2003; Foster et al. 2009),

to some extent due to habitat degradation and modifica-

tion. Flowever, C. alleganiensis still survives in near

historic numbers in some locations, and some habitats

modified by siltation and agricultural development still

support substantial numbers of C. alleganiensis. Flow-

ever, the recruitment of C. alleganiensis has failed for de-

cades over a substantial part of its range due to unknown

causes, and many of these declining populations are now

comprised of only a few old individuals (D. McGinnity,

pers. comm.).

Cryptobranchus alleganiensis is subject to many

ongoing surveys; however, these research activities have

not revealed the cause of poor recruitment (Wheeler et al.

2003; Foster et al. 2009). Addressing this problem will

require targeting the life history stage where the failure of

recruitment occurs, from mating success through fertil-

ization, to egg development, and larval and juvenile sur-

vivorship. Surveys will need to correlate recruitment to

different life history stages with environmental variables

such as pollutants. Attempts to reproduce C. alleganien-

sis in captivity for restocking are in the early stages of

development, and no larvae have been produced. Flow-

ever, the production of large numbers of individuals from

wild eggs has been successful and their release to natural

habitats is underway. The cryopreservation of sperm is

now being undertaken to perpetuate the genetic varia-

tion of populations with poor or no recruitment (National

Geographic 2010; Michigan State University 2010). In

addition, research has been initiated to provide a suite of

reproduction technologies to produce genetically compe-

tent individuals (D. McGinnity, pers. comm.).

Cryptobranchids present particular survey chal-

lenges because of their large variation in body size, from

three cm larvae to 1.5 m adults. Additional challenges

include the wide variation in their aquatic habitats (deep

turbulent water, shallow riffles, pools, lakes) and varied

microhabitats (crevices, large rocks, pebble bed in rif-

fles) (Nickerson and Krysko 2003; Tao et al. 2004; Oka-

da et al. 2008). The habitats of A. japonicus and C. alle-

ganiensis are relatively accessible, but, the habitat of A.

davidianus includes difficult to survey, rugged, remote,

fast-flowing interior rivers in the mountainous areas of

central China (Tao et al. 2004).

Effective survey methods depend on associating

the life stages of target species with their microhabi-

tats. Adult Cryptobranchids live in cavities, under large

rocks, and in bank-side dens. Because of the low popula-

tion densities of the relictual populations of A. davidi-

anus, recent surveys have relied on the observation of

adults, electrofishing and the use of bow hooks (Wang

et al. 2004). Surveys for adult and subadult A. japonicus

in their habitats of slow flowing rivers have largely re-

lied on direct observation with some netting (Okada et

al. 2008). In contrast, surveys of adult and subadult C.

alleganiensis have used a wide variety of techniques, in-

cluding rock turning while snorkeling or, in deeper water,

scuba diving or trapping (Nickerson and Krysko 2003;

Foster et al. 2008). Recent innovations in survey tech-

niques for C. alleganiensis include the use of artificial

spawning sites to reveal reproductive success. The use of

video cameras has the potential to increase observations

of mating, brooding by males, and the development of

oocytes and larvae. Environmental DNA (eDNA) detec-

tion (Goldberg et al. 2011) has the potential to both detect

Cryptobranchids and to estimate their standing biomass

and population. Radiotelemetry offers an opportunity to

survey the movements and survival of an increasing size

range of Cryptobranchids over an extended period (Ken-

ward 2001).

Andrias japonicus and C. alleganiensis larvae and

early juveniles are encountered less frequently than adults

due to their particular microhabitats and to the low larval

recruitment of C. alleganiensis in some regions (Nicker-

son and Krysko 2003; Okada et al. 2008). In contrast, the

larvae of A. davidianus were commonly found in surveys

of shallow mountain streams in the Qin Ling Mountains

until their populations rapidly declined in the early 1980s

(Zhang et al. 2002). Okada et al. (2008) found recently-

hatched larvae of A. japonicus in pools under leaf litter

or undercut banks, whereas more developed A. japonicus

larvae were found under rocks and in gravel beds. Adults

can be found in bunk burrows or among deeper rocks or

branches. Although little is known about the microhabi-

tat of the larval stages of C. alleganiensis, observations

suggest that both larvae and small juveniles inhabit inter-

stitial spaces under river gravel in riffles (Nickerson and

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December 2011 | Volume 5 | Number 4 | e34

Browne et al.

Krysko 2003; Foster et al. 2008). Juvenile and subadult

C. alleganiensis most frequently occur in clean, rock-

based streams, although they are also found in deeper

pools with rocks, vegetation, and snags (Nickerson and

Krysko 2003).

The efficacy of survey methods can vary through the

interaction of climate and season with diel activity cy-

cles. For example, the nocturnal activity of C. allegani-

ensis in streams of southeastern North America is posi-

tively correlated with high water levels (Humphries and

Pauley 2000). Nocturnal surveys are most productive in

late spring and early summer, whereas wire mesh baited

traps were most efficient from early winter to late spring

(J. Briggler, pers. comm.). Recent survey innovations

for C. alleganiensis include the use of artificial breeding

dens for adults, egg masses, and larvae, and the place-

ment of natural rocks to provide habitat. Safeguarding

the health and reproductive success of Cryptobranchids

is critical when choosing survey techniques. Techniques

necessitate minimal disturbance to the habitat, the use of

sanitary procedures to prevent pathogen dissemination,

and the protection of nest sites. If possible, several sur-

vey techniques should be used concurrently to improve

survey accuracy and minimize sampling bias (Nickerson

and Krysko 2003).

Survey design needs to incorporate the recogni-

tion of potential biases through the choice of technique,

surveyed microhabitat, species, and life stage (Dodd

2009). Nowakowski and Maerz (2009) tested the effi-

cacy of surveys of larval stream salamanders by com-

paring the mark-recapture success of passive leaf litter

trapping and dip netting. Significant size bias occurred,

with traps capturing a higher proportion of large indi-

viduals and dip netting yielding a greater proportion of

smaller size classes. The survey efficiency of first and

second order streams was greater at low salamander den-

sities with time-constrained opportunistic sampling, but

greater with quadrat sampling when salamanders were

at high densities (Barr and Babbitt 2001). Nowakowski

and Maerz (2009) concluded that the physical dynamics

Figure 4 . Cryptobranchus alleganiensis has been the subject of

the most diverse and innovative survey methods of all Crypto-

branchids. Image Dale McGinnity.

Figure 5. Natural rock placed in stream to provide habitat and

sampling locations for C. alleganiensis . Image Kenneth Roblee.

of water bodies and geographic region are primary con-

siderations when selecting the most promising season for

surveying different life stages.

An important consideration when surveying Cryp-

tobranchids and other aquatic Caudata is the prevention

and spread of infectious diseases. Chytridiomycosis

(Chytrid; Batrachochytrium dendrobatidis) is an in-

fectious disease of particular conservation concern for

amphibians. Chytrid is an emerging pathogen that can

regionally extirpate up to 90% of species and 95% of in-

dividuals in naive populations, at least among frogs (Lips

et al. 2005). However, the effect of chytrid on Crypto-

branchids has not been significant. One strain of chytrid

has been suggested as endemic to populations of A. ja-

ponicus (Goka et al. 2009), and an undetermined strain

of chytrid is found on mainland Asia in South Korea and

may eventually impacts, davidianus (Yang et al. 2009).

Chytrid has been shown to be pathogenic in cap-

tive populations of C. alleganiensis (Briggler et al. 2007,

2008), although with apparently few, if any, pathological

effects on natural populations. Nevertheless, good sani-

tation is a primary consideration in surveying Crypto-

branchids, and other amphibians as a precaution against

spreading chytrid. The same sanitary procedures will also

prevent the spread of pathogens to other species of ani-

mals and plants. Another main pathogen currently threat-

ening Cryptobranchids and other amphibians is Rana-

virus (Geng et al. 2011). To prevent the spread of both

amphibian chytrid and Ranavirus , equipment should be

thoroughly sanitized when moving among aquatic sys-

tems, including all instruments, containers (e.g., measur-

ing boards, weighing containers, and other instruments

and equipment used), human body parts (hands), and

clothing (especially, boots and waders) that come into

contact with amphibians and their environment.

We review and compare the types and application

of survey techniques for Cryptobranchids and other

aquatic Caudata from a conservation and animal welfare

perspective. Reviews or comparative studies of survey

techniques for Ciyptobranchids include Nickerson and

amphibian-reptile-conservation.org

December 2011 | Volume 5 | Number 4 | e34

Survey techniques for giant salamanders

Krysko (2003; C. alleganiensis ), Wang et al. (2004; A.

davidianus), Okada et al. (2008, 2006; A. japonicus), and

Dodd (2009) for general survey techniques of amphib-

ians.

Survey techniques we review include: 1) Wading,

turning substrate, netting, and snorkeling, 2) Scuba/

hookah diving, 3) Nocturnal spotlighting, 4) Bow -hooks/

trot-lines, 5) Questionnaires, 6) Electrofishing, 7) Under-

water camera systems, 8) Passive integrated transpon-

ders (PIT tags) and mark-recapture, 9) Radiotelemetry,

10) Modular artificial spawning dens and rock substrate

placement, 11) Wire mesh baited traps, 12) Population

genetic techniques, and 13) Environmental DNA (eDNA)

detection.

Review of survey techniques

1. Wading , turning substrate, netting, and

snorkeling

Wading and turning substrate, coupled with snorkeling

and downstream netting and seining, are widely used

techniques for surveying C. alleganiensis and other

Cryptobranchids (Taber et al. 1975; Peterson et al. 1983,

1988; Nickerson and Krysko 2003). These techniques

are considered the most effective techniques in relatively

clear shallow streams and pools less than one meter in

depth with a substrate of rocks and other loose shelters

(Nickerson and Krysko 2003). Cryptobranchids can be

surveyed through blind searches by reaching beneath

large rocks or within hollow logs or holes in banks. These

techniques have resulted in the detection of hundreds to

thousands of C. alleganiensis in some surveys (Taber et

al., 1975; Peterson et al. 1983, 1988).

Snorkeling is another common technique for survey-

ing C. alleganiensis (Nickerson and Krysko 2003) and

other salamanders and is most effective in clear waters

from 0.5 to < 3.0 m in depth. This method has proved

more efficient than wading and turning substrate in sur-

veys of C. alleganiensis in the gilled larval stage (Nick-

erson et al. 2002).

Foster et al. (2008) turned rocks to survey for adult

and larval C. alleganiensis and captured 157 in 317 per-

son hours (0.5 individuals per person hour (pph)). Bank

searching through turning substrate within four meters of

the stream bank yielded 14 juveniles in 55 person hours

(0.25 pph). Bank searches of four of the seven inhabited

sites yielded no C. alleganiensis, but at three sites bank

searching was more efficient than rock turning (Foster

et al. 2008). Capture rates of C. alleganiensis in four

streams in the White River drainage, Missouri, varied

from zero to 2.5 pph (Trauth et al. 1992). Okada et al.

(2008) used diurnal wading and substrate surveys with

one to three people searching under piled rocks or leaves

(by hand or with dip-nets) to observe 227 A. japonicus at

a rate of 1 .4 pph.

Figure 6. Turning heavy rocks, combined with snorkeling with

face masks and nets is an effective means to survey juvenile

and adult C. alleganiensis. Image Robert Browne.

2. Scuba/hookah diving

Deep water habitats have not generally been well sur-

veyed for Cryptobranchids, although standard scuba div-

ing equipment and surface-based air compressor systems

(hookah dive systems) are being used increasingly for

surveying C. alleganiensis in fast-flowing, deep water

two to nine meters in depth. Scuba diving allows for

prolonged submergence giving the diver the capability

to systematically check all available cover and often cap-

ture all individuals observed.

Standard scuba diving equipment provides unlim-

ited mobility in terms of the area a worker can survey. In

contrast, divers using a stationary anchored boat, canoe,

or bank-side hookah system are limited by air line length.

Figure 7. Snorkeling and turning small substrate is a good tech-

nique for surveying small to large C. alleganiensis in water of

moderate depth. Image Robert Browne.

amphibian-reptile-conservation.org

December 2011 | Volume 5 | Number 4 | e34

Browne et al.

Nevertheless, free-floating hookah systems are available

that allow hookah divers to work in moderately fast wa-

ters with unlimited mobility as the compressor floats

freely behind the divers. If conditions are not favorable

for use of a free-floating hookah system, then a boat or

canoe can be used to provide a semi-mobile platform for

a stationary hookah compressor.

Boat-mounted hookah systems enable dives of one

hour (hr) to more than 1 .5 hr duration, and can be used at

multiple sites during a full day of fieldwork without the

need to refuel. Hookah systems require the use of a dive

harness fitted with lead weight (usually 20-25 kg) suffi-

cient to hold a diver in place in fast currents. The stream-

lined profile of hookah systems reduces the fatigue expe-

rienced by divers using standard scuba equipment. Divers

also must be capable of working in fast moving water

and have the physical strength to move large cover ob-

jects to successfully locate Cryptobranchids. For safety

reasons, all diving requires a minimum of two divers, so

that a “buddy system” is in place. If using a hookah dive

system, a topside operator is required to monitor condi-

tions and equipment. All divers must have appropriate

certification and must surface when air cylinder pressure

drops to 500 psi.

3. Nocturnal spotlighting

Nocturnal spotlighting has the advantage of producing

minimal substrate disturbance, as rocks are lifted after

the protruding heads of C. alleganiensis are observed.

Spotlighting also allows observation of migratory and

other behaviors. A spotlight survey of C. alleganiensis in

West Virginia, USA, showed that increased nocturnal ac-

tivity is correlated with high water levels, and suggested

that spotlight surveys for mature adults are best conduct-

ed in May and June in this region (Humphries and Pauley

2000). Kawamichi and Ueda (1998) used nocturnal sur-

veys combined with wading for A. japonicus in stream-

beds, and this technique, without substrate turning, is the

most common survey technique for A. japonicus.

Figure 8. Artificial spawning dens for C. alleganiensis are used

to increase the number of nesting sites and allow monitoring

of egg production and larval survival. Image Noelle Rayman.

Nocturnal snorkeling/scuba surveys follow the same

protocol as wading surveys, except that the observers

are swimming and using dive lights to spot salamanders.

Nocturnal snorkeling/scuba surveys have been conduct-

ed with some success in Missouri and Arkansas, USA,

especially during the spawning period. Boats with halo-

gen spotlights powered by generators have been used to

survey for C. alleganiensis in Missouri (Wheeler 2007;

Nickerson and Krysko 2003).

4. Bow-hooks/trot-lines

Bow-hooks or trot-lines can be an efficient survey tech-

nique in detecting the presence of Cryptobranchids at

low population densities (Wang et al. 2004; Liu et al.

1991). Wild populations of A. davidianus have declined

dramatically during the past 40 years, and in many re-

gions bow-hooks may provide the most practical survey

technique (Liu 1989; Wang 1996; Zhang and Wang 2000;

Zhang et al. 2002).

Wang et al. (2004) surveyed A. davidianus us-

ing bow-hooks made of small pieces of bamboo fitted

with four or five sharp hooks. In this study, only one A.

davidianus was captured with the bow-hooks, whereas

none were observed during night surveys and eight were

captured by electrofishing. Bow-hooks were found to be

an effective survey technique for A. davidianus in the

remote and rugged Huping Mountain National Nature

Reserve, an area of particular conservation significance

(Zhang et al. 2002; Tao et al. 2004). Protection now

forbids the use of hooks for surveying A. japonicas, al-

though they can be captured without a hook by using bait

on a stick (Tochimoto 2005). Bottom-set bank lines have

been used in surveys of C. alleganiensis in sections of

river with no rocks or logs, or that were unsuitable for

wading and substrate turning (Dundee and Dundee 1965;

Wortham 1970; Nickerson and Krysko 2003).

5. Questionnaires

Questionnaire surveys were conducted by Wang et al.

(2004) with local fisheries managers and villagers to

analyze the past and present distribution and status of

A. davidianus. A total of 72 answered questionnaires

concluded 1 ) A. davidianus were abundant prior to the

1980s, when individuals could be found easily and cap-

tured, 2) populations have since dramatically declined,

and it is now difficult to capture A. davidianus , and 3) the

main reasons for declines are excessive poaching, habi-

tat fragmentation, and pollution. Responses to question-

naires also suggested that A. davidianus inhabited areas

where 82 subsequent nocturnal surveys failed to detect

them, so questionnaire results were neither verified nor

discredited.

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Survey techniques for giant salamanders

In another example of questionnaire survey, Tochi-

moto et al. (2008) collated data using questionnaires on

the past distribution of A. japonicus in Hyogo Prefecture,

western Honshu, Japan. A distribution map of A. japoni-

cus was produced from the combined responses of oral

interviews, answers to written questionnaires, and data

from previous publications. Oral interviews were con-

ducted with 17 people from fishermen’s associations, two

people from the nature conservation society in Hyogo

Prefecture, and 21 people recommended by the fisher-

men’s associations as veiy familiar with A. japonicus.

The interviews were supported by information obtained

through written questionnaires provided by the Boards of

Education of 44 municipalities.

6. Electrofishing

Electrofishing requires a backpack voltage generator,

connected to two submersible electrodes, earned by a

researcher walking slowly through a stream. Amphib-

ians and other aquatic vertebrates are first attracted to

the electrical field of the electrodes and then temporarily

paralyzed (Reynolds 1983).

Williams et al. (1981) considered electrofishing

with seining effective for surveying C. alleganiensis.

However, subsequent studies have not supported this

conclusion (Bothner and Gottlieb 1991; Nickerson and

Krysko 2003). In extensive river sections where large

populations were found using other survey techniques,

electrofishing failed to reveal C. alleganiensis (Nicker-

son and Krysko 2003). Electrofishing failed to locate C.

alleganiensis during surveys on the Susquehanna drain-

age in New York, whereas turning rocks was successful

(Soule and Lindberg 1994). Substantial rock cover and

poor water currents can result in shocked C. alleganien-

sis not moving from beneath rocks during electrofishing

(Nickerson and Krysko 2003).

A two-year population study of another large aquatic

salamander, the Common mudpuppy ( Necturus macu-

losus ), concluded that electrofishing was ineffective in

surveying sites with large populations (Matson 1990).

Nevertheless, there are examples of successfiil electro-

fishing for aquatic salamanders, especially when sala-

mander abundance is being associated with other species

abundance including fish. Maughan et al. (1976) used

electrofishing to successfully survey the Pacific giant sal-

amander ( Dicamptodon ensatus ), and Nakamoto (1998)

exhaustively surveyed both fish and D. ensatus using

multiple passes with backpack electrofishing. Occa-

sionally, C. alleganiensis are incidentally captured with

electrofishing by fisheries biologists during late summer/

early autumn.

Because of its potential to harm salamander health

and reproduction the use of electrofishing for surveys is

not generally recommended, and should be confined to

occupancy surveys of special conservation significance

where other techniques are not effective. Electrofishing

is well known for causing spinal injuries and mortality in

fish (Cho et al. 2002; Wang et al. 2004), and there is po-

tential for electric shock to reduce salamander reproduc-

tive success (particularly during the breeding season) and

to damage the immune system (Nickerson and Krysko

2003). Electrofishing can seriously affect the health of

critically endangered fish such as the Chuanshan taimen

{Hucho bleekeri), and electrofishing is banned in the

range of H. bleekeri in Taibai, Shamixi Province, China

(W. Zhenghuan, pers. comm.)

Nevertheless, electrofishing may be the best tech-

nique for occupancy surveys in some difficult habitats

where the detection of threatened salamanders is of ma-

jor conservation significance (Nickerson and Krysko

2003). Wang et al. (2004) reported the capture of eight

A. davi dianus with electrofishing, whereas nocturnal sur-

veys revealed none and bow-hooks only one (Zhang and

Wang 2001).

7. Underwater camera systems

The use of waterproof video systems for surveys mini-

mizes habitat disturbance, and video systems can locate

den sites, record reproduction and behavior, and provide

other valuable information on Cryptobranchid biology.

Waterproof video systems are very effective where Cryp-

tobranchids utilize heavy large rocks or bedrock crevices

for shelter.

Black and white cameras have been used success-

fully. However, suitably small underwater color cameras

are now available. Although color cameras are less light

sensitive than black and white, the use of color is more

efficient at revealing salamanders and eggs. We are not

aware of an “off the shelf’ video camera system opti-

mal for surveying all Cryptobranchid species, or one

that incorporates all features needed for efficient aquatic

surveys. However, there are two relatively inexpensive

systems available suitable for surveys of aquatic sala-

manders: 1) fishing video systems, and 2) inspection

cameras.

Fishing video systems (12 volt) can easily be modi-

fied for surveys of Cryptobranchids. However the water-

proof charged couple device (CCD) cameras associated

with these systems are too large to access many crevices.

These cameras are also relatively bulky and better suited

to use from a small boat or canoe. Inspection cameras

are very lightweight, and with small camera heads, have

proven effective for surveying C. alleganiensis. A limita-

tion of both systems is that standard monitors are rela-

tively small and are not waterproof.

Video systems are being developed by researchers

that are waterproof, lightweight, and incorporate a wire-

less camera system, digital recorder, and video goggles.

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Browne et al.

The video recorder, battery pack, and wireless compo-

nents are placed inside waterproof bags and worn in a

backpack. Improved waterproofing of video goggles and

some components of wireless inspection cameras would

provide greater flexibility in using these systems.

In addition to utilizing video camera systems for ac-

tive surveying, cameras may be left in the field as a pas-

sive survey technique, if connected to a 12 V (volt) sur-

veillance digital recorder. Batteries for the recorder need

replacement, and data must be retrieved approximately

once a week, depending on battery size and data storage

capabilities of the recorder. Batteries are heavy and trans-

port for recharging is arduous, but solar panels could be

used to provide electricity in remote but secure locations.

8. Passive integrated transponders (PIT) and

mark-recapture

PIT tags are small, waterproof, glass-encased capsules

containing an alphanumeric code read with a portable

reader. PIT tags are generally inserted sub-dermally with

a syringe and needle, have life spans of at least 10 years,

and are relatively inexpensive. PIT tags are available as

read-only tags containing unique factory-set alphanu-

meric codes or as read-write tags that can be changed

to any value. The new read/write PIT tags enable details

to be recorded, retrieved or changed using the receiver,

including the GPS location, habitat, tagger’s name, and

contact information. Gorsky et al. (2009) used 23 mm

read/write PIT tags to assess Atlantic salmon ( Scilmo sa-

lar ) migratory path selection. Although the size of PIT

tags has steadily decreased, the detection range increases

with PIT tag size. The standard reader ranges for read-

only PIT tags are 3-8 cm for the smallest microchips (1.5

x 7 mm) and 15-45 cm for the largest (34 mm). Fish less

than 55 nun have been successfully tagged using 11.5

mm PIT tags that weigh 0.1 g, and the smallest PIT tags

now available should be suitable for all but the smallest

Caudata.

A promising new technique, for surveying and locat-

ing salamanders in shallow water habitats is the use of

submersible antennae and larger PIT tags that have been

detected up to 90 cm through water (Hill et al. 2006)

and detection range should further increase through im-

provements in antenna technology (Hamed et al. 2008).

Cucherousset et al. (2008) showed that detecting Pyre-

nean brook salamanders ( Calotriton asper ) using PIT te-

lemetry was 30% more efficient for individual sampling,

and four times as efficient in sampling over time, than

direct sampling through visual searching and rock turn-

ing. The efficiency of PIT telemetry was negatively cor-

related with the presence of large stones that blocked the

PIT signal, and positively correlated with the number of

easily sampled spring inlets and undercut banks (Cucher-

ousset et al. 2008).

Figure 9. Trap used to capture C. alleganiensis in the Allegh-

eny River drainage during the summers of 2004 and 2005. Bait

(White sucker, Catostomus commersonii) was attached to the

inside of the hinged door of a wire mesh cage. The bait cage

was later removed and replaced using plastic zip ties. From

Foster et al. 2008. Used with permission from Herpetological

Review.

Bub et al. (2002) showed that when PIT tags were

hidden within different stream microhabitats, more than

80% were subsequently located with portable antennas.

Hill et al. (2006) tested specialized “PIT pack” antenna

systems and found that design modifications and reduced

equipment weight made PIT packs easy to use. The read

range of optimized PIT packs approached 90 cm when

the PIT tag was submerged in water. Breen et al. (2009)

found a detection efficiency of 76% with PIT-tagged fish

using a portable antenna investigating displacement,

mean movement distance, and home range of Mottled

sculpins ( Cottus bairdii).

Prior to PIT tagging, photographs of head or tail

spotting patterns were used to identify post metamor-

phic individual A. japonicus for mark-recapture studies

(Kawamichi and Ueda 1998; Tochimoto 1991; Tochi-

moto et al. 2005). PIT tagging is the most common tech-

nique for mark-recapture studies. For example, Tochi-

moto et al. (2005) recorded 1204 individual salamanders

in the Ichi River, Hyogo Prefecture, between 1975 and

2004, with 588 of these PIT tagged between 1998 and

2004. Okada (2006) tagged more than 500 individuals in

Tottori Prefecture between 2001 and 2008.

Wheeler (2007) used the BioMark® submersible

antenna with a detection distance of up to 30.5 cm to sur-

vey for previously PIT tagged C. alleganiensis. Of six

C. alleganiensis marked using PIT tags, surveyors were

able to detect only two the following day. A search of

the area with rock turning did not detect any additional

C. alleganiensis. The four undetected C. alleganiensis

had either moved into water deeper than the reach of the

detector wand antenna (two meters) or moved under the

cobble substrate (Wheeler 2007).

Automatic systems to survey movement have been

used with PIT tags in fisheries research. These consist of

remote antenna arrays spanning water bodies. Meynecke

et al. (2008) successfully used remote PIT technology

to monitor fish movement for 104 days in a mangrove

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Survey techniques for giant salamanders

Table 1 . The advantages and disadvantages of survey techniques.

Survey technique

Advantages

Disadvantages

1. Wading, turning substrate,

netting, and snorkeling.

Low equipment costs. Simple and rapid surveying.

Snorkeling provides better vision and a closer proximity to

exposed C. alleganiensis. Rocks can be tilted more easily

due to buoyancy and water currents can provide “lift” of

rocks.

Cannot sample deep water, surveyor strain and fatigue are high,

and there is considerable habitat disturbance. Risks of blind

searches include bites and cuts and rock turning can result in be-

ing held under water by a trapped arm. Some institutions will not

allow surveying alone due to risk of injury. Costs for wetsuits,

mask, snorkel, dive boots, and other equipment. Transporting

heavy equipment (along shallow mountain streams) and working

in high velocity areas can produce increased surveyor strain and

fatigue.

2. Scuba/hookah diving

Deeper water habitats can be surveyed that are not acces-

sible to other methods besides traps and trot-lines. Diving

enables prolonged submergence, with less fatigue than

snorkeling, at depths of one to two meters. Systematic

checking of all cover and ensuring the capture of all

exposed Caudata.

Surveying multiple sites requires the transport and handling

of many air cylinders. Refilling air cylinders when at remote

survey sites requires extensive transportation time. Requires

substantial equipment costs including scuba or hookah equip-

ment and sometimes boats, and extensive training time and costs.

Diving is more dangerous than other surveying methods. It is

time consuming to sanitize snorkeling, scuba and hookah diving

equipment.

3. Nocturnal spotlighting

Nocturnal lighting creates little habitat disturbance,

and enables the simultaneous survey of other nocturnal

amphibians.

Potential costs of equipment (lights and boats), limited visibility

through poor water clarity, and increased safety concerns.

4. Bow-hooks/trot-lines

Efficient for the detecting of the presence/absence and

population assessment of Cryptobranchids at low popula-

tion densities.

Bow -hooks (using fishing hooks) can cause injuries to sala-

manders, increase salamander stress over hand collecting, and

increase predation risk. Bow-hook lines should be made too short

to reach the esophagus and possibly cause injuries.

5. Questionnaires

Regional assessment of occupancy.

Relies on credibility of respondents.

6. Electrofishing

Presence/absence and population surveys in difficult habi-

tats of major conservation significance.

Electrofishing for surveys is not generally recommended because

of its potential to harm salamander health and reproduction and

its use should be confined to occupancy surveys of special con-

servation significance where other techniques are not effective.

Electrofishing has high equipment costs, a number of particular

safety concerns, and requires several surveyors working together.

7. Underwater camera sys-

tems

Minimal habitat disturbance, location of den sites, record-

ing of reproduction and behavior, and provision of other

information on Cryptobranchid biology. Video camera

systems can provide a passive survey technique in combi-

nation with a digital recorder.

Problems with waterproofing, battery charging and supply, lim-

ited water depth, and viewing monitors in bright sunlight. Costs

can be high with this method for camera, recorder, and monitor,

and only a single site can be monitored per camera.

8. Passive integrated tran-

sponders (PIT) and mark

recapture

Recorded information can be retrieved from tagged

salamanders (with limited habitat disturbance) enabling

calculation of movement and dispersal. Allows tracking of

confiscated animals.

Only previously tagged animals are detectable, a relatively short

detection range, the workable water depth being limited by wand

length, and detection range limited by shelter type and depth.

PIT tag surveys using hand readers are economical; however,

optimized antenna systems are costly. PIT tags can be lost.

9. Radiotelemetry

Monitoring of individuals to study movements, habitat

use, and survival. Smaller, lighter, longer-lived, and more

reliable units have increased the efficacy of radio-tracking

with increasingly smaller individuals.

Surveys can be costly due to the initial expense of transmitters,

antennas and receiver. Surgical implant is required for attaching

transmitters to salamanders.

10. Modular artificial spawn-

ing dens and rock placement

Modular artificial spawning dens provide efficient means

to support critical spawning habitat, enable monitoring of

egg and larval survival, and survey male and female occu-

pancy and movement. Further development of the capacity

to provide camera surveillance will increase all the above.

Modular artificial spawning dens are relatively easy to construct

but there are material and labor costs. They are heavy and require

vehicular transport and a team to place in selected locations.

Their stability under exceptionally high stream velocities, in

comparison to natural rock dens, is untested.

11. Wire mesh baited traps

Trap surveying is not hampered by deep, turbid, or cold

water. There are low levels of habitat disturbance, and sites

with very heavy rocks and ledges can be surveyed.

Material and labor costs for trap construction, and supplying a

large amount of fresh bait. Setting traps is labor intensive and

transporting traps to remote areas may be prohibitive. Trapping

should not be performed during the breeding season because

females may spawn in the traps, and trapped males cannot guard

dens. Flooding may carry away traps. Lost traps may be a hazard

to wildlife. As with all unguarded equipment, theft or vandalism

may be a problem.

12. Population genetic tech-

niques

Minor tissue sampling enables ongoing studies of the

number and significance of genetic subpopulations, loss

of genetic variation, migration and dispersal, effective

population size, and parentage. Samples can be sub-

divided and provide material indefinitely for future work

and comparison.

Contamination and poor storage of samples limits analysis.

Cryptobranchids and some other Caudata have low genetic varia-

tion, which can limit the use of techniques. More sophisticated

genetic techniques are expensive.

13. Environmental DNA

(eDNA) detection

Inexpensive, no habitat disturbance, can be used in streams

difficult to monitor by other methods, shows occupancy.

Targeted primers need to be designed to amplify a species-spe-

cific short DNA fragment. Laboratory costs per sample and the

need for several samples to exclude false positives or negatives.

Efficiency depends on DNA shedding rates, population demog-

raphy, water temperature, and thermal properties, to estimate

population size.

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Browne et al.

stream and recorded more than 5000 detections with a

recapture rate of 40%. River monitoring systems for fish

commonly use four different types of antennas: pass-

through, flat plate, crump weir, and circular culvert an-

tennas. Flat plate detectors appear ideal for salamanders

as they can be up to six meters in size, are buried slightly

in the streambed, and can detect salamanders up to 45 cm

above the plate.

The problem of PIT tag loss can be substantially re-

duced by careful application and sealing of the insertion

site (Christy 1996). A coincidental value of PIT tagging

to conservation is that resource managers and interna-

tional border inspectors can utilize PIT tags to identify

home locations of confiscated salamanders.

9. Rodiotelemetry

Radiotelemetry can consistently be used to monitor indi-

vidual animals and has been used to study movements,

habitat use, and survival of many vertebrate species

(Kenward 2001). Radio transmission can be received

in turbid waters, stream flows, or depths that preclude

traditional survey techniques (e.g., rock turning and vi-

sual searches). Surveys using radio-telemetry with C.

alleganiensis have investigated dispersal (Gates et al.

1985b), site fidelity, and frequency and timing of move-

ments (Coatney 1982; Blais 1996; Ball 2001). These

surveys have revealed the use of unique microhabitats

including bedrock ledges, root masses, and bank crev-

ices (Blais 1996) as well as the location of den sites and

causes of mortality (C. Bodinof, pers. comm.).

Monitoring by radiotelemetry requires attachment

of a very high frequency (VHF) radio transmitter to the

target salamander. Each transmitter is tuned to a unique

frequency and emits a pulsed radio signal allowing an

observer to locate individual salamanders. Optional sen-

sors to detect motion, pressure, depth, or temperature can

be incorporated into radio transmitters. To extend battery

life, microcontrollers have been developed to turn trans-

mitters on and off at preset times (Rodgers 2001). Tech-

nological advances have resulted in smaller, lighter, lon-

ger-lived, and more reliable units. Such advances have

increased the efficacy of radio-tracking in increasingly

smaller organisms while minimizing concern for adverse

effects of transmitter attachment.

Several methods of transmitter attachment have

been used with varying success for Cryptobranchids, in-

cluding 1) coelomic implant (Blais 1996), 2) subcutane-

ous implant (Blais 1996), 3) force-feeding (J. Briggler,

pers. comm.), 4) neck collar (Wheeler 2007), and 5) su-

turing through the tail (Olcada et al. 2006; Wheeler 2007;

Blais 1996).

Wheeler (2007) observed poor retention with exter-

nal tail attachments, as well as collars fastened around

the neck of C. alleganiensis. However, Okada et al.

(2006) reported that transmitters attached externally (su-

tured through the tail) to large A. japonicus were retained

for two to four months and caused minimal injuries. Ra-

dio transmitters were force fed and retained for 1 8 to 30

days (Coatney 1982), and 16 to 25 days (Blais 1996), in

C. alleganiensis with no harm. Force-feeding transmit-

ters may be useful for detecting untagged Cryptobran-

chids, which aggregate during a relatively short breeding

season. Surgical implantation of transmitters should be

performed by an experienced veterinarian or biologist

(Fuller et al. 2005), and amphibians should be given am-

ple recovery time from effects of anesthesia and surgery

before release (Byram and Nickerson 2008).

A recommendation to minimize the effect of trans-

mitter attachment is the use of the smallest possible tag.

Transmitters also should not exceed 3-5% body mass and

researchers should use the least conspicuous attachment

technique (Withey et al. 2001). Jehle and Amtzen (2000)

used very small transmitters of 0.5 g to track individual

Tritnrus spp. above a minimum acceptable body mass of

8.0 g. PIT tag tracking may be useful for salamanders

smaller than 8.0 g, but radio tracking antenna systems

are cheaper, and radio tracking has a much greater range

than PIT tags. Different sizes, battery life, outputs, and

ranges of these and various other transmitter models

have been used for radio-tracking Caudata. While trade-

offs exist among unit weight, detection range, and bat-

tery life, many small units offer > six months of battery

life. Resources providing an overview of radio-tracking

technology and study design include Fuller et al. (2005),

Millspaugh and Marzluff (2001), and White and Garrott

(1990).

Radiotelemetry studies of Caudata include T. crista-

tus, T. marmoratus (Jehle and Amtzen 2000), Ambysto-

ma maculatum (Madison 1997; Faccio 2003 ),A.jefferso-

nianum (Faccio 2003), A. californiense (Trenham 2001),

C. a. alleganiensis (Gates et al. 1985a; Blais 1996; Ball

2001), C. a. bishopi (Coatney 1982), and A. japonicus

(Okada et al. 2006).

10. Modular artificial spawning dens and rock

substrate placement

A recent innovation in survey techniques for Crypto-

branchids is development of modular artificial spawn-

ing dens. Bankside artificial dens have been used for A.

japonicus in channelized habitat (where suitable sites

were lacking), and in artificial streams for reproduction

during fanning of A. davidianus. The Ozark Hellbender

Working Group developed modular spawning dens for

C. alleganiensis that proved highly successful in attract-

ing C. alleganiensis and providing spawning sites. Dens

made of ferrocement are light, simple, and economical

to construct. Artificial dens offer the possibility of incor-

porating underwater video systems giving discrete and

continuous monitoring of occupancy and activity. Rocks

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Survey techniques for giant salamanders

have been placed in streams to similarly provide habitat

and increase survey efficiency for C. alleganiensis.

11. Wire mesh baited traps

Cryptobranchus alleganiensis have been surveyed over

several years using baited traps in deep water habitat of

some larger (7 th order) rivers (including the Gasconade

River, Missouri, USA). Such habitats have proved dif-

ficult to survey without trapping due to their depth (> 5

m maximum) and often very turbid waters (lateral Sec-

chi Disk <1.0 meters visibility). The efficiency of baited

traps varies with water temperature (Nickerson 1980);

trapped C. alleganiensis in deep rivers in Missouri were

greatest during the peak foraging period in spring and

very low during the summer breeding season. When wa-

ter temperatures reached above 22 °C, capture rates were

veiy low. Besides seasonal effects, trapping is highly de-

pendent on how the trap is set. Foster et al. (2008) had

greatest success when bait was fresh and the trap was

flush with the substrate.

Wire mesh baited traps have been widely used to

survey Cryptobranchids using a variety of baits. Cryp-

tobranchus alleganiensis can detect baits from consid-

erable distances (Townsend 1 882; Nickerson and Mays

1973), and smelly, fresh baits are most successful in

trapping. Traps baited with chicken livers proved unsuc-

cessful with C. alleganiensis (Soule and Lindberg 1994).

Foster et al. (2008) used similar traps successfully when

baited each day with fresh fish; fresh meat bait proved

unsuccessful. Kern (1984) successfully captured C. al-

leganiensis using hoop-nets baited with fresh sucker fish

(Carpi odes sp.). Trapping with crab traps baited with

strong smelling saltwater baits (such as sardine, mack-

erel, or squid) was effective for catching adult A. japoni-

cas (S. Okada, pers. comm.). When surveying Crypto-

branchids, the bait bags should be strong enough to resist

tearing from salamander bites and the possible ingestion

of bag material. Trapping should not be performed dur-

ing the breeding season because females may spawn in

the traps, and trapping can prevent males from guarding

nests.

The Missouri Department of Conservation, USA,

has a major survey program for C. a. alleganiensis us-

ing traps in habitats unsuitable for other methods. Trap

design was modified from those used by Foster et al.

(2008; Figure 8) by placing a funnel on both ends and

making the traps collapsible to reduce storage space.

Numerous bait types (chicken liver, crayfish, carp, and

Gizzard shad) were used as bait, but fresh Gizzard shad

(Dorosoma cepedianum ) was the most successful bait.

Besides the bait used, the general success of trapping is

also highly dependent upon how the trap is set.

Trapping is a valuable sampling technique used for

C. alleganiensis. In a comparative study, Foster et al.

(2008) reported on three techniques of surveying Hell-

benders: rock turning, bank searches, and trapping. Rock

turning had the highest capture efficiency but damaged

the habitat; bank searches were effective at finding juve-

niles. Besides its use in habitat accessible to other tech-

niques, trapping was useful for water slightly exceed-

ing the maximum depth possible with other techniques

and in areas with unmovable rocks or difficult-to-access

ledges. Trapping may be more effective for capturing the

largest size classes (Figure 10; Foster et al. 2008). Trap-

ping is similarly effective for catching adult A. japonicus

(S. Okada, pers. comm.). Snorkeling, scuba, or hookah

diving combined with trapping would enable better trap

placement, especially at greater depths.

12. Population genetic techniques

Genetic information can guide conservation breeding

programs determining the number and significance of

genetic subpopulations. Using increasingly sophisticated

genetic techniques, evolutionary phylogeny, paleoge-

ography, species status, migration, effective population

size, parentage, and population bottlenecking can be as-

certained. Surveys using molecular techniques to assess

population genetic structure, variation, and migration

patterns have rapidly progressed over the last 1 0 years.

This progress has been largely driven by improved se-

quencing and computer analysis, Information Technol-

ogy systems, and a growing bank of genetic techniques

and resources (GenBank Database 2009).

Mitochondrial techniques are useful for understand-

ing relationships among and historical changes within

populations (Sabatino and Routman 2009), however,

mitochondria are maternally inherited and only track fe-

male lineage.

Genomic microsatellite markers, together with mito-

chondrial DNA information, may provide the most infor-

mative phylogenetic information. Microsatellite markers

have the advantage of requiring very little tissue (even

less than used in mitochondrial sequencing techniques)

and this allows for noninvasive sampling such as buc-

cal swabs. Polymorphic microsatellite markers have very

recently been published for C. a. bishopi (Johnson et al.

2009) and C. a. alleganiensis (Unger et al. 2010).

13. Environmental DNA (eDNA) detection

Environmental DNA (eDNA) has recently been con-

firmed as a sensitive and efficient tool for inventorying

aquatic vertebrates in lotic and lentic aquatic habitats.

Under the Amphibian Research and Monitoring Initia-

tive, U.S. Geological Survey scientists and their partners

developed an efficient protocol for detecting eDNA from

two amphibian species that occur in low density, fast-

moving stream water; the Idaho giant salamander (Di-

camptodon aterrimus ) and the Rocky Mountain tailed

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Browne et al.

frog (Ascaphus montanus). Environmental DNA analy-

sis costs approximately US$30. Sampling efficiency in-

creases in comparison with fieldwork, for example, by

20 times for D. citerrimus and 11 times for A. montanus

(direct survey population estimates of 0. 1 6 and 0.04 indi-

viduals per m 2 , respectively). With Asian carp, sampling

cost efficiencies increase from 16 to 100 times when

compared to field searches. The sensitivity of an eDNA

test depends on the sampling of five to 10 litres of wa-

ter, the amount of DNA shed by the target species, and

the thermal and chemical properties of the water. False

negative rates can be estimated using repeated sampling,

and the probability of false positives can be excluded by

careful primer design and protocol testing using related

non-target species (Goldberg et al. 2011).

Conclusion

Cryptobranchids are iconic amphibians that provide

a range of conservation challenges. Of all the aquatic

amphibians, Cryptobranchids appear to offer the great-

est potential to link amphibian conservation with water-

shed management. They also offer the greatest potential

to apply a suite of modem and innovative techniques to

conservation strategies. Their long-term survival is high-

ly dependent on the effectiveness of these survey tech-

niques to elucidate population stmcture and demography,

bottlenecks in recruitment, threats, and critical habitat

components.

There is a wide variety of survey techniques to de-

tect, capture, and track Cryptobranchids and other aquat-

ic Caudata. However, these techniques vary widely in

efficacy, and a combination of several techniques will

prove most effective at providing critical information

on occupancy and status. Each survey technique has ad-

vantages, disadvantages, and biases depending on survey

objectives (Nickerson and Krysko 2003).

When choosing survey techniques, a primary con-

cern is animal welfare. The preservation of nest sites and

other critical habitat is essential, as is limiting the spread

of pathogens. Suitable C. alleganiensis nesting sites are

increasingly scarce in many locations, and in some lo-

cations siltation is destroying the sites that remain. Un-

derwater camera systems are the only survey techniques

that do not disturb habitat, especially when used with

artificial spawning dens. Only radiotelemetry, PIT tag-

ging with long-range detection, and environmental DNA

(eDNA) detection enable ongoing sampling without fur-

ther habitat disturbance (Nickerson and Krysko 2003).

Wading shallow water and turning substrate, includ-

ing leaves and gravel, is a simple way to survey Crypto-

branchids and may be efficiently combined with surveys

of larvae and juveniles. Survey efficiency for adult and

larval Cryptobranchids, and other Caudata through rock

turning, is improved by the use of downstream seines.

Scuba or hookah diving are the only techniques that de-

tect all sizes of gilled larvae and multiple age groups of

non-gilled and adult Cryptobranchids within short sur-

vey periods, but they are one of the most expensive and

training-intensive methods. The use of eDNA promises

the most rapid and cost effective survey technique for the

inventory of Caudata.

Final remarks: Cryptobranchids are one of the most

endangered groups of Caudata, having highly specialized

habitat requirements at different life stages. Various sur-

■ Rock Turning

<11 11 -20 21 -30 31 -40 41 -50 51 -60 >60

Size Class

Figure 10. The relative success of three capture techniques in locating various size classes of C. alleganiensis. From Foster et al.

2008. Used with permission from Herpetological Review.

amphibian-reptile-conservation.org

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December 2011 | Volume 5 | Number 4 | e34

Survey techniques for giant salamanders

vey techniques offer a range of advantages and disad-

vantages, and surveys should include several techniques

to reduce bias. Cryptobranchids’ high site fidelity and

reliance on easily damaged critical habitat components

make them vulnerable to survey techniques that require

disturbing habitat structure. Therefore, the choice of sur-

vey technique should always include minimum habitat

disturbance and potential to affect salamander health.

Equipment must be sanitized when moving among sites

to limit the spread of pathogens.

Acknowledgments. — We thank Takeyoshi Tochimoto

for advice on the manuscript, A1 Breisch for trap design,

and Ken Roblee, Robin Foster, and Noelle Rayman for

their dedication to Cryptobranchid conservation. We also

thank the Cryptobranchid Interest Group (CIG), and the

“Hellbender Symposiums” in the US for their contribu-

tion to Cryptobranchid research. This work was sup-

ported by core funding from the Flemish Government.

Special thanks to Ken Dodd for his comments on this

manuscript.

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Published: 11 December 2011

ROBERT BROWNE is co-editor of Amphibian and Reptile Conservation having a wide range of

academic and practical experience in many research fields supporting herpetological conservation

and environmental sustainability. Robert designs and produces the ARC website.

HONG LI received her M.Sc. in 2003 in microbiology from West China Normal University, People’s

Republic of China (PRC). Hong then worked in the USA and the PRC with endangered amphibians

including the critically endangered, Wyoming toad (Bufo baxteri ) and Chinese giant salamander

( Andrias davidianus).

DALE MCGINNITY has a wide experience in herpetology and currently works as Curator of Ec-

totherms at Nashville Zoo at Grassmere, Tennessee, USA. Dale has worked with species as diverse

as the Komodo dragon and was featured in a documentary about them. Dale designed the impres-

sive Herpetarium at Nashville Zoo. Dale is currently managing conservation breeding populations

of Galliwasps and initiated the first program to perpetuate the genetic variation of any amphibian

through the spenn cryopreservation of Cryptobranchus alleganiensis alleganiensis.

SUMIO OKADA is a post-doctoral research associate at Tottori University, Japan. He conducts

research focused on ecology and conservation biology of amphibians and reptiles, especially, the

Japanese giant salamander ( Andrias japonicus). He serves as Vice President of the Japanese Giant

Salamander Society.

KELLY J. IRWIN works at the Arkansas Game and Fish Commission. He grew up in northeastern

Kansas, USA where he developed an avid interest in the local amphibians and reptiles. He has

written or co-authored more than 75 popular articles and peer-reviewed papers on herpetology and

vertebrate paleontology.

CATHERINE M. BODINOF is broadly interested in stream ecology and currently employed as a

resource staff scientist with the Missouri Department of Conservation, USA. She recently completed

her M.S. which focused on the first attempt to augment Cryptobranchus alleganiensis bishopi popu-

lations via release of captive-reared individuals.

ZHENGHUAN WANG is an associate professor in the School of Life Sciences, East China Normal

University, Shanghai, PRC. In 2001 he became involved with conservation biology programs aimed

at protecting wild population of the Chinese giant salamander ( Andrias davidianus).

AMY MCMILLAN trained as a population geneticist at the University of Kansas in Lawrence,

Kansas, USA. She is presently in the Biology Department at Buffalo State College in Buffalo, New

York, USA (http://www.buffalostate.edu/biology/). Her current research with Cryptobranchus al-

leganiensis involves the genetic variation and structure of populations.

JEFF BRIGGLER has been the herpetologist for the Missouri Department of Conservation since

2000. He received his M.S. and Ph.D. degrees from the University of Arkansas, Fayetteville, Arkan-

sas, USA. Jeff promotes, protects, and monitors amphibian and reptile populations in Missouri, and

has been leading hellbender conservation efforts in Missouri since 2001.

NOTE: Expanded author bios can be accessed on the ARC website under Author Biographies at: http://www.redlist-arc.org/Authors-biographies.html

amphibian-reptile-conservation.org

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December 2011 | Volume 5 | Number 4 | e34

mons Attribution-NonCommercial-NoDerivs 3.0 Unported License, which permits unrestricted use for non-com-

mercial and education purposes only provided the original author and source are credited.

Amphibian & Reptile Conservation 5(4):17-29.

The giant salamanders (Cryptobranchidae): Part A.

palaeontology, phytogeny, genetics, and morphology

Robert K. Browne, 2 Hong Li, 3 Zhenghuan Wang, 4 Paul M. Hime, 5 Amy McMillan, 6 Minyao Wu, 7 Raul

Diaz, 8 Zhang Hongxing, 9 Dale McGinnity, and 10 Jeffrey T. Briggler

1 Center for Research and Conservation, Royal Zoological Society of Antwerp, Antwerp, BELGIUM '-Polytechnic Institute of New York University,

New York, USA 3 School of Life Sciences, East China Normal University, Shanghai, PEOPLE’S REPUBLIC OF CHINA 4 University of Kentucky,

Department of Biology, Lexington, Kentucky, USA 5 Buffalo State College, Buffalo, USA 6 Shaanxi Normal University, Xi ’an, Shaanxi Province, PEO-

PLE’S REPUBLIC OF CHINA 1 University of Kansas Medical Center, Kansas City, Kansas, USA * Shaanxi Institute of Zoology, Shaanxi Institute

of Endangered Zoology Species, Xi ’an, Shaanxi Province, PEOPLE ’S REPUBLIC OF CHINA 9 Nashville Zoo at Grassmere, Nashville, Tennessee,

USA 10 Missouri Department of Conservation, Jefferson City, Missouri, USA

Abstract. — The Cryptobranchidae, commonly called the Giant Salamanders, are the largest surviv-

ing amphibians and comprise two extant genera, Andrias and Cryptobranchus . There are three

cryptobranchid species, the Chinese giant salamander ( Andrias davidianus ; 180 cm, 59 kg), the

Japanese giant salamander (A. japonicus; 155 cm, 55 kg), and the North American giant salaman-

der (Cryptobranchus alleganiensis ; 74 cm, 5.1 kg). Because of their iconic status as the world’s

largest amphibians and their biopolitical significance, all cryptobranchids are subject to major and

expanding initiatives for their sustainable management. Cryptobranchids are biologically similar

in many ways; however, within these similarities there are differences in their habitats, diet, size,

reproductive behavior and seasonality, fecundity and egg size, paternity, and growth and develop-

ment. These characteristics are a consequence of their palaeontology, phylogeny, genetics, and

morphology. Cryptobranchid conservation genetics reveal the evolutionary significant units (ESUs)

toward which conservation and research efforts must be directed to provide genetically competent

individuals for rehabitation or supplementation programs. Knowledge of these scientific fields in

concert with cultural, political, and economic factors all contribute to cryptobranchid conservation

biology and the formulation of optimal strategies for their sustainable management. However, there

has previously been no comparative review of the numerous scientific fields contributing to the

knowledge of cryptobranchids, and little peer-reviewed material on A. davidianus and A. japonicus

has been published in English. Here we present the first article in a series about cryptobranchid

salamanders, “The giant salamanders (Cryptobranchidae): Part A. paleontology, phylogeny, genet-

ics, and morphology.”

Key words. Giant salamander, cryptobranchid, palaeontology, phylogeny, genetics, morphology, conservation, sus-

tainable management, Cryptobranchidae

Citation: Browne RK, Li H, Wang Z, Hime PM, McMillan A, Wu M, Diaz R, Hongxing Z, McGinnity D, Briggler JT. 2012. The giant salamanders (Crypto-

branchidae): Part A. palaeontology, phylogeny, genetics, and morphology. Amphibian & Reptile Conservation 5(4):17-29(e54).

Introduction

“The giant salamanders (Cryptobranchidae): Part A. pal-

aeontology, phylogeny, genetics, and morphology” is the

first of a series of three review articles that have been

produced to review the biology and sustainable man-

agement of giant salamanders. Although there has been

much published on giant salamanders, the information

has previously been scattered within articles on each of

the three species largely in languages of their biopolitical

regions: Mandarin Chinese, Japanese, and English.

To maximize the potential for the sustainable manage-

ment of these species, the public and scientific communi-

ty must have access to accurate knowledge about them to

direct policy and provide for Internet-based information

and news portals. Consequently, “The Giant Salaman-

ders (Cryptobranchidae)” suite of articles, review and

discuss a broad range of biological data known for gi-

ant salamanders, which have been collected globally by

researchers and enthusiasts over a period of four years.

Different authors have made varying contributions to

each article depending on their area of expertise. Howev-

er, due to the complexity of rewriting and contributing to

Correspondence. Email: hvbert.browne@gmail.com (corresponding author) 2 pandakittylhong@gmail.com : 'zhwang@bio.ecnu.

edu.cn 4 paul.hime@uky.edu 5 MCMILLAM@buffalostate.edu 6 minyaowu@snnu.edu.cn 1 lissamphibia@gmail.com % kunyang@pub.

xaonline.com 9 dmcginnity@nashviUe.org 10 Jeff Briggler@mdc.mo.gov

amphibian-reptile-conservation.org

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Figure 1 . A North American giant salamander ( Cryptobranchus alleganiensis ) shows the characteristic morphology of the crypto-

branchids; large robust dorso-ventrally flattened head and body, small eyes, thick legs with stubby digits, lateral folds of skin for

respiration, and sensory papillae for detecting water movement and prey (laterally flattened tail not shown). Image and copyright

by Ray Miebaum.

the suite of articles as it has progressed over many years,

we have included all authors on all articles. The major

contributing authors to “The giant salamanders (Cryp-

tobranchidae): Part A. palaeontology, phytogeny, genet-

ics, and morphology” are Am y McMillan and Paul Hime

(genetics), Raul Diaz (palaeontology, genetics), and Paul

Hime (phytogeny).

The caudate superfatnily, Crytobranchoidea is one of

the most ancient amphibian clades and comprises two

families Cryptobranchidae and Hynobiidae, totalling 5 1

species. The family Cryptobranchidae derives its name

from the Ancient Greek, “kryptos” (hidden) and “bran-

chos” (gill), which originally referred to the gills which

must be hidden in adults as they lack external gills, un-

like most aquatic vertebrates (larvae have external gills).

The Cryptobranchidae, or “Giant Salamanders,” are the

largest surviving amphibians and comprise two genera,

Andrias and Cryptobranchus. There are only three extant

cryptobranchid species, the Critically Endangered, Chi-

nese giant salamander {Andrias davidianus Blanchard,

1871), the Near Threatened, Japanese giant salamander

(A.japonicus Temminck, 1936), and the North American

giant salamander {Cryptobranchus alleganiensis Daudin,

1803) which exists as two formally named subspecies, C.

a. alleganiensis and C. a. bishopi (Petrankal998).

The Crytobranchoidea, along with probably (Larson

2003) the fully aquatic caudate family Sirenidae are ex-

ceptional within the Caudata (salamanders) in having the

reproductive mode of external fertilization (Duellman

and Trueb 1994). As giant salamanders are the largest

amphibians in their respective major biopolitical regions,

they are conservation icons, not only for threatened am-

phibians but also, for the sustainable management of wa-

tersheds. Sustainable management requires providing the

broadest range of educational material that relates to both

public interest and species conservation. This knowledge

can then be used by field, conservation breeding, and

culturally engaged conservationists, to provide the best

technical approaches to species conservation, and pro-

vide a background for the required political and financial

support.

A critical part of this knowledge is the paleontologi-

cal history and phytogeny to show a species’ evolution-

ary significance, and how a species fits into the tree of

life; while conservation genetics shows its evolutionary

significant units (ESUs) for directing conservation and

research efforts. However, there has been no comparative

review of the conservation biology of cryptobranchids

and associated scientific fields, and little peer-reviewed

information of the conservation biology of A. davidianus

and A. japonicus has been published in English.

Here we review “The giant salamanders (Crypto-

branchidae): Part A. paleontology, phytogeny, genetics,

and morphology” in concert with “The giant salaman-

ders (Cryptobranchidae): Part B. range and distribution,

demography and growth, population density and size,

habitat, territoriality and migration, diet, predation, and

reproduction” and “The giant salamanders (Cryptobran-

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Giant salamanders: palaeontology, phylogeny, genetics, and morphology

Figure 2. Fossil salamanders strongly support an east Asian (red ellipse) origin for the Cryptobranchoidea. The continents were

distributed very differently in the Mid- Jurassic (170 MYA) before continental drift moved them to their present locations. However,

Eurasia and North America remained in the Northern Hemisphere. By the Late Pliocene (3 MYA) the continents had moved to

their present positions. Image courtesy of palaeos site: http://palaeos.com/mesozoic/jurassic/midjura.html. Adapted from Gao and

Shubin, 2003.

chidae): Part C. etymology, cultural significance, conser-

vation status, threats, sustainable management, reproduc-

tion technologies, aquaculture and conservation breeding

programs, and rehabitation and supplementation.”

Palaeontology and phylogeny

The Cryptobranchoidea is comprised of the giant sala-

manders, family Cryptobranchidae (found in China, Ja-

pan, and eastern North America), and the Asiatic sala-

manders, family Hynobiidae (found throughout Asia

and European Russia). From fossil evidence in Asia, the

evolutionary origins of the Cryptobranchidae extend to

at least the Mid- Jurassic (160 million years ago [MYA];

Gao and Shubin 2003), with their fossils later being

known from Europe, Asia, and North America. Fossils of

more recent cryptobranchids from the Late Eocene (40

MYA) to the Early Pliocene (5.3 to 3.6 MYA) are known

from two genera and two or three species from over 30

Eurasian localities (Bohme and Ilg 2003). Molecular and

morphological studies strongly suggest an Asian origin

for cryptobranchids with subsequent expansions into Eu-

rope and North America by the Upper Paleocene (3.6 to

2.5 MYA). The expansion into North America was prob-

ably facilitated by the resumption of ice ages creating a

land bridge between Asia and North America during the

Late Pliocene-Early Quaternary glaciation that started

about 2.6 million years ago (Kruger 2008).

This basal caudate salamander family has experi-

enced remarkable morphological stasis throughout its

evolution, with ancient and modem Cryptobranchids

being morphologically very similar. The Late Oligocene

(23.0 MYA) to Early Pliocene (5.3 MYA) species A.

scheuchzeri was distributed from Central Europe to the

Zaissan Basin on the border of Kazakhstan and China.

Vasilyan et al. (2010) considered from fossil and paleo-

climatological evidence that both fossil and extant An-

drias species occur in regions with annual precipitation

from 90 to 130 cm.

The monophyly of the Cryptobranchoidea (Hynobi-

idae + Cryptobranchidae) has not been a point of conten-

tion (Gao and Shubin 2003; Larson and Dimmick 1993;

Larson et al. 2003; Frost et al. 2006; Roelants et al. 2007;

Pyron and Wiens 2011), though the base of the salaman-

der phylogeny, relative to the placement of widely ac-

cepted clades, has been contentious for many decades,

specifically due to the placement of Sirenidae and the re-

lationship of other paedomorphic taxa (see: Wiens et al.

2005; Vieites et al. 2009). Salamanders have displayed a

relatively conserved tetrapod body plan, at least since the

Jurassic Period (Vieites et al. 2009). The independent-

ly derived paedomorphic morphology (a heterochronic

change where sexually mature adults retain several as-

pects of the larval body plan) displayed by several rec-

ognized families, has played a central role in discussions

of salamander morphology, and whose morphological

characters have been considered to play a substantial

confounding role in phylogenetic reconstmction.

Fossil cryptobranchids from the Late Eocene to the

Early Pliocene are known from two genera and two or

three species from over 30 Eurasian localities (Bohme

and Ilg 2003; Milner 2000). Phylogenetic and paleonto-

logical evidence suggests an East Asian origin for cryp-

tobranchids by, at latest, the Cretaceous (135-100 MYA),

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Figure 3. The Late Oligocene to Early Pliocene (23.0 to 5.3 MYA) species A. scheuchzeri was distributed from Central Europe to

the Zaissan Basin on the border of Kazakhstan and China. Fossil room II, Teylers Museum, The Netherlands Andrias scheuchzeri

Oeningen. Courtesy of: http://en.wikipedia.org/wiki/Andrias_scheuchzeri

with subsequent expansions into Europe and North

America by the Upper Paleocene (Milner 2000) via

the Asian-American interchange (Duellman and Trueb

1994), though an alternate scenario has been proposed

but not widely accepted (Naylor 1981). This basal cau-

date family has experienced remarkable morphological

stasis throughout its evolution, with ancient and modem

cryptobranchids appearing very similar, and neoteny be-

ing present since the time of early salamander origins

(Gao and Shubin 2001; Gao and Shubin 2003). Andrias

are morphologically conservative and their skeletons are

so similar thatH. davidianus has been considered a junior

synonym of A. scheuchzeri (Westphal 1958).

Currently recognized fossil cryptobranchids include

Chunerpeton tianyiensis (Gao and Shubin 2003), the ear-

liest crown-group member, Crvptobranchus ( =Andrias ?)

saskatchewanensis (Naylor 1981), and Piceoerpeton

willwoodensis (Meszoely 1967; described from a single

vertebra). Cryptobranchus guildavi (Holman 1977) was

also described, based on limited samples and whose va-

lidity had previously been questioned (Estes 1981; Nick-

erson 2003), but whose apomorphies have recently been

dismissed due to as yet undescribed intraspecific skeletal

variation for C. alleganiensis, and the misidentification

of the ceratohyal, which was actually a sacral rib; this

taxon is thus synonymous with C. alleganiensis (Brede-

hoeft 2010). Andrias matthewi has also been described

from Nebraska from a single mandible (Cook 1917; see

Estes and Tihen 1964; and Naylor 1981). Zaissanurus

beliajevae has been described from the Eocene/Oligo-

cene of Mongolia and Russia while Aviturus exsecratus

and Ulanurus fractus have been described from the Pa-

leocene of Mongolia (Gubin 1991; Milner 2000).

Cryptobranchoid salamanders (Hynobiidae + Cryp-

tobranchidae) share several synapomorphies including:

high chromosomal counts (Hynobiidae: 2 n [diploid num-

ber] = 40-78 and Cryptobranchidae: 2 n = 60); extremely

large nuclear genomes (Hynobiidae: 15.2-46.5 Gbp

[Giga base pairs] and Cryptobranchidae: 45.5-53.8 Gbp)

(Gregory 2012. Animal Genome Size Database, http://

www.genomesize.com [Accessed: 12 June 2012]); pres-

ence of a hypoglossal foramen and nerve (Fox 1957; Fox

1959); fusion of the first hypobranchial and first cerato-

branchial into a single structure, as well as the fusion of

the M. pubotibialis and M. puboischiotibialis (Duellman

and Trueb 1994); and retention of a separate angular bone

in the lower jaw (Fox 1954; Fox 1959; Zhang et al. 2006;

Vieites et al. 2009). Members of the Cryptobranchoidea

display other primitive features such as external fertil-

ization (also present in Sirenidae) and the production of

eggs either as paired clusters (hynobiids) or strings (cryp-

tobranchids), with one set from each oviduct (Duellman

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and Tmeb 1994). Cryptobranchid salamanders are spe-

cialized for an aquatic habitat of cold, fast flowing, rocky,

and oxygen rich streams (Petranka 2010).

Extensive epidermal folds (with a dense subsurface

capillary network) are present along the flanks of the

trunk and limbs to increase surface area, serving as a

body length “gill” for oxygen exchange, with the lungs

thought to function only for buoyancy (Guimond and

Hutchison 1973). Larval cryptobranchids have a dorsal

tail fin and short external gills as do the majority of trans-

forming salamanders. Adult Cryptobranchus maintain a

single pair of gill clefts, while all are closed in Andrias

(Duellman and Trueb 1994; Dunn 1922; Meszoely 1966;

Rose 2003). The development of an angular bone and

lack of a septomaxilla, lacrimal, and os thyroideum are

shared skeletal characters of cryptobranchids (Fox 1954,

1959; Rose 2003), while diagnostic generic differences

are the presence of four bones contributing to the border

of the naris in Cryptobranchus (premaxilla, maxilla, na-

sal, and frontal), with a lack of the frontal bone contact-

ing the naris in Andrias (Dunn 1922; Meszoely 1966).

Cryptobranchus also fails to resorb the third and fourth

ceratobranchials (Rose 2003). Other skeletal and ontoge-

netic differences can be found in Rose (2003).

Cryptobranchoidea, from genetic inference, are con-

sidered to have evolved during the Middle to Late Ju-

rassic (Gao and Shubin 2003; Roelants et al. 2007; San

Mauro et al. 2005; Zhang et al. 2005; Mueller 2006;

Wiens 2007; Zhang and Wake 2009), while some re-

searchers estimate early Cretaceous (Marjanovic and

Laurin 2007; San Mauro 2010). Mitochondrial and

nuclear DNA analysis shows the family Cryptobranchi-

dae is a monophyletic group (e.g., Weisrock et al. 2005;

Matsui et al. 2008; Zhang and Wake 2009) and that the

two genera within this family, Cryptobranchus (North

America) and Andrias (Asia) diverged between the Late

Cretaceous to the Paleocene (around 70 MYA; Matsui et

al. 2008; Zhang and Wake 2009). The sister taxa A. ja-

ponicus and A. davidianus likely diverged in the Pliocene

(about 4.3 MYA) and are considered separate species de-

spite a small degree of genetic differentiation (Matsui et

al. 2008). The root of the Cryptobranchus mtDNA tree

likely lies on the branch leading to the Current, Eleven

Point, and New Rivers, and a common ancestor in the

southern Ozarks and/or southern Appalachians is hy-

pothesized to have given rise to all other populations,

which is consistent with a Pleistocene refuge for this spe-

cies as ice sheets covered the more northern regions until

approximately 1 1 ,000 Before Present (BP) (Sabatino and

Routman 2009).

In a recent study by Wiens et al. (2005), it was re-

vealed that not simply the “presence” of “paedomorphic”

characters, but rather the lack of clade synapomorphic

characters were what misled phylogenetic analyses. This

plasticity in the development of adult/terrestrial charac-

ters has allowed for convergence toward morphologi-

cal/ecological specialization in the larval aquatic envi-

ronment (which secondarily misleads reconstructions).

Variation in the “larval” traits in these groups presents

a special problem in that not all paedomorphic traits are

shared across all clades/species (Wiens et al. 2005), with

cryptobranchids presenting an adult skull more similar to

those of other fully transformed salamanders (Duellman

and Tmeb 1994; Rose 1999; Rose 2003; Wiens et al.

2005).

Early morphology-based systematic studies placed

Cryptobranchoidea as sister to all remaining salaman-

ders, with the exception of the Sirenidae which are placed

as basal on the phylogeny (Duellman and Tmeb 1994).

The classic study by Larson and Dimmick (1993), com-

bining both molecular and morphological data, placed

Sirenidae as sister to all extant salamanders and the early

rRNA molecular dataset of Larson (1991) placed Sireni-

dae nested within the salamander tree. Current support

for the basal placement of Cryptobranchoidea has come

from molecular, morphological, and mixed datasets (Gao

and Shubin 2001 ; Gao and Shubin 2003; San Mauro et al.

2005*; Wiens et al. 2005*; Zhang et al. 2005*; Frost et al.

2006; Marjanovic and Laurin 2007; Mueller 2006; Wang

and Evans 2006; Roelants et al. 2007; Vieites et al. 2009;

Pyron and Wiens 2011;* = subsets of analyses presented

these relationships), while the basal placement for Sireni-

dae has come from morphology and some reconstructed

phytogenies comprised of molecular and mixed datasets

(Duellman and Trueb 1994; Larson and Dimmick 1993;

San Mauro et al. 2005 §; Wiens et al. 2005 §; § = subsets

of analyses presented these relationships).

Recent studies utilizing whole mitochondrial genome

sequences (Zhang and Wake 2009) and mitochondrial

genome and nuclear sequences (albeit, with limited

taxon sampling; San Mauro 2010) placed Sirenidae as

sister to all salamander families. San Mauro et al. (2005)

placed (Sirenidae + Cryptobranchoidea) as sister to all

other extant salamanders based on sequence from the 3’

end of Rag-1. The characters analyzed (i.e., inclusion or

exclusion of reproductive morphology and “paedomor-

phic” traits) and methodology used for phylogenetic re-

construction have played significant roles in the affecting

the output of relationships; for this article we follow the

Cryptobranchoidea placed basal on the phylogeny and

Sirenidae sister to all other extant lineages (as in Vieities

et al. 2009, Roelants et al. 2007, and Pyron and Wiens

2011). Nonetheless, we emphasize that deep salamander

relationships are not clearly resolved at present.

Conservation genetics — Species and

Evolutionary Significant Units

The basis of conservation genetics is identifying the ge-

netic variation within a clade and within its comprising

species, and consequently defining species and their ge-

netic sub-populations in conservation categories as Evo-

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MYA 336 294 252 210 16S 126 84 42 0

CAECILIANS

HYNOBIIDAE

CRYPTOBRANCHUS

ANDRIAS

SIRENIDAE

ALL OTHER

SALAMANDERS

FROGS AND TOADS

Figure 4. Phylogenetic tree showing ancestry of cryptobranchids and their hypothesized relationships to other amphibians. Adapt-

ed from Roelants et al. 2007.

lutionary Significant Units (ESU; sensu Wood and Gross

2008). This knowledge in combination with geography

defines the range and distribution of species and their

ESUs. This knowledge can then be used to perpetuate the

genetic variation of the species through a range of prac-

tices based on the primary management unit, the ESU.

An increasing focus on cryptobranchid conservation, and

recent advances in genetic technologies, has resulted in a

rapid increase in our knowledge of cryptobranchid con-

servation genetics.

The molecular techniques used to assess population

structure, migration patterns, and their relationship to

genetic variation, have rapidly progressed over the last

10 years. This progress has been largely driven by more

rapid and cheaper sequencing and computer analysis, In-

formation Technology systems, and a growing bank of

molecular techniques and resources (GenBank 2012).

Genetic variability in cryptobranchids has been defined

with several types of molecular markers including al-

lozymes, mitochondrial DNA (mtDNA) sequencing and

Figure 5 a, b. Taking tissue samples from tail clips (Image: Amy McMillan ) or blood samples (Image: Jeff Briggler ) enables con-

servation geneticists to assess an individual’s relationship to other individual cryptobranchids and the relationship of its population

to other populations of the same species.

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Restriction Fragment Length Polymorphisms (RFLP),

Amplified Fragment Length Polymorphisms (AFLP),

and microsatellites. Older techniques used to estimate

genetic structure and diversity, such as allozyme assays,

required sampling whole organisms and may have nega-

tively impacted population numbers. More recent Poly-

merase Chain Reaction (PCR) based techniques includ-

ing AFLP, mitochondrial sequencing, and micro satellite

markers take advantage of very small amounts of tissues

that can be sampled without harm (Tanaka-Ueno et al.

2006).

For example, Foster (2006) collected small amounts

of shed blood (amphibian erythrocytes are nucleated)

when PIT tags were inserted subcutaneously, or sampled

a small tail clip from C. alleganiensis that quickly re-

generated. Blood samples also can easily be taken from

the caudal veins of larger salamanders (see figure 5a).

Tanaka-Ueno et al. (2006) found buccal swabbing was

the most efficient non-invasive technique for sampling

genetic material from caudata. Newer, non-invasive

techniques, including environmental DNA (eDNA) sam-

pling, have proven successful for detecting amphibian

species in streams (Goldberg et al. 2011) and may prove

useful for detection of cryptobranchids in natural habitats

(Browne et al. 2011).

Mitochondrial markers have been used to resolve

both inter- and intra-specific phylogenetic relationships

as well as assess broad-scale population genetic struc-

ture. However, mtDNA is maternally inherited and so

only tracks female lineages. Polymorphic microsatellite

loci are typically found in non-coding or neutral regions

within the genomic DNA, and their markers are currently

the most commonly used genetic marker for studies of

fine-scale population genetic structure in cryptobran-

chids. However, emerging methods for high-throughput

genetic analysis promise to expand the scope of crypto-

branchid conservation genetics to a genome-wide scale.

Many areas of cryptobranch id research are likely to ben-

efit greatly from ongoing efforts to obtain genome-wide

nuclear sequence data, including transcriptome analysis

(P. M. Hime, data not shown) and genomic analysis (R.

L. Mueller, data not shown) in Cryptobranchus.

Polymorphic microsatellite markers can be robust

and easily detected on either acrylamide gels or with

fluorescence-based detection methods and are available

for Cryptobranchus a. alleganiensis (Unger et al. 2010),

C. a. bishopi (Johnson et al. 2009), Andrias davidianus

(Meng et al. 2008; Yoshikawa et al. 2011), and A.japoni-

cus (Yoshikawa et al. 2011). However, as the field of con-

servation genetics enters the genomic era, genome-wide

molecular datasets will become increasingly available for

cryptobranchids. These will enable deeper insights into

their evolutionary history and cryptobranchid conserva-

tion genetics. Through using increasingly sophisticated

genetic techniques phylogeny, paleogeography, species

status, migration, effective population size, parentage,

and population bottlenecking can eventually be known.

Andrias davidianus: Allozyme assays and mitochon-

drial DNA sequences revealed more variability in A. da-

vidianus than in A. japonicus (Murphy et al. 2000). Tao

et al. (2005) sequenced the mitochondrial control region

of A. davidianus from the Yangtze, Yellow, and Pearl

River regions and found low nucleotide and haplotype

diversity within regions, especially the Yangtze River.

Both of these studies showed very little differentiation in

A. davidianus between regions. The population from the

Huangshan area in China was genetically distinct from

other areas, which suggests localized divergence, prob-

ably due to genetic drift and a lack of gene flow between

this and other populations (Murphy et al. 2000). Despite

the low genetic diversity, Muiphy et al. (2000) found

substantial substructure among A. davidianus popula-

tions but poor geographic correlation, even between the

three major river systems in China. Nevertheless, Tao et

al. (2005) discovered significant phylogeographic differ-

ences between the Pearl and Yangtze River regions, and

between the Pearl and Yellow River regions. The genetic

patterns discovered in these studies suggest that A. david-

ianus have a much higher gene flow between populations

than either A. japonicus and Cryptobranchus allegani-

ensis (see below). Extensive human-mediated movement

of A. davidianus may have begun over 3,700 years ago

before the advent of historic Chinese Civilization by the

Zhang Dynasty (3782-3058 BP; Ebrey 1996); the use of

A. davidianus for medicine and food may have led to its

human mediated transportation and thus may have facili-

tated this higher gene flow (Murphy et al. 2000).

Andrias japonicus : Early allozyme assays revealed

little genetic diversity within A. japonicus (Matsui and

Hayashi 1992). Mitochondrial DNA sequence variation

is also relatively low but nevertheless indicates genetic

subdivisions into central and western clades (Matsui et al.

2008). Matsui et al. (2008) noted that the low genetic dif-

ferentiation in A. japonicus contrasted strongly with that

of sympatric and also totally aquatic Hynobius species

(Cryptobranchoidea). They suggested that the reduced

genetic variability in A. japonicus may be attributed to

polygyny by gigantic males with late sexual maturity and

high longevity, a stable aquatic environment as habitat,

as well as bottleneck effects during Quaternary glacia-

tions (1.8 MYA to 20,000 BP). They suggested that the

low genetic variation of A. japonicus may make the spe-

cies prone to increased risk of extinction. Matsui and

Tominaga (2007) found some nuclear genomic diversity

in A. japonicus in a study of AFLPs but were not able to

differentiate any geographic groups not identified with

mtDNA methods.

Cryptobranchus alleganiensis: Early allozyme assays

revealed very little genetic diversity across the range

of C. alleganiensis (Merkle et al. 1977; Shaffer 1989).

However, mtDNA RFLP and mtDNA sequencing stud-

ies revealed enough genetic diversity in C. alleganiensis

to detect putative clades or management units (Rout-

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Figure 6. An early figure of Japanese giant salamander, An-

drias japonicus , showing the dorso-ventrally flattened tail, the

very broad head, and massive bulk of the Andrias species. The

skeleton has remained almost unchanged for tens of millions of

years. Image from G. Mdsch, Der Japanische Riesensalaman-

der und der fossile Salamander von Oeningen, Neujahrsblatt

der NGZH Nr 89, 1887. Cryptobranchus japoniens Y. deHoev.

(Japanischer Riesensalamander.) Nach einer Photographie

gezeichnet, in etwas mehr als 1/3 der naturlichen Grosse.

man 1993; Routman et al. 1994; Sabatino and Routman

2009), a finding that was recently supported by nuclear

microsatellite DNA markers (Tonione et al. 2011).

The monotypic genus Cryptobranchus has tradition-

ally been divided into two distinct subspecies based on

morphology and geography. The Ozark hellbender (C. a.

bishopi ) is only found in the Ozark Highlands of Mis-

souri and Arkansas, whereas, the Eastern hellbender (C.

a. alleganiensis ) ranges throughout eastern North Ameri-

ca; from eastern New York and Pennsylvania to the north

and east, Mississippi, Alabama, and Georgia to the south,

and Missouri to the west (Conant and Collins 1998).

Cryptobranchus a. bishopi is characterized by large dark

blotches on the dorsum and dark mottling along the chin,

while C. a, alleganiensis has small spots on the dorsum

and a uniform chin pattern (Petranka 1998). Cryptobran-

chus a. bishopi was described as a separate species by

Grobman (1943), but current taxonomy recognizes the

Ozark hellbenders as a subspecies.

Recent mitochondrial and microsatellite analyses

have shown greater than previously recognized genetic

variation in Cryptobranchus. These analyses suggest that

this group is paraphyletic with respect to the currently

recognized subspecies designations, and may potential-

ly harbor unrecognized diversity. However, the species

status of genetically distinct entities within this genus

has yet to be examined in a comprehensive framework.

Crowhurst et al. (2011) used nuclear microsatellite loci

to show that C. a. bishopi is genetically distinct from C.

a. alleganiensis, but that within the Ozark region there

are two strongly supported groups that are as genetically

distant from each other as each is from all C. a. allegani-

ensis samples combined. When the Ozark and Eastern

hellbender samples were analyzed separately, the eastern

samples resolved as two groups, albeit with weaker sup-

port than the Ozark sample distinction. This finding is

not trivial for Cryptobranchus conservation. The Ozark

subspecies was listed on the US Fish and Wildlife Endan-

gered Species List in November, 2011 (US Government,

2011 No. FWS-R3-ES-2009-0009) and both subspecies

have been included on Appendix III of the Convention on

International Trade in Endangered Species of Wild Fauna

and Flora (CITES).

Work by Sabatino and Routman (2009) using mito-

chondrial sequencing, and by Tonione et al. (2011) us-

ing microsatellite markers, recovered eight independent

groups of C. alleganiensis which the authors advocated

should be treated as separate ESUs. These are the North-

ern Ozarlcs, Ohio, and Susquehanna Rivers, Tennessee

River, Copper Creek, North Fork of the White River,

Spring River, New River, and Current/Eleven Point Riv-

ers. These studies show that gene flow is severely re-

stricted or non-existent among these eight major groups

(as measured by the markers under investigation), and

potentially among populations (rivers) within groups.

Use of highly polymorphic microsatellite markers allows

assignment of individual samples to specific manage-

ment units. For example, Crowhurst et al. (2011) cor-

rectly assigned Ozark samples >91% of the time and a

new Hellbender population in Georgia had an 84% prob-

ability of membership with an adjacent Tennessee River

(Albanese et al. 2011).

Morphology and morphometries

Andrias : The heads of Andrias are wide and flat reach-

ing 1/5- 1/4 of the snout-vent length. On their heads and

necks, A. davidianus has paired small tubercles arranged

in rows and A. japonicus large, single, and scattered

tubercles. With both species tubercles are interspersed

with abundant tiny sensory neuromasts that detect wa-

ter movement and the presence of prey (Lamioo 1987).

Their snouts are rounded with small nostrils near the

snout tip, and their eyes are small and without eyelids.

A labial fold is prominent at the posterior of the upper

jaw. Their tongue with free lateral margins adheres to the

mouth floor. Thick skin folds are present at the lateral

side of the body and there are 12-15 costal grooves. All

four limbs are short and stout with four fingers and five

toes and lack skin folds or prominent interdigital web-

bing.

Tail length is between 59 and 80% of the snout- vent

length. The dorsal fin of the tail is prominent and the ven-

tral fin only conspicuous nears the vent (Fei et al. 2006).

Coloration exhibits great variation. The skin of A. davidi-

anus is dark brown, black or greenish and A. japonicus

is reddish-brown with a paler venter; irregularly blotched

and marbled with dusky spots (Chang 1936; Thom 1969).

Juveniles often have lighter coloration with small black

flecks. Albinos (white or golden) have been recorded (Fei

et al. 2006). There is no obvious sexual dimorphism in

cryptobranchids, except during the breeding season when

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Giant salamanders: palaeontology, phylogeny, genetics, and morphology

mature males have an enlarged cloaca and females have a

swollen belly when gravid (Niwelinski 2007). The larvae

of A. davidianus have longer gills, their fingers and toes

are more pointed, and their color darker than the larvae of

A. japonicus. External gills disappear when total length

reaches 170-220 mm (Fei et al. 2006).

Cryptobranchus : The head is strongly flattened, with

small eyes and wrinkled fleshly folds of skin along each

side of the body for respiration. Coloration exhibits great

variation. The base coloration of C. alleganiensis ranges

from grayish-black to tan and olive-green across the ma-

jority of the body (Nickerson and Mays 1973). The Ozark

form Cryptobranchus a. bishopi, has many black blotch-

es on the dorsum and the lower lips, while the dorsum of

C. a. alleganiensis bears black spots rather than blotch-

es, and the throat region may have pale spots (Petranka

2008). Albinos and morphs (orange to red patterns) have

been occasionally observed (Dyrkacz 1981; Nickerson

and May 1973; Fautli et al. 1996). Cryptobranchus re-

tains a single pair of gill slits as adults unlike Andrias.

Sexual dimorphism (enlarged cloaca in males and swol-

len belly in gravid female) is only obvious during the

late summer to autumn breeding season. The larval stage

of C. alleganiensis lasts 1-1.5 years during which they

grow to 12.5 cm in length, gradually lose their external

gills, and develop internal gills and a circular opening

on each side to provide water for respiration, as well as

development of fleshly fold along the sides of the body

for respiration.

Acknowledgments. — We thank Helen Merdith, Zoo-

logical Society of London, and Sumia Okada for their

invaluable contribution. The Hellbender Symposiums,

USA, provided not only a gateway to current knowledge

but also encouragement and enthusiasm for this review

series. Finance for core funding from the Flemish Gov-

ernment.

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Received: 14 June 2012

Accepted: 01 July 2012

Published: 30 September 2012

Robert Browne is co-editor of Amphibian and Reptile Conservation having a wide range of academic

and practical experience in many research fields supporting herpetological conservation and environmental

sustainability.

Hong Li received her M.Sc. in 2003 in microbiology from West China Normal University, Peoples Repub-

lic of China (PRC). Hong then worked in the USA and the PRC with endangered amphibians including the

critically endangered, Wyoming toad (Bufo baxteri ) and Chinese giant salamander ( Andrias davidianus).

Zhenghuan Wang is an associate professor in the School of Life Sciences, East China Normal University,

Shanghai, PRC. In 2001 he became involved with conservation biology programs aimed at protecting wild

population of the Chinese giant salamander ( Andrias davidianus).

Paul Hime is broadly interested in speciation, population genetics, phylogenetics, and genome evolution

in vertebrates. Paul previously worked on the conservation of the Ozark hellbender at the Saint Louis

Zoological Park. He is currently a Ph.D. student at University of Kentucky where he elucidates genomic

approaches to delimiting species boundaries in Cryptobranchus and the design of a genetic sex assays for

cryptobranchids.

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Giant salamanders: palaeontology, phylogeny, genetics, and morphology

Amy McMillian trained as a population geneticist at the University of Kansas in Lawrence, Kansas, USA.

She is presently in the Biology Department at Buffalo State College in Buffalo, New York, USA (http://

www.buffalostate.edu/biology). Her current research with Cryptobranchus alleganiensis involves the ge-

netic variation and structure of populations.

Minyao Wu is a Professor at the College of Life Science, Shaanxi Normal University, Xi’an, PRC. He re-

searches gene transfer, stem cells, and wild animal breeding and reproduction for threatened species such as

the giant panda, Chinese giant salamander, golden takin and ibis, and also in amphibian disease diagnostics

and mitigation strategies.

Raul Diaz is currently a Ph.D. candidate at the University of Kansas Medical Center/Stowers Institute

for Medical Research. His research includes the developmental genetics and evolution of the vertebrate

skeleton, focusing on the reptile cranial and appendicular skeletons, gastrulation, embryology, and trying to

unweave the genetics of species morphological divergence and adaptation through the use of genomics and

classic morphological techniques. Raul has since worked in over 20 countries and hopes to bridge the field

of biomedical research with the study of biodiversity/evolution and conservation.

Zhang Hongxing is a Professor at the Shaanxi Institute of Endangered Zoology Species, Xi’an, Shaanxi

Provence, Peoples Republic of China.

Dale McGinnity has a wide experience in herpetology and currently works as Curator of Ectotherms at

Nashville Zoo at Grassmere, Tennessee, USA, where he designed the impressive Herpetarium at Nashville

Zoo. Dale is currently managing conservation breeding populations of Galliwasps and initiated the first

program to perpetuate the genetic variation of any amphibian through the sperm cryopreservation of Cryp-

tobranchus alleganiensis alleganiensis.

Jeffrey T. Briggler has been the herpetologist for the Missouri Department of Conservation since 2000.

He received his M.S. and Ph.D. degrees from the University of Arkansas, Fayetteville, Arkansas, USA.

Jeff promotes, protects, and monitors amphibian and reptile populations in Missouri, and has been leading

Hellbender conservation efforts in Missouri since 2001.

amphibian-reptile-conservation.org

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September 2012 | Volume 5 | Number 4 | e54

Attribution-NonCommercial-NoDerivs 3.0 Unported License, which permits unrestricted use for non-commer-

cial and education purposes only provided the original author and source are credited.

Amphibian & Reptile Conservation 5(4): 51-63.

In vitro culture of skin cells from biopsies from the Critically

Endangered Chinese giant salamander, Andrias davidianus

(Blanchard, 1871) (Amphibia, Caudata, Cryptobranchidae)

^arah Strauli, 2 ’ 3 ’ 5 Thomas Ziegler, Christina Allmeling, ^erstin Reimers, 2 Natalie Frank-Klein,

4 Robert Seuntjens, and 4 Peter M. Vogt

l Ambystoma Mexicanum Bioregeneration Center, Department of Plastic, Hand- and Reconstructive Surgery, Hannover Medical School, Carl-Neu-

berg-Str. 1, 30625 Hannover, GERMANY 2 Cologne Zoo, Riehler Strafie 173, 50735 Koln, GERMANY 3 Cologne Biocenter, University of Cologne,

Zulpicher Strasse 47b, 50674 Cologne, GERMANY 4 Berlin Zoo, Hardenbergplatz 8, 10787 Berlin, GERMANY

Abstract . — We established a primary skin cell culture of the Critically Endangered Chinese Giant

Salamander, Andrias davidianus , from small biopsies using minimal invasive methodologies. Bi-

opsies were taken from three animals simultaneously with assessment of two biopsy sampling

techniques using samples from the tail tip. Cell culture was performed in a wet chamber at room

temperature. Several culture media and supplementations were tested as well as culture contain-

ers and surface coatings. The handling of A. davidianus in a landing net, without transfer out of the

tank, allowed easier biopsy withdrawal. Best outgrowth of cells from explants was achieved in 60%

DMEM/F12 medium with supplementation. Cells started to grow out as monolayer within the first 12

hours, and after three weeks formed pigmented multilayers, then died after 10 weeks. Primary cul-

tures of Andrias skin cells, as well as other amphibian primary cell cultures, can be used in future

studies to evaluate effects of disease, pollution, regeneration, wound healing, and could provide

cells for use in reproduction technologies such as cryopreservation to preserve gene lines in this

and other Critically Endangered species with minimal harm to the animals.

Key words. Caudate cell culture, skin tissue explants, skin biopsy, biopsy withdrawal, amphibian skin cell culture,

regeneration, wound healing

Citation: Straufi S, Ziegler T, Allmeling C, Reimers K, Frank-Klein N, Seuntjens R, Vogt PM. 2013. In vitro culture of skin cells from biopsies from the

Critically Endangered Chinese giant salamander, Andrias davidianus (Blanchard, 1871) (Amphibia, Caudata, Cryptobranchidae). Amphibian & Reptile

Conservation 5(4): 51-63 (e66).

Introduction

The Chinese giant salamander (Andrias davidianus ) is

the largest extant amphibian, with a total length of up

to 1 80 cm. Together with the Japanese giant salamander

(A. japonicus ) from central and southern Japan, and

the North American Hellbender ( Cryptobranchus

alleganiensis), these species form the sole members of

the giant salamander family Cryptobranchidae, which is

thought to be a basal family among caudate amphibians

(Gao and Shubin 2003; review Browne et al. 2011). This

family might be a survivor of a lineage that was already

present in the Jurassic (Gao et al. 2003). The Chinese

giant salamander is widespread in central, south-eastern

and southern China, although its range is now very

fragmented. The species inhabits streams and rivers in

mountainous forested areas, at elevations from 100 to

1,500 m above sea level. Once common, the species has

declined catastrophically over the last decades in their

natural habitats while millions of these animals are bred in

farms. Wild harvesting for human consumption is a major

threat to A. davidianus , along with habitat destruction and

degradation (IUCN 2012). Consequently, A. davidianus

is now very rare in nature. Andrias davidianus is listed in

Appendix I of the Convention on International Trade in

Endangered Species of Wild Fauna and Flora (CITES)

and is also listed as Critically Endangered on the IUCN

Red List of Threatened Species (IUCN 2012).

Research on diseases and other issues in salamanders,

including A. davidianus, often involves sacrifice of the

animals at the end of the experiments (e.g., Geng et

al. 2011). An alternative to whole animal experiments

that would minimize destruction of the animals is the

use of in vitro cell cultures. Such assays have already

been described for fishes. For example, Estepa et al.

(1993) described a cell culture model to study the viral

haemorraghic septicaemia virus in fin cells of rainbow

trout Oncorhynchus mykiss (Estepa 1993). For this assay

primary cultures from tissue explants of trout fins were

established and infected with the virus in vitro.

The purpose of the present study was to determine

whether it is possible to establish primary in vitro cultures

of the skin cells of A. davidianus from small biopsies

of tail tip tissue. Various cell culture media, surface

Correspondence. Email: 5 ziegler@koelnerzoo.de (Corresponding author)

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Fig. 1. Biopsy method one performed in the zoo of Berlin. The animal was housed in an exhibition tank. It was captured and trans-

ferred in a tub, resulting in aggressive reactions of the animal, which made the biopsy procedure very difficult. A: animal housing.

B: animal transferred into tub. C: handling of the animal to perform biopsies.

coatings and types of plastic containers were checked for

cell outgrowth and long term survival. We find that this

technique could serve as a feasible alternative to studies

that require the destruction of individual animals.

Material and Methods

Three adult A. davidianus were used for this study.

Sexing was done via ultrasound. One male was housed

at the Berlin Zoo in an aqua-terrarium (L340 x W160

x H220 cm) with 50 cm water depth with shelter and

decorative objects provided (Fig. 1 A). Water temperature

is 20 °C and water quality maintained by a sand-pressure

filter, and partial daily, and complete weekly water

changes. The remaining two adult A. davidianus were

housed at the Cologne Zoo Aquarium. The couple is

held in two concrete tanks (each LI 50 x W190 x H60

cm) with 50 cm water depth. The water (flowing water

system) is connected to a cooling system and an external

filter (OASE pond filter, Type Biotec Screenmatic)

with a capacity of 10,000 L/h. Water parameters are as

follows: temperature 20 °C, pH 7.3, conductivity 740 pS,

carbonate hardness 7, and total hardness 16. Illumination

is provided by T 26 fluorescent tubes (3x58 Watt). Tank

roofing consists of stainless steel fence (1 cm mesh size),

with one half being shaded each by styrofoam mats. Both

tanks can be connected through a sliding gate (W60 x

H60 cm) consisting of stainless steel wire (1 cm mesh

size). The ground substrate consists of gravel and sand

mixture with large roots. As hiding possibility, each tank

contains a shelter (female tank: L80 x W50 x H50 cm;

male tank: L125xW50xH50 cm) with entrance in front

and exit at the rear side (each opening arched, W36 x

HI 8 cm). Another adult male (not used for this study)

is held in a tank in the public area of the Cologne Zoo

Aquarium (L350 x W126 x H85 cm; temperature 14 °C,

pH value 7.3, conductivity 668 pS, carbonate hardness 7,

and total hardness 16; illumination: HQI spotlight, 400

Watt).

Biopsies

In order to keep the biopsy procedure as stress-free

and efficient as possible two methods were tested.

Method one (conducted at Berlin Zoo): 1) capture of

the salamander, and 2) placing it in a tub with water and

then taking biopsies from the tail tip (Fig. 1). Method

two (conducted at Cologne Zoo): 1) Capture of the

salamander in a landing net, and keeping it in its housing

tank and taking biopsies (Fig. 2). Minimally-invasive

biopsies were perfonned by using biopsy punches

(Stiefel GmbH, Coral Gables, USA), with 4 and 6 mm

biopsies taken from the tail tips of two males (Berlin

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In vitro culture of skin cells from the Chinese giant salamander

Fig. 2. Biopsy method two performed in the zoo of Cologne. Animals remained calm during the whole procedure and showed no

reactions regarding handling of their tails. A and B: capture of the animal in a landing net. C and D: biopsy procedure at tail tip by

use of biopsy punches. E: tissue inside a punch. F: transfer of tissue in tube with amphibian ringer solution for rinsing.

Table 1. Contents of Modified Amphibian Ringer Solution.

NaCI

100 mM

KCI

1.8 mM

MgCI 2

1 mM

CaCI 2

2 mM

HEPES

5 mM

and Cologne Zoo) and one female (Cologne Zoo). The

procedure was performed without anesthesia as pain

of biopsy is negligible, and consequently the risks of

anesthesia too high. The procedure was classified as

minimally invasive and perfonned in consent with the

veterinary commissioner of the Cologne Zoo and the

zoo’s veterinarians. Giant salamanders are noted for

their regenerative capacity, and consequently wound

medication was not perfonned.

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Fig. 3. Andrias tails nine months after biopsy procedures. Lost tissue was completely regenerated without scar formation or dyspig-

mentations. A: overview of tail. B and C: detail of tail tip.

Table 2. Cell culture media, supplements and coatings. Green highlight: optimal conditions for culture of Andrias skin cells.

Medium

Supplements

Coating

ITS

Sodium-P

NEA

A2P

P/S

Genta

HEPES

Collagen

PLL FS

none

Williams

Medium E

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

Leibovitz

L-15

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

DMEM/F12

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 U/ml

0.1 mg/ml

1 mM

50 pg/ml

50 U/ml

0.05 mg/ml

5 mM

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In vitro culture of skin cells from the Chinese giant salamander

Tissue Preparation

To reduce microbial contamination of cell cultures,

biopsies were rinsed in 60% (v/v) PBS (phosphate

buffered saline) or Amphibian Ringer Solution (see Table

1). These were salt solutions adapted to the osmolarity of

amphibian cells, at pH 7. The solutions were supplemented

with 50 U/ml penicillin/streptomycin (Biochrom) and

0.05 mg/ml gentamicin (Biochrom) to further support

reduction of microbial contamination. Samples of one

male A. davidianus (Berlin) were transported in cell

culture media without supplementation for four hours.

Samples of one male and one female animal (Cologne)

were rinsed carefully, directly processed for cell culture

without use of a cell culture workbench and after

adherence transported to their storage place within three

hours.

Biopsies were processed by cutting them into small

(1-2 mm) pieces. As only small tissue samples were

available, we decided to perfonn cell culture in small

containers. The choice was between flasks that could

be sealed thus making them suitable for transport of

the culture from the zoo to the lab and multi well plates

which are commonly used for cell culture assays. So 25

cm 2 tissue culture flasks were used especially for the

starting cultures and 24 and 12 well plates were tested as

Fig. 4. Cell outgrowth from tissue maintained in three types

of culture media. Within the first days no differences of cell

outgrowth in media types was observed. Pictures were captured

using phase contrast light microscopy on day 3. A: DMEM/

F12; scale bar 100 pm. B: Leibovitz L-15; scale bar 100 pm. C:

Williams Medium E; scale bar 100 pm.

well. Biopsy pieces were placed in plastic tissue culture

dishes, with or without coatings (see Table 2). Medium

(see Table 2) was added three minutes later. The volume

of medium was adjusted to size of the culture well or

flask, so tissue pieces were slightly immersed. Culture

containers were stored in a wet chamber under sterile

conditions at room temperature. Final concentration of

non-essential amino acids is provided in Table 3 and used

abbreviations and suppliers in Table 4.

Culture Containers

Following containers were examined for cell culture:

• 12 well plates, attachment surface of 3.6 cm 2 /well

(#92012, TPP, Trasadingen, Switzerland).

• 24 well plates, attachment surface 1.9 cm 2 /well (#

92024, TPP, Trasadingen, Switzerland).

• Microflask, attachment surface 10 cm 2 (#91234, TPP,

Trasadingen, Switzerland).

• Miniflask, attachment surface 25 cm 2 (# 90025 and

90026, TPP, Trasadingen, Switzerland).

• 24 well plates, attachment surface 1.9 cm 2 /well

(#CC7682, Cyto One, USA).

• Miniflask, attachment surface 25 cm 2 (#7.690, Greiner

Bio One, Frickenhausen, Germany).

Depending on manufacturer's production processes

adhesion surfaces of the containers might be treated

differently (e.g., plasma treatment of surfaces with

varying protocols), resulting in vaiying adhesion

conditions. As from mammalian primary cell culture is

known that not every cell type adheres on every type of

culture plastic, containers of various manufacturers were

examined for cell culture of Andrias skin tissue explants.

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Fig. 5. Melanophores migrating from tissue. Skin epithelial cells and melanophores after

two weeks of cultivation. The morphology of resident Andrias melanophores (A and B)

appeared similar to observations of Billingham et al. in cytology of pigmented guinea

pig skin. (C). Pictures were captured using phase contrast light microscopy. A: over-

view, migrating melanophores appeared rounded, whereas resident appear in the typical

dendritic form; scale bar 500 pm. B: resident melanophore; scale bar 50 pm. C: resident

melanophore (Billingham 1948).

Results

Biopsy in A. davidianus

Table 3. The final concentrations of non-essential amino acids

and ITS in cell culture pg/ml.

L-alanine

8.9

L-asparagine*H 2 0

L-aspartic acid

13.3

L-glutamic acid

14.7

Glycine

7.5

L-proline

11.5

L-serine

10.5

Insulin

Transferrin

5.5

Selenium A

0.0067

Media , Supplements and Coatings

All cell culture media were diluted to 60% (v/v) with

sterile distilled water to achieve appropriate osmolarity.

Media, supplementations and plastic coatings are listed

in Table 1 .

Cell culture material was coated by dropping solutions

on the surfaces and drying under sterile conditions under

a workbench, followed by three rinsing steps with sterile

distilled water. Afterwards surfaces were dried again

under sterile conditions. Coated surfaces were stored

under sterile conditions at 4 °C for a maximum of one

week. Media were changed twice a week. Cell outgrowth

was digitally photographed with an inverse microscope

and Cell D software (Olympus).

Method one resulted in aggressive reactions of the male

that made taking of the biopsy difficult (Fig. 1). With

method two both salamanders remained calm and did

not react to the biopsy taking, which took less than five

minutes (Fig. 2). Directly after biopsy the wounds bled

sparsely or not at all, and inflammation and/or infection

of the wounds did not occur. Healing took about two

months; the lost tissue was completely regenerated

without scar formation (Fig. 3).

Cell Culture

Cell culture was performed in a wet chamber at room

temperature. Initially, technical difficulties had to be

overcome resulting from low rates of adherence of the

tissue fragments. In 12 and 24 well plates and microflasks,

the tissue fragments adhered only in small proportions

(5%), whereas more than 80% of the fragments adhered

on the plastics of both types of miniflasks (Greiner and

TPP). Cells started to grow out from adhered tissue

under all culture conditions within 12 hours (Fig. 4).

Beside skin epithelial cells also melanophores grew out.

The melanophores appeared rounded during migration

processes whereas resident cells showed typical dendritic

morphology (Fig. 5).

Surface coatings did not result in better adherence or

enhanced outgrowth. Interestingly, outgrowth from the

female tissue appeared to be faster and spatially extended

more than those from the males. Whether this observation

is a general phenomenon or just occasional should be

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In vitro culture of skin cells from the Chinese giant salamander

Fig. 6. Comparison of male (A) and female (B) tissue after three days of cell culture. Note that more outgrowing cells were observed

in the female samples. A: male tissue at day three; scale bar 500 pm. B: female tissue at day three; scale bar 500 pm.

examined in further studies with higher numbers of

tested individuals. In our study female cells grew out

earlier and covered greater areas indicating faster rates

of migration (Fig 6). Additionally, male cells became

senescent earlier.

Influence of media conditions was tested in long-

term culture. Cells in Leibovitz or WilliamsE cell culture

media survived only for two weeks whereas cells with

DMEM/F12 survived for 10 weeks. Cells grown in

DMEM/F12 with full supplementation (see Table 2,

green highlight) generally showed best results (Fig. 7).

Cells grew out, formed complete monolayers and started

to form tissue-like structures with pigmentation (Fig.

7 and 8). After six weeks multi nucleic cells occurred

more frequently (Fig. 9), these cells stopped growing

and finally died after 10 weeks. Dead cells broke away

from the adhesion surface and floated in big sheets in

the containers. Medium supplementation with HEPES

(4-(2-hydroxy ethyl)- 1 -piperazineethanesulfonic acid)

resulted in pH stabilization (visualized by phenol red

indicator in cell culture media). Without this buffer

medium’s pH changed after less than one hour in the

wet chamber as C0 2 fumigation was not available. With

HEPES pH remained stable for up to two days. This short

time of stability was caused by the low concentration of

HEPES (5 mM) and small volumes of medium applied

to the cells. Usually a concentration of 10 mM is used to

stabilize media, but this concentration was found to be

harmful to the cells of the giant salamander.

Problems with contamination by a fungus (white

appearance, no detennination of species perfonned)

occurred in cell culture from one male animal (Cologne)

and were treated with amphotericine B (Biochrome).

This treatment stopped fungus growth, but cells started to

age after two days of antifungal treatment. The cultures

of the female (Cologne) and the other male (Berlin)

tissues remained uncontaminated during the culture

process. Repeated preparations from further biopsies of

Cologne animals at later time points resulted again in

fungal contaminations of male cultures.

Discussion

The large size and weight of adult Andrias davidianus

make handling of the animals difficult and cause stress

and possibly injury for both animals and researchers

(e.g., bites, Beckstein 2009). To minimize such risks, we

recommend using a landing net to restrain the animals

in the housing tank for biopsy procedures as the animals

stayed absolutely calm and apparently oblivious to the

procedure (cf. Nickerson 2003; Mutschmann 2009).

We could find no literature concerning the cell

culture of A. davidianus or any other cryptobranchid

species in Western literature, or from correspondence

through Chinese literature. Based on the cold freshwater

physiological conditions experienced by A. davidianus ,

cell culture could be expected to be most successful

with lower temperatures than with mammalian cells.

Other conditions to consider with the establishment

of A. davidianus cell cultures, in respect to those of

mammals, are a lower osmolarity of body fluids in A.

davidianus (Albert et al. 1987; Chemoff et al.l 990), and

particular cell culture coatings for optimal cell adherence

and proliferation, as shown with Xenopus laevis and

Ambystoma mexicanum primary cells (Nishikawa et

al. 1990; Chemoff et al.1990). We assessed the use of

different cell culture containers and various treated

plastics (according to manufacturer’s datasheets)

combined with various media conditions and surface

coatings.

We found that the size of cell culture containers was

important for the successful outgrowth of cells, and tissue

pieces were more likely to stick when small flasks were

used instead of multiwell plates. This might be explained

by the tendency of small pieces of tissue to float on the

surface of solutions toward the containers wall thus

preventing adhesion to the bottom of the container.

Cells from multicellular organisms communicate with

each other by release of messenger substances into the

extracellular fluids, e.g., the culture medium, or by direct

cell-cell contacts. To accomplish sufficient concentrations

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Fig. 7. Picture time line of cell outgrowth in DMEM/F12 with full supplementation. Images in overview and detail show represen-

tative examples of long term outgrowth of cells under full supplementation. Cells grew out in dense layers (A). At the migration

front cells filopodia formation was observed (B). Outgrowing cells proliferated (C, indicated by arrow). No visual evidence for

senescence was observed at day 18 to 25 (D, E, and F). After three weeks cells started to form pigmented tissue-lilce structures (E

and F). Pictures were captured using phasecontrast lightmicroscopy. A: cells at day three; scale bar 500 pm. B: cells at day three;

scale bar 100 pm. C: cells at day seven; scale bar 100 pm. D: cells at day 18; scale bar 50 pm. E: cells at day 21; scale bar 500 pm.

F: cells at day 25; scale bar 500 pm.

of bioactive molecules by cellular release of substances

like growth factors (e.g., vascular endothelial growth

factor, keratinocyte growth factor, fibroblast growth

factor), enzymes (e.g., lipoxygenases) and cytokines

(e.g., interleukines) to their culture medium, low volume

for small cell numbers is recommended. Too low

concentrations of these substances lead to cell death in

vitro as cells are missing paracrine stimulation. So the

choice of cell culture container size means a balancing act

between low surface curvature (implying use of greater

culture containers) and low medium volume (implying

use of smaller culture containers).

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In vitro culture of skin cells from the Chinese giant salamander

Fig. 8. Multilayer formations after six weeks of cultivation. Outgrowing cells tended to form pigmented multilayers with tissue-like

appearance which became thicker with prolonged cultivation time. A: tissue-like structure after six weeks; scale bar 500 pm. B:

tissue-like structure with pigmentation after six weeks of cultivation; scale bar 500 pm.

Fig. 9. Cell aging. After six weeks in DMEM/F12 multinuclear cells were observed more frequently. Pictures show representative

examples and were captured using phasecontrast lightmicroscopy. A: overview (multinuclear cell indicated by arrows); scale bar

200 pm. B: detail of A; scale bar 50 pm.

Table 4. List of abbreviations and suppliers.

Williams Medium E

PAA, Colbe, Germany

Leibovitz L-15

PAA, Colbe, Germany

DMEM/F12

PAA, Colbe, Germany

Ascorbate-2 -phosphate

A2P

Sigma Aldrich, Taufkirchen, Germany

Insuline-Transferrine-Selenium A

ITS

Gibco

Non-essential aminoacids

NEA

Biochrom, Berlin, Germany

Sodium-Pyruvate

Sodium-P

Biochrom, Berlin, Germany

Penicilline/ S treptomycine

P/S

PAA, Colbe, Germany

Gentamicine

Genta

Biochrom, Berlin, Gennany

Amphotericine B

Ampho

Biochrom, Berlin, Germany

(4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid

HEPES

PAA, Colbe, Germany

Collagen

Biochrom, Berlin, Germany

Poly-L-Lysine

PLL

Biochrom, Berlin, Germany

Fish Serum

own production from trout blood

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The influence of the adhesion surface on adhesion

rates, cell migration, cell growth or the culture survival

time seems to be negligible as no correlation to the cell

culture material or surface coatings was observed. This

is contrary to data from the literature describing culture

of various amphibian cell types from X. laevis and A.

mexicanum on developmental or regenerative aspects

as well as toxicological studies (Albert at al. 1987;

Nishikawa et al. 1990; Chemoff et al.1990; Goulet et

al. 2003 et al.; Ferris 2010). In those studies cell culture

vessel plastics were coated with fibronectin, collagen,

matrigel and other matrices to encourage cell adhesion.

As nutrition media MEM, FI 2, MCDB151 or

combination of these diluted to 70% with sterile distilled

water were used (Nishikawa 1990). Culture media were

supplemented with insulin, transferrin and EGF. Skin

explant cultures obtained from Ambystoma mexicanum

can be grown in 60% DMEM under supplementation

with 10% fetal bovine serum and ITS (insulin transferrin,

selenium A) (Ferris et al. 2010).

Culture survival appeared to be more dependent on

the stabilization of culture medium pH than on surface

coatings; mammalian primary cells usually need a

stable pH around 7 to remain vital in vitro . Cells of A.

davidianus were veiy sensitive to the supplementation

with HEPES while the commonly used concentration of

HEPES of 10 mM was toxic to the cells and led to cell

death. A concentration of 5 mM resulted in stabilization

of the pH as well as no detectable toxic influence on A.

davidianus cells. High sensitivity to HEPES was also

shown with a blastema model of A. mexicanum (Guelke

et al. submitted). Previous publications on amphibian

cells did not mention the use of HEPES in the culture

media. Alternatively to HEPES, an incubator with CO,

fumigation can be used to stabilize the pH (Chemoff

et al.1990; Nishikawa et al. 1990; Ferris et al. 2010).

Without pH stabilization cell outgrowth and survival was

greatly reduced in our study as well as in other studies

using C0 2 fumigation.

The benefit of the use of antibiotic supplements in

amphibian cell culture may be negated by decreased

survival. As caudates do not live in a sterile environment

and need a certain skin flora, thus a problem rises with

the transfer of tissue to cell culture; the culture medium

offers good growth conditions for the target cells and

simultaneously for microorganisms. Bacteria and fungi

accrete faster than the cells and cause cell death by

release of toxic substances. In our study, cells tolerated

50 U/ml of penicillin/streptomycin mix (p/s) which

is sufficient to avoid infections of already established

cultures. Therefore 0.05 mg/ml gentamicin was thus

added. The common antibiotic supplementation of cell

culture media contains 0. 1 mg/ml of gentamicin, but this

concentration resulted in early senescence and cell death

of A. davidianus cells. There is no comparative research

in Western scientific publications on the use of antibiotics

in amphibian cell culture media.

The fungal contamination of the Cologne Zoos male’s

cell culture appeared to be from the skin microflora.

Contaminations during tissue processing seem an

unlikely cause as culture contaminations occurred

under a wide range of preparation conditions including

sanitized conditions. Further research is planned to

identify the type of fungus and to assess its possible

influence on outgrowth of cells from the tissue explants.

In vitro treatments with amphotericin B for this fungus

resulted in early senescence and cell death. Causes

for this toxic effect remain unclear as amphotericin B

(Fungizone) is commonly used in fish and amphibian cell

cultures and known to be not toxic to cells so far. There

is only one publication mentioning possible toxic effects

of amphotericin B (Fungizone) on tadpoles of Alvtes

cisternasii (Martel et al. 2011).

Based on cell morphology we consider that outgrowing

cells were skin epithelial cells and melanophores.

Migrating melanophores appeared rounded while

resident cells showed typical dendritic forms as these

cells are from dendritic origin (Rawles 1948; Billingham

1948). In light microscopic imaging melanophores ofrt.

davidianus appeared equal to those of guinea pigs shown

in the study of Billingham (1948) which are compared

in Fig. 5.

Interestingly, cell outgrowth from female tissue

appeared to be faster than from male (Fig. 6). As we tested

only samples of three animals so far, these observations

need to be confirmed by repeating trials with other giant

salamanders. From MRL mice it is known that females

heal wounds better and faster than male animals due to

sexually dimorphic genes (Blankenhom et al 2003) and

also with human cutaneous tissue (Gilliver et al. 2007),

however we could find no published information on this

phenomenon in fish or amphibians.

Cells did not only form a monolayer as known from

primary cells in general, but tended to form pigmented

multilayers in long term cultivation (Fig. 7F and 8)

after three weeks. Usually mammalian primary cells

stop proliferation when reaching confluence in vitro

due to contact inhibition by cell-cell and cell-substrata

interactions (Qi et al. 2008). In contrast most cancer

cells or immortalized cell lines are refractory to contact

inhibition and can continue to proliferate (Hanahan

et al. 2000). Cell cultures from Xenopus skin explants

only grew out as monolayer stopped expanding after

six to eight days (Reeves et al. 1975). This raises the

question whether the observed multilayer formation of

Andrias skin explants could be related to the regenerative

capacity of caudate amphibians.

Senescence is a well-known process in mammalian

primary cells. Due to their limited proliferation capacity

(Hayflick index) mammalian cells become senescent

after certain time of in vitro cultivation in contrast to

immortalized cell lines. Literature regarding life span

of amphibian primary cells is limited and described

results are ambiguous. While Nishikawa reports ageing

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In vitro culture of skin cells from the Chinese giant salamander

of Xenopus skin cells in vitro (Nishikawa 1990), Kondo

et al. (1983) describes a growth crisis (senescence) in

melanophores followed by a spontaneous transformation

to an immortalized cell line derived from Rana

catesbeiana (Kondo et al. 1983). In our study skin cells

became senescent and did not undergo a spontaneous

transformation and eventually died.

The creation of an immortal Andrias skin cell line could

possibly be achieved by: 1) spontaneous transformation

of cells as a small number of them undergoes a set of

genetic alterations which lead to unlimited life span. This

means, however, that very high numbers of primary cells

may have to be cultivated over a long period of time until

some of them start unlimited proliferation. 2) expression

of telomerase reverse transcriptase (TERT) which e.g., is

available as eukaryotic expression plasmid from ATCC

(MBA- 141). The use of method one is well documented

in anurans (Kondo et al. 1983) as well as in fishes (review

Lalcra et al. 2011) while for method two only literature

regarding fish cell lines is available (review Lakra et al.

2011 ).

Conclusion

This study examined the basic needs of primary

cultures for A. davidianus skin cells raised from small

skin biopsies. These cells seem to have no exceptional

culture needs when cell culture is performed in a wet

chamber except for specific medium osmolarity and pH

stabilization with HEPES buffer.

Primary cultures of Andrias skin cells, as well as

other amphibian primary cell cultures can be used in

future studies to evaluate effects of; 1) diseases and

effects of medication, 2) toxicity tests of pollutants and

other substances as already described for fishes (Dayeh

2005) and anurans (Goulet 2003), 3) for the study of

regeneration, and 4) the role of gender specific hormones

on wound healing. The use of active or cryopreserved

cell cultures, in conservation programs for threatened

amphibians is being increasingly recognized. These cells

can provide for the banking of cells and organelles, and

their genetic material for use in reproduction technologies

(Browne et al.). The next steps in the establishment of an

in vitro cell culture model will be on the one side the

development for cryopreservation cells do not have to

be immortalized; they can be stored and cultivated we

predict as mammalian primary cells.

A further contribution to cryptobranchid conservation

of cell lines is their use for establishing of a karyogram

based sex determination. Because of the large size of

cryptobranchids sexing is often performed by ultrasonic

examination, and due to the size of adult Andrias is an

elaborate procedure. During ultrasonic examination

which is usually done without anesthesia, also injury

risks, both for animals and human beings, must be

considered. Sexing with ultrasound is also most effective

during the breeding period, when gonads are distinct and

may effect reproduction. Based on the study of Zhu et

al. (2002) A. davidianus may be distinguished by their

sex chromosomes and this technique would enable a new

less stressful sexing of these salamanders. Karyotyping

also offers the opportunity to screen the animals for

chromosomal aberrations to distinguish salamanders

that may be unsuitable for use in conservation breeding

programs. However, skin cell karyograms can only

provide insights into chromosomal aberrations of somatic

cells and not those induced by failures in the germ line.

Examination of wound closure processes resulting

from biopsy withdrawal in vivo and cell outgrowth in

vitro could give information about the regenerative

capacities of A. davidianus. Using cell culture models

for A. davidianus research would reduce the number of

experimental animals and provide new research horizons

and benefit conservation breeding programs.

Acknowledgments . — We thank Sabine Ommer and

Bodo Lang (Cologne) for fruitful discussions and for

reviewing a previous version of the manuscript. Thanks

also to the veterinarians Dr. Andre Schiile (Berlin) and

Dr. Olaf Behlert (Cologne) for kindly supporting the

biopsy procedures.

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Received: 05 February 2013

Accepted: 13 May 2013

Published: 29 June 2013

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In vitro culture of skin cells from the Chinese giant salamander

Sarah StrauiJ has been working since 2008 as a biologist at the Ambystoma Mexicanum Bioregeneration

Center (AMBC) which is part of the Department of Plastic-, Hand- and Reconstructive Surgery, Hannover

Medical School. The AMBC combines species-preserving captive breeding and biomedical research — benefit

for human and animal according to the WHO claim “health for all.”

Thomas Ziegler has been the curator of the Aquarium/Terrarium Department of the Cologne Zoo since 2003.

He completed his herpetological Ph.D. in the year 2000 at the Rhineland Friedrich Wilhelms University Bo nn .

Thomas so far has conducted herpetological field work in South America (Paraguay) and South East Asia

(Vietnam, Laos). Since 1994, he has published 272 papers and books, mainly dealing with herpetodiversity.

His main research interests include diversity, systematics, and zoo biology of amphibians, geckos, monitor

lizards, snakes, and crocodiles. Since February 2009, he has been an associate professor at the Zoological

Institute (Biocentre) of Cologne University.

Christina Allmeling is responsible for captive care and breeding at the Ambystoma Mexicanum Bioregenera-

tion Center. She is author of the Axolotl captive care management paper, defining the European guidelines for

these animals in laboratories. She is a member of the AG Urodela which is a working group of the German

Society for Herpetology and Herpetoculture.

Kerstin Reimers leads the research division of the Department of Plastic-, Hand- and Reconstructive Surgery

at Hannover Medical School. Since 2011, she is a W2 professor for regenerative biology in plastic surgery.

Her research focuses on tissue engineering and regenerative processes in mammals and amphibians — espe-

4LL L cially the axolotl. In 2008 she and her team identified the AmbLoxe as a signal key to the axolotls regeneration

capacity.

Natalie Frank-Klein started her career at the Aquarium/Terrarium Department of the Cologne Zoo in Sep-

tember 1997. She finished her apprenticeship as a zoo keeper in 2000. Her focus of expertise is freshwater

fishes and aquatic urodelans. Since 2011 she has been section keeper in the freshwater department of Cologne

Zoo’s Aquarium.

Robert Seuntjens has been the head zoo keeper of the insect and amphibian section at the Zoo Aquarium of

Berlin since 1994.

Peter M. Vogt has been head of the Department of Plastic-, Hand- and Reconstructive Surgery at Hannover

Medical School since 2001. In the same year he founded the research division of his department. His research

activities focus on tissue engineering, regenerative medicine, reconstructive surgery, bum medicine and criti-

cal wound care. Among other things he is head of the German Society of Plastic-, Reconstmctive and Aesthetic

Surgery (DGPRAC) and the German Society of Wound Healing and Wound Treatment (DGFW).

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