Amphibian reptile conservation_5a

amphibian-reptile-conservation.org elSSN: 1525-9153 Editor Craig Hassapakis USA Berkeley, California, Associate Editors Raul E Howard Diaz University of Kansas, USA O Clark, Garcia and Associates, Erik R Wild Jr USA University of Wisconsin-Stevens Point, USA Assistant Editors Daniel D Fogell Alison R Davis University of California, Berkeley, USA Southeastern Community College, USA Editorial Review Board David C Blackburn California C Bill Academy of Sciences, USA Kenneth Dodd, University of Florida, Harvey B Peter V Texas USA USA Jodi J L R Pakistan USA Rohan Pethiyagoda VENEZUELA Australian Rowley Museum, AUSTRALIA Virginia SAUDIA ARABIA Rafaqat Masroor Museum of Natural History, PAKISTAN Elnaz Najafimajd Ege University, TURKEY Mushinsky University of South Florida, A Ibrahim Ha’il University, Julian C Lee New Mexico, USA Henry Lindeman Jaime E Pefaur Australian Adel A&M University, USA Taos, Edinboro University of Pennsylvania, Universidad de Los Andes, Jelka Crnobrnja-Isailovc IBISS University of Belgrade, SERBIA SOUTH AFRICA Lee A Fitzgerald Jr USA Lillywhite University of Florida, Branch Museum, Port Elizabeth Nasrullah Rastegar-Pouyani Museum, AUSTRALIA Peter Uetz Commonwealth University, Razi University, IRAN Larry David Wilson USA instituto Regional de Biodiversidad, USA Advisory Board Aaron M Bauer Allison C Alberts Zoological Society of San Diego, Michael USA Villanova University, James Hanken Harvard University, USA B Eisen Public Library of Science, USA USA Royal Ontario Museum, Antonio W Salas Environment and Sustainable Development, R Erdelen Roy W McDiarmid USGS Patuxent Wildlife Research Center, USA Robert W Murphy Russell A Mittermeier Conservation International, Walter UNESCO, FRANCE USA Eric R Pianka CANADA University of Texas, Austin, USA Dawn S Wilson AMNH Southwestern Research Station, USA PERU Honorary Members Carl C Joseph T Collins (1939-2012) Gans (1923-2009) Cover : Color varieties of the Chinese Giant Salamander (Andrias davidianus) from aquaculture farming operations in China Photo Sumio Okada Amphibian & Reptile Conservation — Worldwide Community-Supported Herpetological Conservation (ISSN: 1083-446X; elSSN: 1525-9153) is & Reptile Conservation as full issues at least twice yearly (semi-annually or more often depending on published by Craig Hassapakis/Amphibian needs) and papers are immediately released as they are finished on our website; http://amphibian-reptile-conservation.org; email: arc.publisher@gmail.com Amphibian & Reptile Conservation is published as an open access journal Please visit the official journal website at: http://amphibian-reptile-conservation.org Instructions to Authors : Amphibian & Reptile Conservation accepts manuscripts on the biology of amphibians and conservation, sustainable management, and biodiversity Topics in these areas can include: bution, conservation, species profiles, ecology, natural history, sustainable reptiles, taxonomy and phylogeny, species management, conservation breeding, with emphasis on inventories, distri- citizen science, social network- and any other topic that lends to the conservation of amphibians and reptiles worldwide Prior consultation with editors is suggested and if you have any questions and/or concerns about submissions Further details on the submission of a manuscript can best be obtained by consulting a current published paper from the journal and/or by accessing Instructions for Authors at the Amphibian and Reptile Conservation ing, important website: http://amphibian-reptile-conservation.org/submissions.html © Craig Hassapakis!Amphibian & Reptile Conservation Copyright: © 2011 Browne et al This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are Amphibian & Reptile Conservation 5(4): 1-1 credited Survey techniques for giant salamanders and other aquatic Caudata "DALE MCGINNITY, SUMIO OKADA WANG ZHENGHUAN, "CATHERINE M BODINOF, KELLY J IRWIN, “AMY MCMILLAN, AND "JEFFREY T BRIGGLER 'ROBERT K BROWNE HONG LI, , , Center for Research and Conservation, Royal Zoological Society ofAntwerp, Antwerp, BELGIUM Polytechnic Institute of New York University, New York, New York 11201, USA 3Nashville Zoo, Nashville, Tennessee 37189, USA 4Laboratory of Biolog}’, Department ofRegional Environment, Tottori University, Tottori 680-8551, souri, Department ofFisheries and Buffalo State College, Buffalo, JAPAN Wildlife, New School of Life Sciences, East China Normal University, 200062, Shanghai, CHINA University ofMisColumbia, Missouri 65211, USA ''Arkansas Game and Fish Commission, Benton, Arkansas 72015, USA York 14222, USA Missouri Department of Conservation, Jefferson City, Missouri 65109, USA Abstract.— The order Caudata (salamanders and newts) comprise ~13% of the ~6,800 described amphibian species Amphibians are the most threatened (~30% of species) of Caudata are the most threatened (~45% of species) amphibian order The vertebrates, and the all 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 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, environmental threats, and genetic subpopulations Survey techniques are constrained by logistical considerations 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 large Key words Survey techniques, Citation: Browne RK, Hong L, giant salamander, amphibian, Caudata, Cryptobranchid, conservation 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 institutional support for water catchment management 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 In Japan, A japonicus has the Caudata are the fully aquatic Cryptobranchids (fam- village conservation programs Similarly, in the People’s Cryptobranchidae; suborder Cryptobranchoidea) All Republic of China, the release of A davidianus from three Cryptobranchids are fully aquatic and include the farm stock has received widespread government support world’s largest amphibians: the Critically Endangered, and formal public recognition, and Chinese giant salamander (Andrias davidianus), the Near ing a symbol for watershed conservation There Threatened, Japanese giant salamander (A japonicus ), increasing and the North American giant salamander ( Cryptobran ensis as an icon for watershed conservation in the chus alleganiensis), commonly known as the Hellbender (Browne et al 2012a, b) However, in addition ily (CNAH education and environmental awareness campaigns, and The conservation potential of Cryptobranchids extends beyond their immediate conservation needs As er aquatic iconic species, Cryptobranchids offer an ideal opportu- their Correspondence bio earn edu cn; a national symbol, attracting publicity including parades with large floats, 2011) nity to develop public awareness become momentum toward this species is establishing C allegani- to public USA and government and Caudata conservation size, habitat relies upon genetics, knowledge of population demography scientific and microhabitat variables, reproduc- Emails: jxindakittylhong@gniaiI.com; dmcginnity@nashville.org; 4shichibu@mocha.ocn.ne.jp; bodinofc@missouri edu; also an support, the conservation of Cryptobranchids and oth- and government and is becom- kirwin@agfc state ar us; % mcmillam@buff(dostate edu; zhwang@ Jeff Briggler@mdc.mo.gov; 'robert.browne@gmail.com (Corresponding author) amphibian-reptile-conservation.org 01 December 2011 | Volume | Number e34 | Browne Figure weight Image Robert Browne tion Andrias davidianus and life the largest and is et al most threatened Cryptobranchid, and can reach 200 The most appropriate survey techniques to achieve this knowledge will depend on survey objectives in concert also with logistical constraints including the type of habitat bin 1999) of genetically to enable the provision Consequently, the major conservation needs of A davidianus, besides watershed restoration, limiting wild interacting factors, including the species’ life stages, be assessed competent individuals for release (Reisenbichler and Ru- surveyed (Dodd 2009) The choice of survey techniques autecology, targeted length and 59 kg in other Cryptobranchids Their population genetics must stage survival, and environmental threats must consider cm in total and season, as well as harvest, and pathogen management, are assessing the water depth, velocity, and clarity (Dodd 2009) Survey presence of relictual populations and their conservation techniques must minimize environmental disturbance genetics, and possible negative on the health of the targeted effects and then matching the genetics of released stock with those found in nature When these requirements survey focus must include selecting are individuals and populations through the spread of patho- satisfied, the gens and trauma to individuals able release 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- ongoing assessment of the survival and reproduction of sites, suit- then release of juveniles or adults, and 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 2002) However, surveys can identify remaining ber of relict populations distributed throughout China et al The few remaining populations (in lowland fairly genetically homogenous, probably due areas) are populations, provide genetic samples, and assess the suc- to anthro- cess of restocking programs suitable habitats al (Tochimoto et al 2005), and ongoing molecular studies may reveal even ponicus was harvested in the past, Murphy, pers comm.) and in place to prevent this species finer population structure (R further Evolutionarily Significant Units (Crandall et relies on the main- still remain in 2008) Although A.jastrict from protection exploitation now How- is and other an- ever, threats include habitat modification al 2000 ) thropogenic changes, including pollutants, and the introduction of A davidianus in some systems Consequently, Andrias davidianus has a considerable aquaculture potential, cilities 2004) tenance of the populations that generally civilization Nevertheless, there are genetically distinct populations remaining (Tao et et al The conservation of A japonicus pogenic transport and the building of canals over China’s ~6,000 year history of (Wang and more than 1000 licensed aquaculture are in production in China with up the conservation needs of A japonicus include surveying fa- to 106 indi- viduals in stock In concert with aquaculture, there are an increasing culture is number of restocking programs using aqua- brood stock However, aquaculture brood stock subject to genetic drift, a process that reduces genetic diversity over generations Additionally, the source of the unknown, and examples such as the unmanaged release and escape of aquaculture stock of Pacific salmon ( Oncorhynchus spp.) have result- aquaculture brood stock is often 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 the presence of relictual populations to avoid reveal Figure Genetic drift and selection for color traits in A da- vidianus have resulted in orange, piebald, and albino strains compro- Image Robert Browne mising the long-term conservation of A davidianus and amphibian-reptile-conservation.org 02 December 2011 | Volume | Number e34 | Survey techniques salamanders for giant reproduction technologies to produce genetically compe- McGinnity, pers comm.) tent individuals (D Cryptobranchids present survey particular chal- lenges because of their large variation in body size, from cm three larvae to 1.5 m adults Additional challenges include the wide variation in their aquatic habitats (deep turbulent water, shallow pools, lakes) and varied riffles, microhabitats (crevices, large rocks, pebble bed in (Nickerson and Krysko 2003; Tao fles) da et al 2008) The et al habitats of A japonicus rif- 2004; Oka- and C alle- ganiensis are relatively accessible, but, the habitat of A davidianus includes rugged, remote, difficult to survey, fast-flowing interior rivers in the mountainous areas of central China (Tao et al 2004) on associating Effective survey methods depend Figure Andrias japonicus cm chid and reaches 150 is the second largest Cryptobran- in total length the life stages of target species with their microhabi- and 44 kg in weight Adult Cryptobranchids tats Image Sumio Okada live in cavities, rocks, and in bank-side dens under large Because of the low popula- population densities and demography, habitat variables tion densities of the relictual populations of A davidi- including channelization and watershed characteristics, anus, recent surveys have relied on the observation of assessing the effects of obstacle removal to migration, adults, electrofishing such as dams, and the provision of survival and recruitment (Browne artificial habitats et al 2012a, on bow hooks (Wang 2004) Surveys for adult and subadult A japonicus et al in their habitats b) and the use of of slow flowing rivers have largely some re- The conservation needs of C alleganiensis include identifying the most enigmatic threat to any Cryptobran- lied chid and perhaps any amphibian species Cryptobran- alleganiensis have used a wide variety of techniques, in- chus alleganiensis has generally been declining over cluding rock turning while snorkeling most of its range (Wheeler et scuba diving or trapping (Nickerson and Krysko 2003; to some tion al 2003; Foster et al al 2009), extent due to habitat degradation and modifica- Flowever, C alleganiensis still netting (Okada numbers in some locations, and some habitats modified by siltation and agricultural development still support substantial numbers of C alleganiensis Flow- et 2008) In contrast, surveys of adult and subadult C al or, in deeper water, 2008) Recent innovations in survey tech- niques for C alleganiensis include the use of survives in near spawning sites to reveal reproductive success artificial The use of video cameras has the potential to increase observations of mating, brooding by males, and the development of oocytes and larvae Environmental of C alleganiensis has failed for de- cades over a substantial part of its range due to direct observation with Foster et historic ever, the recruitment on unknown tion (Goldberg et al DNA (eDNA) detec- 2011) has the potential to both detect now Cryptobranchids and to estimate their standing biomass comprised of only a few old individuals (D McGinnity, and population Radiotelemetry offers an opportunity to causes, and pers many of these declining populations are survey the movements and survival of an increasing size comm.) Cryptobranchus alleganiensis is subject to ongoing surveys; however, these research many activities et al al Andrias japonicus and C alleganiensis larvae and 2009) Addressing this problem will early juveniles are encountered less frequently than adults require targeting the life history stage where the failure recruitment occurs, from mating success through ization, to ward 2001) have not revealed the cause of poor recruitment (Wheeler et 2003; Foster range of Cryptobranchids over an extended period (Ken- egg development, and larval due of fertil- and juvenile sur- to their particular microhabitats and to the low larval recruitment of C alleganiensis in some regions (Nicker- son and Krysko 2003; Okada 2008) In contrast, the et al commonly found in surveys vivorship Surveys will need to correlate recruitment to larvae of A davidianus were different life history stages with environmental variables of shallow mountain streams in the Qin Ling Mountains such as pollutants Attempts to reproduce C alleganien- until their populations rapidly declined in the early sis in captivity for (Zhang restocking are in the early stages of development, and no larvae have been produced Flowever, the production now is of large numbers of individuals from underway The cryopreservation of sperm been amphibian-reptile-conservation.org (2008) found recentlylitter more developed A japonicus 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- is no recruitment (National initiated to et al larvae tat Geographic 2010; Michigan State University 2010) In addition, research has Okada or undercut banks, whereas being undertaken to perpetuate the genetic varia- tion of populations with poor or 2002) hatched larvae of A japonicus in pools under leaf wild eggs has been successful and their release to natural habitats et al 1980s of the larval stages of C alleganiensis, observations suggest that both larvae and small juveniles inhabit inter- provide a suite of stitial 03 spaces under river gravel in December 2011 | riffles Volume (Nickerson and | Number e34 | Browne et al Krysko 2003; Foster et al 2008) Juvenile and subadult C alleganiensis most frequently occur in clean, rockbased 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 cycles For example, the nocturnal activity of C alleganiensis in streams of southeastern North America is positively 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 Figure dens for adults, egg masses, and larvae, and the place- ment of Natural rock placed in stream to provide habitat and sampling locations for C alleganiensis Image Kenneth Roblee natural rocks to provide habitat Safeguarding the health and reproductive success of Cryptobranchids is critical when choosing survey necessitate minimal disturbance of water bodies and geographic region are primary con- techniques Techniques to the habitat, the use siderations of sites If selecting the surveying different An sanitary procedures to prevent pathogen dissemination, and the protection of nest when most promising season life stages important consideration when surveying Cryp- tobranchids and other aquatic Caudata possible, several sur- for is the prevention 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- and spread of infectious diseases Chytridiomycosis tion of potential biases through the choice of technique, regionally extirpate up to surveyed microhabitat, species, and 2009) life Nowakowski and Maerz (2009) stage Batrachochytrium dendrobatidis) (Chytrid; is an in- fectious disease of particular conservation concern for amphibians Chytrid (Dodd is an emerging pathogen that can 90% of species and dividuals in naive populations, at least tested the effi- et al among 95% of in- frogs (Lips 2005) However, the effect of chytrid on Crypto- cacy of surveys of larval stream salamanders by com- branchids has not been significant paring the mark-recapture success of passive leaf has been suggested as endemic to populations of A ja- litter trapping and dip netting Significant size bias occurred, ponicus (Goka with traps capturing a higher proportion of large indi- of chytrid viduals and dip netting yielding a greater proportion of may smaller size classes The survey efficiency of second order streams was greater sities at first and is et al One of chytrid strain 2009), and an undetermined strain found on mainland Asia in South Korea and eventually impacts, davidianus (Yang et al 2009) Chytrid has been shown to be pathogenic in cap- low salamander den- tive populations with time-constrained opportunistic sampling, but of C alleganiensis (Briggler 2008), although with apparently few, if any, 2007, et al pathological when salamanders were and Babbitt 2001) Nowakowski tation is a primary consideration in surveying Crypto- and Maerz (2009) concluded that the physical dynamics branchids, and other amphibians as a precaution against greater with quadrat sampling at high densities (Barr effects on natural populations Nevertheless, good sani- 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 ening Cryptobranchids and other amphibians is threat- Rana- (Geng et al 2011) To prevent the spread of both amphibian chytrid and Ranavirus equipment should be thoroughly sanitized when moving among aquatic sysvirus , 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 Figure Cryptobranchus alleganiensis has been the subject of the most diverse and innovative survey methods of all Crypto- branchids perspective Image Dale McGinnity amphibian-reptile-conservation.org Reviews or comparative studies of survey techniques for Ciyptobranchids include Nickerson and 04 December 2011 | Volume | Number e34 | Survey techniques Krysko (2003; davidianus), Dodd C alleganiensis ), Wang et al for giant salamanders (2004; A Okada et al (2008, 2006; A japonicus), and (2009) for general survey techniques of amphib- ians we Survey techniques review include: and snorkeling, turning substrate, netting, hookah diving, 3) Nocturnal spotlighting, 4) trot-lines, 5) 1) Wading, 2) Scuba/ Bow -hooks/ Questionnaires, 6) Electrofishing, 7) Under- water camera systems, Passive integrated transpon- 8) 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) ders (PIT tags) detection Review of survey techniques Wading, turning substrate, netting, and snorkeling Figure Wading and turning substrate, coupled with snorkeling and downstream netting and seining, are widely used et al Turning heavy rocks, combined with snorkeling with masks and and adult nets is an effective means to survey juvenile C alleganiensis Image Robert Browne alleganiensis and other techniques for surveying C Cryptobranchids (Taber face 1975; Peterson et 1983, al Scuba/hookah diving 1988; Nickerson and Krysko 2003) These techniques 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 Deep water surveyed through blind searches by reaching beneath surveying C alleganiensis in fast-flowing, deep water are considered the large rocks or within hollow logs or holes in banks veyed habitats have not generally been well sur- for Cryptobranchids, although standard scuba div- ing equipment and surface-based air compressor systems (hookah dive systems) are being used increasingly for These two to nine meters in depth Scuba diving allows for techniques have resulted in the detection of hundreds to prolonged submergence giving the diver the capability thousands of C alleganiensis in some surveys (Taber to systematically al., 1975; Peterson et Snorkeling al common technique for survey- < m from 0.5 to more efficient than 3.0 is most ited mobility in terms effective in clear waters in depth This all available cover and often cap- Standard scuba diving equipment provides unlim- ing C alleganiensis (Nickerson and Krysko 2003) and other salamanders and check ture all individuals observed 1983, 1988) another is et contrast, divers using a stationary method has proved wading and turning of the area a worker can survey In anchored boat, canoe, or bank-side hookah system are limited by air line length substrate in sur- veys of C alleganiensis in the gilled larval stage (Nickerson et al 2002) Foster et and al (2008) turned rocks to survey for adult 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 bank yielded 14 juveniles the stream (0.25 pph) sites person hours searches of four of the seven inhabited yielded no C alleganiensis, but searching et al Bank in 55 was more efficient than at three sites bank rock turning (Foster 2008) Capture rates of C alleganiensis in four streams in the White River drainage, Missouri, varied pph (Trauth et al 1992) Okada et al (2008) used diurnal wading and substrate surveys with from zero one to 2.5 to three people searching under piled rocks or leaves (by hand or with dip-nets) to observe 227 A japonicus a rate of Figure at 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 pph amphibian-reptile-conservation.org 05 December 2011 | Volume | Number e34 | Browne et al Nocturnal snorkeling/scuba surveys follow the same Nevertheless, free-floating hookah systems are available that allow ters hookah divers work to in moderately fast with unlimited mobility as the compressor protocol as wading surveys, except that the observers wa- ed with some success in Missouri and Arkansas, hookah system, then a boat or hookah compressor Boat-mounted hookah systems enable dives of one hour (hr) to more than hr duration, and can be used at a stationary need to refuel full USA, especially during the spawning period Boats with halo4 gen spotlights powered by generators have been used to canoe can be used to provide a semi-mobile platform for multiple sites during a dive lights to spot salamanders Nocturnal snorkeling/scuba surveys have been conduct- freely behind the divers If conditions are not favorable for use of a free-floating swimming and using are floats survey for C alleganiensis in Missouri (Wheeler 2007; Nickerson and Krysko 2003) day of fieldwork without the Hookah systems require the use of a dive harness fitted with lead weight (usually 20-25 kg) suffi- Bow-hooks/trot-lines The stream- cient to hold a diver in place in fast currents lined profile of hookah systems reduces the fatigue expe- Bow-hooks rienced by divers using standard scuba equipment Divers nique in detecting the presence of Cryptobranchids must be capable of working in fast moving water and have the physical strength to move large cover objects to successfully locate Cryptobranchids For safety 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 regions bow-hooks may provide the most practical survey technique (Liu 1989; Wang 1996; Zhang and Wang 2000; Zhang et al 2002) also reasons, that a all diving requires a “buddy system” is in place If using a system, a topside operator tions minimum of two is divers, so hookah dive required to monitor condi- and equipment All divers must have appropriate certification drops to 500 and must surface when air cylinder Wang pressure ing psi or trot-lines can be an efficient survey tech- et (2004) surveyed A al davidianus us- bow-hooks made of small pieces of bamboo with four or five sharp hooks In at this study, fitted only one A davidianus was captured with the bow-hooks, whereas were observed during night surveys and eight were captured by electrofishing Bow-hooks were found to be none Nocturnal spotlighting an effective survey technique for A davidianus in the Nocturnal spotlighting has the advantage of producing remote and rugged Huping Mountain National Nature minimal substrate disturbance, as rocks are Reserve, an area of particular conservation significance lifted after the protruding heads of C alleganiensis are observed (Zhang Spotlighting also allows observation of migratory and forbids the use of hooks for surveying A japonicas, al- other behaviors A spotlight survey of C alleganiensis in ed in May and June in this region (Humphries and Pauley Kawamichi and Ueda (1998) used nocturnal surveys combined with wading for A japonicus in stream2000) most common survey technique is 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) mature adults are best conduct- beds, and this technique, without substrate turning, now 2004) Protection et al though they can be captured without a hook by using West Virginia, USA, showed that increased nocturnal activity is correlated with high water levels, and suggested that spotlight surveys for 2002; Tao et al the for A japonicus 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 were abundant prior to the 1980s, when individuals could be found easily and captured, 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, habitat fragmentation, and pollution Responses to questionconcluded ) A davidianus , naires also suggested that A davidianus inhabited areas Figure Artificial to increase the spawning dens for number of nesting C alleganiensis are sites where 82 subsequent nocturnal surveys failed to detect them, so questionnaire results were neither verified nor used and allow monitoring discredited of egg production and larval survival Image Noelle Rayman amphibian-reptile-conservation.org 06 December 2011 | Volume | Number e34 | Survey techniques example of questionnaire survey, Tochi- In another moto et al salamanders for giant occupancy surveys of special conservation significance (2008) collated data using questionnaires on where other techniques are not Hyogo Prefecture, A distribution map of A japoni- the past distribution of A japonicus in is western Honshu, Japan fish effective Electrofishing known for causing spinal injuries and mortality in (Cho et al 2002; Wang et al 2004), and there is po- well cus was produced from the combined responses of oral tential for electric interviews, answers to written questionnaires, and data tive success (particularly during the breeding season) from previous publications Oral interviews were conducted with 17 people from fishermen’s associations, two people from the nature conservation society in Hyogo Prefecture, and 21 people recommended by the fishermen’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 to damage the shock to reduce salamander reproduc- and immune system (Nickerson and Krysko 2003) Electrofishing can seriously affect the health of critically {Hucho endangered bleekeri), fish such as the Chuanshan taimen and electrofishing is banned in the range of H bleekeri in Taibai, Shamixi Province, China (W Zhenghuan, pers comm.) may be Nevertheless, electrofishing the best tech- nique for occupancy surveys in some difficult habitats where the detection of threatened salamanders is of major conservation significance (Nickerson and Krysko Electrofishing 2003) Wang (2004) reported the capture of eight et al A davidianus with electrofishing, whereas nocturnal sur- Electrofishing requires a backpack voltage generator, veys revealed none and bow-hooks only one (Zhang and connected to two submersible electrodes, earned by a Wang researcher walking slowly through a stream ians and other aquatic vertebrates are first 2001) Amphib- attracted to the electrical field of the electrodes and then temporarily Underwater camera systems paralyzed (Reynolds 1983) Williams et al The use of waterproof video systems (1981) considered electrofishing for surveys mini- with seining effective for surveying C alleganiensis mizes habitat disturbance, and video systems can locate However, subsequent studies have not supported den this sites, record reproduction and behavior, and provide conclusion (Bothner and Gottlieb 1991; Nickerson and other valuable information on Cryptobranchid biology Krysko 2003) In extensive river sections where large populations were found using other survey techniques, Waterproof video systems are very effective where Cryptobranchids utilize heavy large rocks or bedrock crevices electrofishing failed to reveal C alleganiensis (Nicker- for shelter son and Krysko 2003) Electrofishing failed to locate C alleganiensis during surveys age in New York, on the Black and white cameras have been used success- Susquehanna drain- fully whereas turning rocks was successful are However, suitably small underwater color cameras now available Although color cameras are (Soule and Lindberg 1994) Substantial rock cover and sensitive than black poor water currents can efficient at revealing result in shocked C alleganien- moving from beneath rocks during (Nickerson and Krysko 2003) sis not Common mudpuppy losus ), concluded that electrofishing ( mal Necturus macu- was that incorporates all features needed for surveys However, there are two Nevertheless, there are examples of successfiil electro- cameras fishing for aquatic salamanders, especially mander abundance is abundance including when Maughan et al are not opti- one efficient aquatic relatively inexpensive 1) fishing video systems, and 2) inspection Fishing video systems (12 volt) can easily be modi- sala- being associated with other species fish We more systems available suitable for surveys of aquatic sala- ineffective in manders: sites salamanders and eggs is for surveying all Cryptobranchid species, or with large populations (Matson 1990) surveying and white, the use of color aware of an “off the shelf’ video camera system electrofishing A two-year population study of another large aquatic salamander, the less light fied for surveys of Cryptobranchids However the water- proof charged couple device (CCD) cameras associated (1976) used many crevices electrofishing to successfully survey the Pacific giant sal- with these systems are too large to access amander (Dicamptodon ensatus ), and Nakamoto (1998) exhaustively surveyed both fish and D ensatus using multiple passes with backpack electrofishing Occa- These cameras are also relatively bulky and better suited sionally, C alleganiensis are incidentally captured electrofishing by fisheries biologists during late from a small boat or canoe Inspection cameras are very lightweight, and with small camera heads, have to use with proven effective for surveying summer/ tion of both systems early autumn Because of tively small its potential to harm salamander that standard monitors are rela- and are not waterproof that are waterproof, lightweight, is not generally recommended, and should be confined to amphibian-reptile-conservation.org A limita- Video systems are being developed by researchers health and reproduction the use of electrofishing for surveys is C alleganiensis less 07 camera system, and incorporate a wire- digital recorder, December 2011 | and video goggles Volume | Number e34 | Browne The video recorder, battery pack, et al 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 tive surveying, cameras may be camera systems left in for ac- 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 Figure used to provide electricity in remote but secure locations 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 mesh inside of the hinged door of a wire Passive integrated transponders (PIT) and was later Foster et mark-recapture cage The bait cage removed and replaced using plastic zip ties From al 2008 Used with permission from Herpetological Review PIT Bub tags are small, waterproof, glass-encased capsules et al (2002) showed that when PIT tags were containing an alphanumeric code read with a portable hidden within different stream microhabitats, more than 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 80% were Hill et read-only tags containing unique factory-set alphanu- equipment weight made PIT packs easy meric codes or as read-write tags that can be changed range of optimized PIT packs approached 90 reader to any value The new read/write PIT tags enable to be recorded, retrieved or changed using the receiver, including the GPS contact information Gorsky et read/write PIT tags al to assess Atlantic lar) migratory path selection details (2009) used 23 the size of The standard reader ranges in water Breen The read cm when et al (2009) fish using a portable antenna investigating displacement, mm mean movement sculpins PIT ( Cottus distance, and home range of Mottled bairdii) PIT tagging, photographs of head or tail spotting patterns were used to identify post metamorPrior to tags has steadily decreased, the detection range increases with PIT tag PIT tag was submerged to use found a detection efficiency of 76% with PIT-tagged salmon ( Scilmo sa- Although the (2006) tested specialized “PIT pack” antenna systems and found that design modifications and reduced name, and location, habitat, tagger’s al subsequently located with portable antennas for read- phic individual A japonicus for mark-recapture studies cm for the smallest microchips (1.5 cm for the largest (34 mm) Fish less (Kawamichi and Ueda 1998; Tochimoto 1991; Tochimoto et al 2005) PIT tagging is the most common tech- than 55 nun have been successfully tagged using 11.5 nique for mark-recapture studies For example, Tochi- mm PIT tags that weigh 0.1 moto size only PIT tags are 3-8 x mm) now and 15-45 g, and the smallest PIT tags available should be suitable for all but the smallest ing salamanders in shallow water habitats cm detected up to 90 is the use of Tottori Prefecture through water (Hill provements in antenna technology (Hamed Cucherousset et al 2006) et al et al 2008) (2008) showed that detecting Pyre- nean brook salamanders ( Calotriton asper) using PIT lemetry was 30% more and four times as C alleganiensis sampling over time, than had sampling through visual searching and rock turnefficiency of PIT telemetry was negatively signal, ousset et al inlets The four undetected A search of any additional C alleganiensis moved into water deeper than the reach of the wand antenna (two meters) or moved under the cobble substrate (Wheeler 2007) cor- Automatic systems used with PIT tags and undercut banks (Cucher- to survey movement have been in fisheries research These consist of remote antenna arrays spanning water bodies Meynecke 2008) et al to amphibian-reptile-conservation.org day either detector and positively correlated with the number of sampled spring two the following the area with rock turning did not detect te- efficient for individual sampling, efficient in between 2001 and 2008 able to detect only related with the presence of large stones that blocked the easily between 1975 and Wheeler (2007) used the BioMark® submersible antenna with a detection distance of up to 30.5 cm to survey for previously PIT tagged C alleganiensis Of six C alleganiensis marked using PIT tags, surveyors were and detection range should further increase through im- PIT Prefecture, 2004 Okada (2006) tagged more than 500 individuals in submersible antennae and larger PIT tags that have been The Hyogo 2004, with 588 of these PIT tagged between 1998 and A promising new technique, for surveying and locat- ing (2005) recorded 1204 individual salamanders in the Ichi River, Caudata direct et al 08 (2008) successfully used remote PIT technology monitor fish movement for 104 days in a December 2011 | Volume | mangrove Number e34 | 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- and structure of populations netic variation 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 Raul Diaz for strategies currently a Ph.D candidate at the University of Kansas Medical Center/Stowers Institute is Medical Research His research includes the developmental genetics and evolution of the vertebrate skeleton, focusing unweave the classic on the reptile cranial and appendicular skeletons, gastrulation, embryology, and trying to genetics of species morphological divergence and adaptation through the use of genomics and 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 Nashville Zoo Zoo Dale program to at Grassmere, Tennessee, in herpetology USA, where he and currently works as Curator of Ectotherms designed the impressive Herpetarium at at Nashville managing conservation breeding populations of Galliwasps and initiated the first perpetuate the genetic variation of any amphibian through the sperm cryopreservation of Cryp- is currently 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, Jeff promotes, protects, and monitors amphibian and reptile populations in Missouri, USA and has been leading Hellbender conservation efforts in Missouri since 2001 amphibian-reptile-conservation.org 029 September 2012 Volume | | Number e54 | © 2013 StrauB et al This is an open-access article distributed under the terms of the Creative Commons Copyright: Amphibian & Reptile Conservation Attribution-NonCommercial-NoDerivs 3.0 Unported License, which permits unrestricted use for non-commercial and education purposes only provided the original author and source are credited 5(4): 51-63 from biopsies from the Critically Endangered Chinese giant salamander, Andrias davidianus (Blanchard, 1871) (Amphibia, Caudata, Cryptobranchidae) In vitro culture of skin cells ^arah Strauli, ’ ’ Thomas ^erstin Reimers, Natalie Frank-Klein, Robert Seuntjens, and Peter M Vogt Ziegler, Christina Allmeling, Department ofPlastic, Hand- and Reconstructive Surgery, Hannover Medical School, Carl-Neuberg-Str 1, 30625 Hannover, GERMANY Cologne Zoo, Riehler Strafie 173, 50735 Koln, GERMANY Cologne Biocenter, University of Cologne, Zulpicher Strasse 47b, 50674 Cologne, GERMANY 4Berlin Zoo, Hardenbergplatz 8, 10787 Berlin, GERMANY l Ambystoma Mexicanum Bioregeneration Center, — Abstract We established a primary skin cell culture of the Critically Endangered Chinese Giant Salamander, Andrias davidianus from small biopsies using minimal invasive methodologies Biopsies 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 containers 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 cultures 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, regeneration, wound healing skin tissue explants, skin biopsy, biopsy withdrawal, amphibian skin cell culture, 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) Citation: Straufi S, Ziegler T, Allmeling C, Reimers K, Frank-Klein N, degradation Introduction is The Chinese (A the now very rare 2012) Consequently, A davidianus in nature Andrias davidianus is listed in 80 cm Together with the Japanese giant salamander 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 japonicus ) from central and southern Japan, and Red giant salamander (Andrias davidianus ) the largest extant amphibian, with a total length of to (IUCN North American up form the sole members of including A davidianus, often involves sacrifice of the which animals is among caudate amphibians (Gao and Shubin 2003; review Browne et al 2011) This thought to be a basal family al present in the Jurassic (Gao et widespread in and southern China, although 2003) al range The Chinese is now 1,500 areas, at elevations alternative to fishes For example, Estepa in fin cells to of rainbow established and infected with the virus in vitro The purpose of the present study was whether it is possible to establish primary farms Wild harvesting for human consumption of the skin major and of Correspondence Email: 5ziegler@koelnerzoo.de (Corresponding amphibian-reptile-conservation.org et al Oncorhynchus mykiss (Estepa 1993) For this assay primary cultures from tissue explants of trout fins were natural habitats while millions of these animals are bred in threat to A davidianus , along with habitat destruction et trout declined catastrophically over the last decades in their a Geng whole animal experiments haemorraghic septicaemia virus m above sea level Once common, the species has is (e.g., (1993) described a cell culture model to study the viral very from 100 An been described for fragmented The species inhabits streams and rivers in mountainous forested 2011) end of the experiments would minimize destruction of the animals is the use of in vitro cell cultures Such assays have already central, south-eastern its at the that family might be a survivor of a lineage that was already is of Threatened Species (IUCN 2012) Research on diseases and other issues in salamanders, ( the giant salamander family Cryptobranchidae, giant salamander List Cryptobranchus Hellbender alleganiensis), these species is 051 tail tip cells tissue to determine in vitro cultures of A davidianus from small biopsies Various cell culture media, surface author) June 2013 Volume | | Number e66 | Strauft et Fig Biopsy method one performed in the al zoo of Berlin The animal was housed in an exhibition tank ferred in a tub, resulting in aggressive reactions of the animal, which made the biopsy procedure very It was captured and trans- 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 H60 cm) find that this technique could serve as a feasible alternative to studies that require the destruction (W60 x cm mesh tanks can be connected through a sliding gate The ground size) of individual animals consisting of stainless steel wire (1 substrate consists of gravel mixture with large roots As and sand hiding possibility, each tank L80 contains a shelter (female tank: x W50 x H50 cm; L125xW50xH50 cm) with entrance in front at the rear side (each opening arched, W36 x male tank: Material and Methods and HI Three adult A davidianus were used for this study is Sexing was done via ultrasound One male was housed at the x Berlin Zoo in an aqua-terrarium (L340 x H220 cm) with 50 cm water depth with decorative objects provided (Fig is cm) Another adult male (not used for this study) and A) Water temperature Zoo held in a tank in the public area of the Cologne Aquarium (L350 W160 shelter exit x W126 x pH value 7.3, conductivity and hardness 16; total H85 cm; temperature 14 °C, 668 pS, carbonate hardness 7, illumination: HQI spotlight, 400 Watt) 20 °C and water quality maintained by a sand-pressure filter, and partial daily, and complete weekly water Biopsies changes The remaining two adult A davidianus were housed at the Cologne Zoo Aquarium The couple held in two concrete tanks (each LI 50 x cm) with 50 system) is cm W190 x In order to keep the biopsy procedure as stress-free is H60 and water depth The water (flowing water connected to a cooling system and an external (OASE pond filter, roofing consists of stainless steel fence (1 possible two methods were tested at Berlin Zoo): 1) capture of the salamander, and 2) placing it then taking biopsies from the tail tip (Fig two (conducted at net, cm mesh size), (Stiefel 2) 1) Method Capture of the it in its housing Minimally-invasive were perfonned by using biopsy punches GmbH, Coral Gables, USA), with and mm biopsies taken from the 052 1) and keeping tank and taking biopsies (Fig biopsies with water and in a tub Cologne Zoo): salamander in a landing with one half being shaded each by styrofoam mats Both amphibian-reptile-conservation.org as Method one (conducted 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 filter efficient tail tips of two males (Berlin June 201 Volume | | Number e66 | In vitro culture of skin cells from the Chinese giant salamander 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 Fig use of biopsy punches E: tissue inside a punch F: transfer of tissue in tube with amphibian ringer solution for rinsing Table Contents of Modified Amphibian Ringer Solution and Cologne Zoo) and one female (Cologne Zoo) The procedure was performed without anesthesia as pain NaCI KCI MgCI CaCI HEPES mM 1.8 mM mM mM mM 100 of biopsy is negligible, anesthesia too high and consequently the The procedure was risks of 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 amphibian-reptile-conservation.org 053 June 201 Volume | | Number e66 | Strauft et Fig Andrias tails nine months mentations A: overview of tail Table Cell culture Medium Williams Medium E Leibovitz L-15 DMEM/F12 after B al biopsy procedures Lost tissue was completely regenerated without scar formation or dyspig- and C: detail of tail tip media, supplements and coatings Green highlight: optimal conditions for culture of Andrias skin Supplements ITS Sodium-P 1% 1% 1% 1% Coating NEA A2P P/S Genta mM 1% 50 U/ml 0.1 mg/ml 1% 50 U/ml 0.1 mg/ml 1% 50 U/ml 0.1 mg/ml mM mM mM 1% 50 U/ml 0.1 mg/ml 1% mM 1% 50 U/ml 0.1 mg/ml 1% 1% 50 U/ml 0.1 mg/ml 1% 1% 50 U/ml 0.1 mg/ml 1% 1% 50 U/ml 0.1 mg/ml 1% 1% 50 U/ml 0.1 mg/ml 1% 1% 50 U/ml 0.1 mg/ml 1% 1% 50 U/ml 0.1 mg/ml 1% 1% 50 U/ml 0.1 mg/ml 1% mM mM mM mM mM mM mM mM 50 U/ml 0.05 amphibian-reptile-conservation.org cells 1% 50 pg/ml 054 mg/ml HEPES PLL Collagen FS none + + + + + + + + + + + + mM + June 201 Volume | | Number e66 | In vitro culture of skin cells from the Chinese giant salamander Fig Cell outgrowth from of culture media Within the tissue maintained in three types first days no differences of cell outgrowth in media types was observed Pictures were captured using phase contrast light microscopy on day DMEM/ A: F12; scale bar 100 pm B: Leibovitz L-15; scale bar 100 pm C: Williams well Medium E; scale bar 100 pm Biopsy pieces were placed in plastic tissue culture dishes, with or without coatings (see Table 2) Medium was added three minutes later The volume of medium was adjusted to size of the culture well or (see Table 2) flask, so tissue pieces were slightly containers were stored in a wet conditions at room immersed Culture chamber under sterile temperature Final concentration of amino acids is provided in Table abbreviations and suppliers in Table non-essential and used Tissue Preparation Culture Containers 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 The solutions were supplemented with 50 U/ml penicillin/streptomycin (Biochrom) and 0.05 mg/ml gentamicin (Biochrom) Following containers were examined for • 12 well plates, attachment surface of 3.6 to further support (# 92024, TPP, Trasadingen, Switzerland) • Microflask, attachment surface 10 cm2 (#91234, TPP, cm2 (# Trasadingen, Switzerland) • Miniflask, attachment surface 25 Samples of one male and one female animal (Cologne) processed for cm2 /well • 24 well plates, attachment surface 1.9 male A davidianus (Berlin) were transported in cell culture media without supplementation for four hours carefully, directly cm2 /well (#92012, TPP, Trasadingen, Switzerland) reduction of microbial contamination Samples of one were rinsed cell culture: 90025 and 90026, TPP, Trasadingen, Switzerland) cell culture • 24 well plates, attachment surface without use of a cell culture workbench and after (#CC7682, Cyto One, USA) adherence transported to their storage place within three • Miniflask, attachment surface 25 hours cm2 1.9 cm2 /well (#7.690, Greiner Bio One, Frickenhausen, Germany) Biopsies were processed by cutting them into small (1-2 mm) available, pieces we As only small tissue samples were decided to perfonn cell culture in Depending on manufacturer's production processes adhesion surfaces of the containers might be treated differently (e.g., plasma treatment of surfaces with small The choice was between flasks that could be sealed thus making them suitable for transport of containers the culture from the zoo to the lab which are commonly used cm varying and multi well plates for cell culture assays conditions So 25 known were used especially for the cultures and 24 and 12 well plates were tested as tissue culture flasks starting amphibian-reptile-conservation.org protocols), in vaiying As from mammalian primary adhesion cell culture is on every type of containers of various manufacturers were that not every cell type adheres culture plastic, examined 055 resulting for cell culture of Andrias skin tissue explants June 201 Volume | | Number e66 | Strauft et Fig al 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: overview, 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) vt Results Biopsy in A davidianus Table The and ITS final concentrations of non-essential amino acids 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 in cell culture pg/ml L-alanine 8.9 L-asparagine*H 15 L-aspartic acid 13.3 not react to the biopsy taking, which took less than five minutes (Fig 2) sparsely or not at Directly after biopsy the all, wounds bled and inflammation and/or infection L-glutamic acid 14.7 of the wounds did not occur Healing took about two Glycine 7.5 months; the L-proline 11.5 without scar formation (Fig L-serine 10.5 Insulin 10 Transferrin 5.5 Selenium A lost tissue was completely regenerated 3) Cell Culture 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, Cell culture 0.0067 Media Supplements and Coatings was performed in a , the tissue fragments adhered only in small proportions All cell culture media were diluted to sterile distilled 60% water to achieve appropriate osmolarity Media, supplementations and plastic coatings are in Table (5%), whereas more than 80% of the fragments adhered on the plastics of both types of miniflasks (Greiner and (v/v) with TPP) Cells started to grow out from adhered tissue listed under Cell culture material was coated by dropping culture conditions within 12 hours (Fig 4) Beside skin epithelial solutions melanophores grew cells also out The melanophores appeared rounded during migration processes whereas resident cells showed typical dendritic morphology (Fig 5) 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 °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) amphibian-reptile-conservation.org all 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 056 June 201 Volume | | Number e66 | In vitro culture of skin cells from the Chinese giant salamander Comparison of male (A) and female (B) tissue after three days of cell culture Note that more outgrowing cells were observed the female samples A: male tissue at day three; scale bar 500 pm B: female tissue at day three; scale bar 500 pm Fig in examined in further studies with higher numbers of tested individuals In our study female cells earlier and covered greater areas indicating of migration (Fig senescent Additionally, 6) male Discussion grew out The faster rates cells became was tested in long- term culture Cells in Leibovitz or WilliamsE media survived only (e.g., bites, and cause two weeks whereas Beckstein 2009) To minimize such recommend using cell culture stress risks, we a landing net to restrain the animals with in the housing tank for biopsy procedures as the animals survived for 10 weeks Cells grown in stayed absolutely calm and apparently oblivious to the with procedure for full cells supplementation (see Table green highlight) generally showed best results (Fig 2, We 7) grew out, formed complete monolayers and started to form tissue-like structures with pigmentation (Fig and 8) After six weeks multi nucleic cells occurred more frequently (Fig 9), these cells stopped growing Cells finally died after 10 weeks Dead from the adhesion surface and floated cells (4-(2-hydroxy ethyl)- -piperazineethanesulfonic acid) pH stabilization (visualized Nickerson 2003; Mutschmann 2009) could find no literature concerning the cell any other cryptobranchid species in Western literature, or from correspondence Based on the cold freshwater physiological conditions experienced by A davidianus cell culture could be expected to be most successful through Chinese literature , in big sheets in HEPES Medium (cf culture of A davidianus or broke away supplementation with the containers resulted in difficult and possibly injury for both animals and researchers Influence of media conditions and and weight of adult Andrias davidianus make handling of the animals earlier DMEM/F12 DMEM/F12 large size with lower temperatures than with mammalian cells Other conditions to consider with the establishment of A davidianus by phenol red mammals, cell cultures, in respect to body are a lower osmolarity of those of fluids in A indicator in cell culture media) Without this buffer davidianus (Albert medium’s pH changed after less than one hour in the wet chamber as C0 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 particular cell culture coatings for optimal cell adherence to the cells stabilize Usually a concentration of 10 mM is cells 1987; Chemoff et and al.l 990), shown with Xenopus laevis and Ambystoma mexicanum primary cells (Nishikawa et al 1990; Chemoff et al.1990) We assessed the use of and used to media, but this concentration was found to be harmful to the et al proliferation, as culture different cell plastics (according containers to and various treated manufacturer’s datasheets) combined with various media conditions and surface of the giant salamander coatings We Problems with contamination by a fungus (white found that the size of was cell culture containers appearance, no detennination of species perfonned) important for the successful outgrowth of cells, and tissue occurred in cell culture from one male animal (Cologne) pieces were and were treated with amphotericine B (Biochrome) more likely to stick used instead of multiwell when plates This small flasks were might be explained This treatment stopped fungus growth, but cells started to by the tendency of small pieces of tissue age after two days of antifungal treatment The cultures surface of solutions toward the containers wall thus of the female (Cologne) and the other male (Berlin) preventing adhesion to the bottom of the container tissues remained uncontaminated during the culture process Repeated preparations Cologne animals at later from Cells from multicellular organisms further biopsies of on the communicate with each other by release of messenger substances into the time points resulted again in extracellular fluids, e.g., the culture fungal contaminations of male cultures amphibian-reptile-conservation.org to float cell-cell contacts 057 medium, or by direct To accomplish sufficient concentrations June 2013 Volume | | Number e66 | Straufl et Fig Picture time line of cell outgrowth in tative examples of long term outgrowth of DMEM/F12 cells under with full full al show represenAt the migration supplementation Images in overview and detail supplementation Cells grew out in dense layers (A) 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 front cells filopodia formation F: cells at was observed (B) day 25; scale bar 500 pm of bioactive molecules by cellular release of substances growth factors like factor, factor), (e.g., for keratinocyte enzymes (e.g., vascular endothelial growth vitro as cells are choice of cell culture container size means a balancing act lipoxygenases) and cytokines between low surface curvature (implying use of greater factor, fibroblast medium, low volume recommended Too low culture containers) and interleukines) to their culture small cell missing paracrine stimulation So the growth growth (e.g., concentrations of these substances lead to cell death in numbers is amphibian-reptile-conservation.org low medium volume (implying use of smaller culture containers) 058 June 2013 Volume | | Number e66 | In vitro culture of skin cells Fig Multilayer formations after six from the Chinese giant salamander 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 Fig Cell aging After six weeks in weeks of cultivation; DMEM/F12 scale bar 500 pm multinuclear cells were observed more examples and were captured using phasecontrast lightmicroscopy A: overview (multinuclear 200 pm B: Table detail List Williams show representative by arrows); scale bar frequently Pictures cell indicated of A; scale bar 50 pm of abbreviations and suppliers Medium E PAA, Colbe, Germany Leibovitz L-15 PAA, Colbe, Germany DMEM/F12 PAA, Colbe, Germany A2P Sigma Aldrich, Taufkirchen, Germany 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 Ampho Biochrom, Berlin, Germany HEPES PAA, Colbe, Germany Ascorbate-2 -phosphate Insuline-Transferrine-Selenium Amphotericine A B (4-(2-hydroxyethyl)- -piperazineethanesulfonic acid Biochrom, Berlin, Germany Collagen Poly-L-Lysine PLL Biochrom, Berlin, Germany Fish Serum FS own production from trout blood amphibian-reptile-conservation.org 059 June 201 Volume | | Number e66 | Straufl et The growth or the culture survival time seems to be negligible as no correlation to the culture material or surface coatings is The fungal contamination of the Cologne Zoos male’s cell culture appeared to be from the skin microflora Contaminations during tissue processing seem an influence of the adhesion surface on adhesion rates, cell migration, cell cell was observed This contaminations culture as occurred under a wide range of preparation conditions including types from X laevis and A cell cause unlikely contrary to data from the literature describing culture of various amphibian al Further research conditions sanitized is planned to mexicanum on developmental or regenerative aspects identify the type of fungus and to assess as well as toxicological influence on outgrowth of cells from the tissue explants Nishikawa al 2003 Chemoff 1990; et al et al.; Ferris studies (Albert at et al.1990; al 1987; Goulet In vitro treatments with amphotericin et for this toxic effect matrigel and other matrices to encourage cell adhesion (Fungizone) MEM, media nutrition MCDB151 FI 2, 70% with combination of these diluted to sterile distilled is insulin, transferrin explant cultures obtained from Ambystoma mexicanum DMEM under supplementation can be grown with 10% fetal in 60% Culture survival appeared to be more dependent on medium pH the stabilization of culture mammalian primary coatings; stable pH around to remain cells vital in vitro a in Fig HEPES while the commonly used concentration of HEPES of 10 mM was toxic to the cells and led to cell with A concentration of the pH as well as davidianus cells mM resulted in stabilization only samples of three animals so no detectable toxic influence on A High sensitivity to HEPES was mention the use of media Alternatively to HEPES HEPES, an need also fumigation can be used to stabilize the Nishikawa al.1990; et al Without pH stabilization cell al C0 The CO, and may outgrowth and survival was primary problem rises the transfer of tissue to cell culture; the culture good growth conditions in fish or with other giant known it is faster than that females male animals due (Blankenhom et al to 2003) and amphibians known from tended to form pigmented cells in general, but weeks Usually mammalian primary when reaching confluence stop proliferation due to contact inhibition by interactions (Qi et with cells or medium for the target cells trials multilayers in long term cultivation (Fig 7F and 8) be negated by decreased flora, thus a these observations Cells did not only form a monolayer as 2010) caudates not live in a sterile environment and need a certain skin offers better after three cell culture As wounds phenomenon benefit of the use of antibiotic supplements in survival MRL mice As we tested human cutaneous tissue (Gilliver et al 2007), however we could find no published information on this fumigation amphibian From 6) tissue also with greatly reduced in our study as well as in other studies using far, be confirmed by repeating sexually dimorphic genes pH (Chemoff 1990; Ferris et to salamanders heal in the culture incubator with from female outgrowth cell appeared to be faster than from male (Fig a blastema cells did not et Interestingly, model of A mexicanum (Guelke submitted) Previous publications on amphibian shown with et al of 2011) of Billingham (1948) which are compared in the study davidianus were veiy sensitive to the supplementation death al from dendritic origin (Rawles 1948; Billingham 1948) In light microscopic imaging melanophores ofrt davidianus appeared equal to those of guinea pigs shown Cells of A (Fungizone) on tadpoles of Alvtes cells are than on surface usually need B 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 2010) et al B only one publication mentioning possible toxic effects cisternasii (Martel et bovine serum and ITS (insulin transferrin, selenium A) (Ferris remain unclear as amphotericin is of amphotericin and EGF Skin Causes commonly used in fish and amphibian cell and known to be not toxic to cells so far There cultures or water were used (Nishikawa 1990) Culture media were supplemented with for this fungus resulted in early senescence and cell death 2010) In those studies cell culture vessel plastics were coated with fibronectin, collagen, As B possible its immortalized inhibition and et al al cell-cell in cells vitro and cell-substrata 2008) In contrast most cancer cell lines are refractory to and can continue to proliferate contact (Hanahan 2000) Cell cultures from Xenopus skin explants simultaneously for microorganisms Bacteria and fungi only grew out as monolayer stopped expanding after accrete faster than the cells and cause cell death six to eight days by (Reeves et al 1975) This raises the release of toxic substances In our study, cells tolerated question whether the observed multilayer formation of 50 U/ml of penicillin/streptomycin mix Andrias skin explants could be related to the regenerative is sufficient to avoid infections cultures which of already established capacity of caudate amphibians Therefore 0.05 mg/ml gentamicin was thus added The culture (p/s) common primary mg/ml of gentamicin, but this (Hayflick index) in Western in amphibian cells There is cell culture after certain no comparative research scientific publications immortalized on the use of antibiotics well-known process results are 060 in mammalian to their limited proliferation capacity mammalian cells become senescent time of in vitro cultivation in contrast to cell lines Literature of amphibian primary media amphibian-reptile-conservation.org cells a Due cell concentration resulted in early senescence and cell death of A davidianus is of antibiotic supplementation media contains Senescence cells is regarding life span limited and described ambiguous While Nishikawa reports ageing June 2013 Volume | | Number e66 | In vitro culture of skin cells from the Chinese giant salamander (Nishikawa 1990), Kondo et al (1983) describes a growth crisis (senescence) in melanophores followed by a spontaneous transformation of Xenopus skin to an immortalized catesbeiana during the breeding period, cells in vitro (Kondo al Rana from derived line cell may became senescent and did not undergo less stressful sexing spontaneous a distinguished by their new of these salamanders Karyotyping chromosomal aberrations The creation of an immortal Andrias skin cell line could 1) et also offers the opportunity to screen the animals for transformation and eventually died possibly be achieved by: may be sex chromosomes and this technique would enable a our study skin cells et al 1983) In and are distinct Based on the study of Zhu effect reproduction (2002) A davidianus when gonads that spontaneous transformation may be programs salamanders distinguish to unsuitable for use in conservation breeding However, skin karyograms can only cell 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 provide insights into chromosomal aberrations of somatic may have to be from biopsy withdrawal of cells as a small some of them cells Examination of wound cultivated over a long period of time until start and not those induced by unlimited proliferation 2) expression vitro closure capacities of A davidianus from ATCC for A davidianus research in use of method one anurans (Kondo Lalcra et al is well documented 2011) while for method two only regarding fish cell lines is Using resulting outgrowth cell cell culture would reduce experimental animals and provide in models number of the new research horizons and benefit conservation breeding programs 1983) as well as in fishes (review et al line could give information about the regenerative e.g., is (MBA- 141) The germ processes and in vivo of telomerase reverse transcriptase (TERT) which available as eukaryotic expression plasmid failures in the literature available (review Lakra et al 2011 ) Acknowledgments —We thank Bodo Lang (Cologne) Sabine Ommer for fruitful discussions and and for reviewing a previous version of the manuscript Thanks Conclusion Andre also to the veterinarians Dr and Schiile (Berlin) Dr Olaf Behlert (Cologne) for kindly supporting the This study examined the needs basic of primary cultures for A davidianus skin cells raised skin biopsies These cells culture needs when chamber except seem HEPES from small have no exceptional performed cell culture is for specific stabilization with to biopsy procedures medium in a Literature Cited wet osmolarity and pH Albert buffer Primary cultures of Andrias skin cells, effects of medication, 2) toxicity tests blastemas by diseases and 1) Boilly B, Courty J, Barritault D 1987 Stimula- tion in cell culture of mesenchymal cells of newt limb as well as other amphibian primary cell cultures can be used in future studies to evaluate effects of; P, EDGF I or II (basic or acidic FGF) Differentiation 21: 63-68 of pollutants and Beckstein R 2009 Gefdhrliche Here Munchen in Menschenhand other substances as already described for fishes (Dayeh Dissertation 2005) and anurans (Goulet 2003), 3) for the study of regeneration, and 4) the role of gender specific hormones muenchen de/ 0779/ /BecksteinRuth.pdf my cells Journal ofAnato- 82: 93-109 Blankenhorn EP, Troutman S, Desquenne Clark L, Zhang XM, Chen P, Heber-Katz E 2003 Sexually dimorphic genes regulate healing and regeneration in MRL mice Mammalian Genome 14: 250-260 Browne RK, Li H, Robertson H, Uteshev VK, Shishova NR, McGinnity D, Nofs S, Figiel CR, Mansour N, Lloyd RE, Agnew D, Carleton CL, Wu M, Gak- their genetic material for use in reproduction technologies The next steps in the establishment of an in vitro cell culture model will be on the one side the development for cryopreservation cells not have to et al.) we hova EN 2011 Reptile and amphibian conservation through gene banking and other reproduction A further contribution to cryptobranchid conservation technologies Russian Journal of Herpetology 18(3): be immortalized; they can be stored and cultivated predict as of Available: http://edoc.ub.uni- Billingham RE 1948 Dendritic 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 (Browne Cell mammalian primary cell lines is their cells use for establishing of a karyogram 165-174 Browne RK, Li H, Wang Z, Hime P, McMillan A, Wu M, Diaz R, Hongxing Z, McGinnity D, Briggler JT 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 elaborate which risks, is procedure During ultrasonic is The an examination palaeontology, phylogeny, genetics, and morphology usually done without anesthesia, also injury both for animals and human considered Sexing with ultrasound amphibian-reptile-conservation.org is Amphibian Dayeh beings, must be also most giant salamanders (Cryptobranchidae): Part A bow effective 061 & Reptile Conservation V, Schirmer K, 5(4): 17-29 Lee LEY, Bols NC 2005 Rain- Trout Gill Cell Line Microplate Cytotoxicity June 2013 Volume | | Number e66 | Strauft et Freshwater Toxicity Investigations Test Small-Scale al Fakra 1:473-503 WS, Swaminathan TR, Joy Kp 2011 Develop- ment, characerization, conservation and storage of Estepa A, Frias D, Coll JM 1993 In vitro susceptibility of rainbow trout fin cells to viral Fish Physiology and Bio- fish cell lines: a review chemistry 37: 1-20 haemorraghic sep- Martel A, van Rooij ticaemia virus Diseases of Aquatic Organisms 15: P, Vercauteren G, Baert K, Van 2010 Colored Atlas of Chinese Waeyenberghe F, Debacker P, Garner TWJ, Woeltjest T, Ducatelle R, Haesebrouck F, Pasmans F 2011 De- Amphibians Sichuan Publishing Group/Sichuan Pub- veloping a safe antifungal treatment protocol to elimi- 35-39 Fei F, Ye CY, Jiang JP House of Science and Technology lishing nate Batrachochytrium dendrobatidis from amphib- [In Chi- Medical Mycology 49(2): 143-149 Mutschmann F 2009 Erkrankungen der Amphibien nese] ians DR, Satoh A, Mandefro B, Cummings GM, Gardiner DM, Rugg EF 2010 Ex vivo generation of a functional and regenerative wound epithelium from axolotl ( Ambvstoma mexicanum) skin Development, Ferris Growth Frost & Differentiation ISBN: 978-3-8304-1097-3 Nickerson MA 2003 Asiatic giant salamanders and hellbenders Pp 343-347 In: Editors, Hutchins MH, Auflage the World: an Michigan, Online Reference Version 5.5 (31 January, 2011) American Museum of Natural New York, USA History, New Isolation, characterization known crown-group vis salamanders Nature 422: 424-428 Ashcroft GS 2007 The hor- monal regulation of cutaneous wound healing Clinics in Dermatology 25(1): 56-62 Goulet BN, Hontel A 2003 Toxicity of cadmium, endosulfan, and atrazine in adrenal steroidogenic cells of two amphibian species, Xenopus laevis and Rana catesbeiana Environmental Toxicology and Chemistry 22(9): S, Bucan a new approach SRC 2013 [Abstract Developmental Biology’ 26: Rawles ME 1948 Origin of melanophores and their role in development of color patterns in vertebrates Physiological Reviews 28: 383M08 Reeves OR, Faskey RA 1975 In vitro differentiation of a homogeneous cell population the epidermis of Xenopus laevis Journal ofEmbryology & Experimental Morphology 34: 75-92 Wiens JJ.2008 Systematics and Herpetology in the Age of Genomics BioScience 58(4): 297-307 Zhao E, Adler K 1993 Herpetology’ of China Society for the Study of Amphibians and Reptiles Contribu- — V, Allmeling C, Radtke C, Vogt PM, Reimers K 2013 Neuronal Guidance Factors: lae- Cell 13(3): 188-192 2106-2113 Guelke E, StrauB Xenopus lar- und Herpetologie Fandbuch- Verlag GmbH Hannover, Feipzig, Feipzig, Germany 466 pp Qi Z, Wanjin H 2008 The Emerging Role of the Hippo Pathway in Cell Contact Inhibition, Organ Size Control, and Cancer Development in Mammals Cancer Andrias davidianus) Journal of Comparative Pathology 145(1): 95-102 JJ, & of tik ( Ashworth in vitro culture 1990 Obst FJ, Richter K, Jacob U 1984 Lexikon der Terraris- ated with morbidity and mortality in farmed Chinese Gilliver SC, In Vitro Cellular F 1128-1134 Geng Y, Wang KY, Zhou ZY, Fi CW, Wang J, He M, Yin ZQ, Fai WM 2011 First report of a ranavirus associgiant salamanders and val and adult epidermal cells of the frog research.amnh.org/vz/heipetology/amphibia/ Earliest USA Nishikawa A, Shimizu-Nishikawa K, Miller York, Electronic database available: http:// Gao KQ, Shubin NH 2003 Verlag Duellman WE Gzrimeks Animal Life Encyclopedia nd Edition) Amphibia Gale Group Inc., Detroit, (2 52: 715-724 DR 2011 Amphibian Species of Thieme to Reconstructive Surgeiy EP- submitted] Hanahan D, Weinberg RA 2000 The Hallmarks of CanCell 100(1): 57-70 tion to Herpetology 10 IUCN (2012) The IUCN Red Fist of Threatened Species 522 pp Zhu B, Feng Z, Qu A, Gao H, Zhang Y, Sun D, Song W, Version 2012.2 Available: http://www.iucnredlist.org Saura A 2002 The kaiyotype of the caudate amphib- [Accessed: 29 October 2012] ian Andrias davidianus Hereditas 136: 85-88 cer Kondo H, Ide H 1983 Fong-term cultivation of amphib- Received: 05 February 2013 ian melanophores: In vitro ageing and spontaneous transformation to a continuous cell line Experimental Accepted: 13 Published: 29 June 2013 Cell Research 149(1): 247-256 amphibian-reptile-conservation.org May 2013 062 June 2013 Volume | | Number e66 | 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 and crocodiles Since February 2009, he has been an associate professor (Biocentre) of Cologne University lizards, snakes, Institute Christina Allmeling tion Center She is is responsible for captive care and breeding at the Ambystoma at the Zoological Mexicanum Bioregenera- management paper, defining the European guidelines for a member of the AG Urodela which is a working group of the German author of the Axolotl captive care these animals in laboratories She is Society for Herpetology and Herpetoculture Kerstin Reimers leads the research division of the Department of Plastic-, Hand- and Reconstructive Surgery 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 especially the axolotl In 2008 she and her team identified the AmbLoxe as a signal key to the axolotls regeneration at — 4LLL capacity Natalie Frank-Klein started her career at the Aquarium/Terrarium Department of the Cologne tember 1997 She finished her apprenticeship as a zoo keeper in 2000 Her focus of expertise fishes and aquatic urodelans Since 2011 she has been section keeper Zoo in Sep- is freshwater 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- 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) cal amphibian-reptile-conservation.org 063 June 201 Volume | | Number e66 | CONTENTS Administration, journal information (Instructions to Authors), and copyright notice Robert K Browne, Hong Li, Dale McGinnity, Sumio Okada, Kelly J Irwin, Amy McMillan, and Jeffrey T Briggler Wang Zhenghuan, — Survey techniques Inside front cover Catherine M Bodinof, for giant salamanders and other aquatic Caudata Robert K Browne, Hong Li, Zhenghuan Wang, Paul M Hime, Amy McMillan, Minyao Wu, Raul Diaz, Zhang Hongxing, Dale McGinnity, and Jeffrey T Briggler The giant salamanders (Cryptobranchi- — dae): Part A palaeontology, phylogeny, genetics, and morphology 17 Sarah Strauss, Thomas Ziegler, Christina Allmeling, Kerstin Reimers, Natalie Frank-Klein, Robert Seuntjens, and Peter M Vogt In vitro culture of skin cells from biopsies from the Critically Endan- — gered Chinese giant salamander, Andrias davidianus (Blanchard, 1871) (Amphibia, Caudata, Cryptobranchidae) 51 Back cover Table of Contents VOLUME 2012 NUMBER ... Instructions for Authors at the Amphibian and Reptile Conservation ing, important website: http:/ /amphibian- reptile- conservation.org/submissions.html © Craig Hassapakis !Amphibian & Reptile Conservation... official journal website at: http:/ /amphibian- reptile- conservation.org Instructions to Authors : Amphibian & Reptile Conservation accepts manuscripts on the biology of amphibians and conservation, sustainable... Hassapakis /Amphibian needs) and papers are immediately released as they are finished on our website; http:/ /amphibian- reptile- conservation.org; email: arc.publisher@gmail.com Amphibian & Reptile