Contents

Greetings ………………………………………..…… P. 1

Information about the Symposium ………………… P. 2

Symposium Program

Amphibian Genomics and Genome Editing …….. P. 4

Amphibian Conservation ………………….....….. P. 5

Poster Presentations ………………………….….. P. 6

Abstracts

Lectures

Amphibian Genomics and Genome Editing …. P. 11

Amphibian Conservation …………………….. P. 21

Poster Presentations ………………………….….... P. 35

List of Participants …………………….…….………. P. 66

Access ……...………………………………………….. P. 68

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Greetings

On behalf of the organizing committee, I would like to welcome you to the Institute for

Amphibian Biology of Hiroshima University’s International Symposium, “Frontiers in

Amphibian Biology: Endangered Species Conservation and Genome Editing,” held in

Hiroshima on March 27–28. The Institute is now engaged in a research project titled

“Pioneering amphibian research: conservation of endangered amphibian species and

development of gene targeting methods,” which is funded by a special education and research

expense from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

The project will conclude at the end of fiscal 2013. The purpose of this symposium is to

introduce the outcomes and findings of the project, as well as promote international exchange

among researchers of amphibian biology.

The symposium’s scientific program comprises oral and poster presentations covering

research areas such as endangered species conservation, landscape genetics, genome editing,

and other topics in amphibian biology. To explore the recent progress in these areas, 10

invited biologists and 4 members of the Institute’s staff will give oral presentations with

chaired discussion. In addition, 46 participants will give poster presentations with free

discussion. The oral presentations will focus on genome editing on March 27 and on

endangered species conservation on March 28.

I hope that this symposium is able to greatly benefit the study of amphibian biology by

inspiring future investigations, stimulating collaborative endeavors, and establishing new

friendships. The organizing committee wishes all participants a productive and enjoyable visit,

and I look forward to seeing you in Hiroshima.

Masayuki Sumida (Ph. D.)

Professor and Director

Institute for Amphibian Biology, Graduate school of Science, Hiroshima University

Chairperson of the Organizing Committee of IABHU International Symposium

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Information about the Symposium

Date 27th (Thu) and 28th (Fri) March 2014

Venue Rooms E002 (Oral presentations) and E203 (Poster presentations), Building E, Graduate School of Science, Hiroshima University, Higashi-Hiroshima Campus

1-3-1 Kagamiyama, Higashi-Hiroshima Hiroshima 739-8526, Japan

Schedule (Please see the back cover for details) 27th March Lectures by invited speakers and institute members

Poster presentations Backyard tour of the Institute (pre-registration required) Social gathering (pre-registration required)

28th March Lectures by invited speakers and institute members Poster presentations

Meeting Fee Participation (including backyard tour) Free Social gathering 5,000 yen (Please pay at symposium check-in)

Poster Presentation Maximum poster size is 80 cm (width) × 160 cm (height). Posters should be set up by 14:40 on 27th March, and be removed by 17:30 on 28th March by poster presenters

Taking Ceremonial Photographs Group photographs of all of the symposium attendances would be taken near the Building E (the symposium venue). Please attention to announce after the third lecture on 27 March (– 12:10).

Backyard Tour of the Institute for Amphibian Biology (pre-registration required) After taking ceremonial photographs, from 12:25 on 27 March, the backyard tour will start. The symposium staff leads the tour attendances to the Institute for Amphibian Biology after taking the ceremonial photo.

Lunch The symposium attendances can take lunches in the buffet (open: 8:00 – 19:30) and restaurant (11:00 – 15:00) or buy their lunches in the shop (10:00 – 17:30) that are located in the North Welfare Center No. 1 (please see the campus map: in the last page) close to the Building E (the symposium venue).

Social Gathering (pre-registration required) The social gathering will be held in the Saijo Hakuwa Hotel. After completion of the final lecture on 27 March (18:05), the symposium staff leads the participants to this hotel. A courtesy bus that gets off the hotel at 20:40 takes attendances to stations and several major hotels in Higashi-Hiroshima.

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Contact Address and Correspondence The Office of IABHU International Meeting, c/o Masayuki Sumida Institute for Amphibian Biology Graduate School of Science, Hiroshima University 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan E-mail: amphsymp@hiroshima-u.ac.jp

Organizing Committee: Masayuki Sumida (Chairperson), Yoshio Yaoita, Atsushi, Kurabayashi (Chair of Secretariat), Keisuke Nakajima, Ichiro Tazawa, Takeshi Igawa, Islam Mohammed Mafizul, Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Japan

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Symposium Program

Amphibian Genomics and Genome Editing 27th March Keisuke Nakajima (et. one coauthor) [Lecture by IABHU Member 1] 9:45 - 10:30 The exploitation of genome editing in Xenopus tropicalis

Enrique Amaya [Invited Lecture 1] 10:30 – 11:15 Will Xenopus remain an important model organism for biomedical research? Hui Zhao [Invited Lecture 2] 11:15 – 12:10 Efficient targeted gene disruption in Xenopus embryos using TALEN and Crispr/Cas9 systems Takuya Nakayama (et. 5 coauthors) [Invited Lecture 3] 15:40 – 16:25 Xenopus tropicalis, a model organism for the new genetics era: from forward to reverse

genetics and now to gene targeting

Hajime Ogino [Invited Lecture 4] 16:25 – 17:10 Evolution of a tissue-specific silencer underlies divergence in the expression of

paralogues

Masanori Taira [Invited Lecture 5] 17:20 – 18:05 The Xenopus laevis genome project: The consequence of allotetraploidization 50 million

years ago.

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Amphibian Conservation 28th March

Mohammed Mafizul Islam [Lecture by IABHU Member 2] 9:00 - 9:35 An ex situ conservation effort for several endangered and near-threaten amphibian species from the Ryukyu Archipelago, Japan: Captive breeding of nine anuran species and cryopreservation of sperm for two of them

Takeshi Igawa [Lecture by IABHU Member 3] 9:35 - 10:10 Conservation genetics of endangered amphibians in Ryukyu Archipelago towards sustainable conservation of the biodiversity hotspot of amphibians

Miguel Vences [Invited Lecture 6] 10:20 – 11:05 Challenges to conserve a megadiverse amphibian fauna in Madagascar, one of the poorest countries of the World

Masafumi Matsui [Invited Lecture 7] 11:05 – 11:50 Current Status of Japanese Amphibians and Their Conservation

Kelly Zamudio [Invited Lecture 8] 13:50 – 14:35 Ecological and Evolutionary dynamics of the amphibian-killing fungus Robert Jehle [Invited Lecture 9] 14:35 – 15:20 Conservation genetics in amphibians: integrating spatial and temporal population processes

Koichi Goka [Invited Lecture 10] 15:30 – 16:15 The Diversity and Endemism of the Chytrid Fungus in Japan

Masayuki Sumida [Lecture by IABHU Member 4] 16:15 – 16:50 A case study of fauna conservation in Japan: Current conservation measures of wild populations, captive breeding, genetic diversity, and potential resource materials in the endangered frog species Odorrana ishikawae and O. splendida

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Poster Presentations (Room E203)

P-01 Artificial Breeding of an Indian Bullfrog (Hoplobatrachus tigerinus): A Case Study on Reproduction and Conservation *Mohammad Shafiqul Alam, Md. Mukhlesur Rahman Khan, Md. Mafizul Islam, Mahmudul Hasan, and Masayuki Sumida

P-02 Surviving chytridiomycosis: Differential anti-Batrachochytrium dendrobatidis activity inbacterial isolates from three lowland species of Atelopus frogs from Colombia, South America *Sandra V. Flechas, Carolina Sarmiento, Edgar Medina, Martha Cárdenas, Silvia Restrepo, and Adolfo Amézquita

P-03 If there’s a silver bullet we can’t find it: options for repatriating chytrid susceptible frogs to the wild are fast evaporating – could genetic modification be our best hope? *Gerry Marantelli, David Hunter, Michael McFadden, Erika Marantelli, Lee Berger, Stephanie Versteegen, and Laura Brannelly

P-04 Battle strategies for a chytrid susceptible species: lessons learnt from ten years sending captive bred Spotted Tree Frogs Litoria spenceri to the frontline *Stephanie Versteegen, *Erika Marantelli, Gerry Marantelli, David Hunter, Matt West, and Glen Johnson

P-05 Conservation of the Japanese giant salamander Andrias japonicus at Toyosaka-cho, Higashi-Hiroshima, Japan Norio Shimizu, Hiromi Yamasaki, Chika Hashimoto, Kenta Tsuchioka, Tetsuo Takamatsu, Susumu Ueda, Katsunori Sato, Kanji Kato, and *Kazushi Kuwabara

P-06 Understanding the origin to preserve in the future: Conservation of Hylid tree frogs in Mesoamerica using an integrative approach *Liliana Solano, Javier Sunyer, and Catherine Walton

P-07 Using advanced molecular techniques in a non-model frog: Lessons for amphibian conservation *Heidi K. Smith-Parker, John B. Wallingford, and Michael J. Ryan

P-08 Fine-scale case study of detecting genetic structure and gene flow restriction of Japanese brown frog caused by recent urbanization. *Soh Kobayashi, Seiya Abe, and Rikyu Matsuki

P-09 Microhabitats of Forest Frogs along Molawin Creek, Mt. Makiling Forest Reserve, Philippines Cabreros, Julie Ann, *de Guzman, Christmas, Corales, Valerie, Molina, Kyla, Moralina, Melchor Jr., Ruiz, Dennis, and Afuang, Leticia

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P-10 Large scale environmental DNA assessment for Japanese and Chinese giant salamanders in Katsura River, Japan So Fukumoto, Atushi Ushimaru, and *Toshifumi Minamoto

P-11 A novel method for detection of the highly endangered cave amphibian Proteus anguinus using environmental DNA and real-time PCR *David Stanković, Špela Gorički, Magda Năpăruş, Matjaž Kuntner, Aleš Snoj, and Gregor Aljančič

P-12 Genetic variation of three different populations of Indian Bull frog, Hoplobatrachus tigerinus for conservation *Md. Rakeb-Ul Islam

P-13 A new species of the Fejervarya limnocharis complex from Japan (Anura, Dicroglossidae) *Djong Hon Tjong, Masafumi Matsui, Mitsuru Kuramoto, Midori Nishioka, and Masayuki Sumida

P-14 Evolution of the Sado-frog, a newly described species, in the small Japanese Island Ikuo Miura, *Mitsuaki Ogata, Kunio Sekiya and Hiromi Ohtani

P-15 Major groups of Hynobius nebulosus in Western Japan *Hiroshi Okawa

P-16 Cytonuclear Discordance and Historical Demography of Two Brown Frogs, Rana tagoi and R. sakuraii (Amphibia: Ranidae) *Koshiro Eto and Masafumi Matsui

P-17 Evolutionary relationships and postmating isolation among Fejervarya species from Lesser Sunda, Indonesia and other Asian countries revealed by mtDNA Cyt b gene sequences and crossing experiments *Mahmudul Hasan, Nia Kurniawan, Aris Soewondo, Mohammed Mafizul Islam, Takeshi Igawa, Wilmientje Marlene Mesang Nalley, Masafumi Matsui, and Masayuki Sumida

P-18 Development and characterization of 27 new microsatellite markers of Indian Bullfrog, Hoplobatrachus tigerinus (Daudin, 1802) and its congeneric species *Nasrin Sultana1, Takeshi Igawa1, Masafumi Nozawa1,2, Mohammed Mafizul Islam1, Md. Mukhlesur Rahman Khan3, Mahmudul Hasan1, Mohammad Shafiqul Alam4 and Masayuki Sumida1

P-19 Inter- and intra-island divergence in Odorrana ishikawae (Anura, Ranidae) of the Ryukyu Archipelago of Japan, with description of a new species *Mitsuru Kuramoto, Naoki Satou, Shohei Oumi, Atsushi Kurabayashi, and Masayuki Sumida

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P-20 New insights into evolutionary history among Asian pond frog species *Shohei Komaki, Takeshi Igawa, Koji Tojo, Atsushi Kurabayashi, Si-Min Lin, Mi-Sook Min, and Masayuki Sumida

P-21 Genetic population structure of an endangered frog species, Babina holsti *Ryosuke Kakehashi, Takeshi Igawa and Masayuki Sumida

P-22 Effect of low temperature on metamorphosis of the salamander Hynobius retardatus in the field and under laboratory conditions *Takuma Midori, Ryoga Kato, Tomohiro Ichikawa, Naoyuki Ushiro, Takuya Sato, Tomoaki Kuwahara, and Naoko Yamashiki1

P-23 Frog fauna around Imori Pond *Naoki Sato and Yoshikazu Takegoshi

P-24 Radiocesium accumulation of frog species in forest floors 2.5 years after the Fukushima Nuclear Power Plant accident *Teruhiko Takahara, Satoru Endo, Momo Takada, Yurika Oba, Wim Ikbal Nursal, Toshihiro Yamada, and Toshinori Okuda

P-25 Effects of female calls on male behavior during the breeding season in frogs, Rana porosa brevipoda and Rana nigromaculata *Makoto Itoh

P-26 Eco-evo-devo study of phenotypic plasticity in the Hokkaido salamander (Hynobius retardatus) *Masatoshi Matsunami, Osamu Kishida, Jun Kitano, Masafumi Nozawa, Takeshi Igawa, Hirofumi Michimae, Toru Miura, and Kinya Nishimura

P-27 Molecular cytogenetic studies on the process of genomic and chromosomal evolution in X. laevis after WGD and the origin and evolution of sex chromosomes in anuran species *Yoshinobu Uno, Chizuko Nishida, Chiyo Takagi, Takeshi Igawa, Naoto Ueno, Masayuki Sumida and Yoichi Matsuda

P-28 Species diversity of hynobiid salamanders of the genus Onychodactylus from Japan *Natsuhiko Yoshikawa and Masafumi Matsui

P-29 Functional analysis of the sex-linked gene SOX3 for female determination in the frog *Ikuo Miura, Mitsuaki Ogata, Yoshinori Hasegawa, and Hiromi Ohtani

P-30 Gene dose, compensation, and anurans *John H. Malone

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P-31 Transient receptor potential channel vanilloid 4 (TRPV4) functions as the hypotonic osmoreceptor in the Japanese tree frog, Hyla japonica *Sho Maejima, Hiroyuki Kaiya, Norifumi Konno, and Minoru Uchiyama

P-32 Discovery and phylogenetic distribution of a short interspersed nuclear element (SINE) subgroup in neobatrachian frogs *Atsushi Kurabayashi, Nobuaki Furuno, Masayuki Sumida, Hideaki Mizuno, Kazuhiko Ohshima, and Miguel Vences

P-33 Molecular domestication of transposons in Xenopus *Akira Hikosaka, Seigo Konishi, Sarina Uno, and Akira Kawahara

P-34 Inbreeding coefficient and genetic relationship of seven strains of Xenopus tropicalis inferred from genome wide genotyping of 54 microsatellite loci *Ai Watanabe, Takeshi Igawa, Akihiko Kashiwagi, Atsushi Suzuki, Atsushi Kurabayashi, Tamotsu Fujii, and Masayuki Sumida

P-35 International Xenopus Resource Network and Educational Activities at Institute for Amphibian Biology *Atsushi Suzuki, Akihiko Kashiwagi, Keioko Kashiwagi, Hideki Hanada, Kimiko Takebayashi-Suzuki, Ichiro Tazawa, Nobuaki Furuno, Atsushi Kurabayashi, Satomi Kobayashi, Junko Takenaka, Yuuna Tamaki, Takeshi Igawa, Takeshi Uto, Chiyo Nanba, Ai Watanabe, Hitoshi Yoshida, Aki Shimada, and Masayuki Sumida

P-36 National BioResource Project (NBRP) Xenopus (Silurana) tropicalis: Standard High Quality Inbred Strains for Biological Research *Akihiko Kashiwagi, Keiko Kashiwagi, Hideki Hanada, Atsushi Suzuki, Kimiko Takebayashi, Atsushi Kurabayashi, Ken-ichi Suzuki, Nobuaki Furuno, Ichiro Tazawa, Keisuke Nakajuma, Takashi Yamamoto and Masayuki Sumida

P-37 Application of CRISPR/Cas system to Xenopus laevis *Minoru Watanabe

P-38 Sex reversibility in amphibians and its applications: sex-hormone-inducible sex reversal in Rana rugosa, sex determination in Silurana tropicalis, and model amphibians as environmental indicators *Minoru Takase

P-39 Bipolar spindle formation during meiosis I required the proper localization cyclin B2 in Xenopus oocytes Yoshitome, S., Prigent, C., Hashimoto, E., and *Furuno, N.

P-40 Effects of Spermine on Neural Morphogenesis in Xenopus laevis Tailbud Embryos *Hitoshi Yoshida, Takeshi Kondo, Kimiko Takebayashi-Suzuki, Atsushi Suzuki, and Koichiro Shiokawa

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P-41 Functional analyses of a novel Insulin-like factor Yoshikazu Haramoto, *Shuji Takahashi, Yasuko Onuma, Yuzuru Ito, and Makoto Asashima

P-42 Establishment of vertebrate body plan via coordinated regulation of dorsal-ventral and anterior-posterior patterning during early Xenopus embryogenesis *Kimiko Takebayashi-Suzuki, Hidenori Konishi, and Atsushi Suzuki

P-43 Induction of homeotic limbs by retinoid treatment on the amputated tails of Japanese anurans *Ichiro Tazawa and Yoshio Yaoita

P-44 Translucent frogs created through crossbreeding, and their inheritance and dermal chromatophore structure Masayuki Sumida, Mohammed Mafizul Islam, Takeshi Igawa, Atsushi Kurabayashi, Yukari Furukawa, Naomi Sano, Tamotsu Fujii, Norio Yoshizaki, and *Midori Nishioka

P-45 The effect of Pituitary hormone and ovaprim in frog ovulation and the difference of food effect to Fejervarya cancrivora tadpole growth Ardyah Ramadhina I.P., Agung Pramana W.M., and *Nia Kurniawan

P-46 Ghrelin stimulates food intake in bullfrog larvae Sunsuke Shimizu, Hiroyuki Kaiya, and *Kouhei Matsuda

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Abstracts

Lectures Genomics and Genome Editing

Invited Lecture 1 (10:30 – 11:15, 27th March)

Enrique Amaya The Healing Foundation Centre, Faculty of Life Science, University of Manchester, Manchester M13 9PT, UK Will Xenopus remain an important model organism for biomedical research?

For over a century, amphibian embryos, larvae and adults have been a very useful model

system in biomedical research. For example, studies in amphibian embryos have helped

elucidate many of the basic principles and mechanisms in cell and developmental biology. A

particular favoured amphibian model organism in the second half of the 20th century was

Xenopus laevis, due to its robustness, ease of breeding and housing, and fecundity. Toward

the later part of the 20th century, Xenopus tropicalis, the only extant member of the genus

with the ancestral diploid state, arrived on the scene as a possible genetically tractable

amphibian model organism, to be used alongside Xenopus laevis. As we enter the

post-genomic age, will Xenopus remain a staple model organism for biomedical research?

The answer to this question depends heavily on the development of advanced technologies,

which will permit the modification of the genome of these animals, such as that afforded by

TALENs. Another critical element will be the widespread adoption and implementation of

these novel technologies by the Xenopus community at large. I will describe what I think are

the major challenges confronting Xenopus researchers in the coming years, and what I believe

needs to be done to maintain Xenopus laevis and Xenopus tropicalis as mainstay model

organisms in biomedical research for decades to come.

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Invited Lecture 2 (11:15 – 12:10, 27th March)

Hui Zhao School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong. Hong Kong SAR, P. R. CHINA Efficient targeted gene disruption in Xenopus embryos using TALEN and Crispr/Cas9 systems Specific gene disruption is essential for studying gene function in biomedical research.

Transcription activator-like effector nucleases (TALENs) have been proven to be effective for

specific gene disruption across animal models. We also reported that TALENs induced

somatic mutations in X. tropicalis embryos with reliably high efficiency. In our study, we

modified the Golden Gate method for TALEN assembly to make the constructs suitable for

microinjection into X. tropicalis embryos. Eight pairs of TALENs were constructed to target

eight X. tropicalis gene loci, and all of them induced indel mutations successfully with high

efficiencies of up to 95.7% at the targeted loci. Given the fact of high gene disruption

efficiency, TALEN may be used to evaluate the phenotype at G0 generation. Furthermore,

mutations induced by TALENs were highly efficiently passed through the germ line to F1

frogs.

In addition to TALEN, the RNA guided Cas9 nuclease is derived from type II of

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system of

bacteria and archaea. The CRISPR/Cas9 emerges as another effective tool for gene disruption

in various animal models including X. tropicalis. Here we utilized CRISPR/Cas9 derived

from Streptococcus pyogenes (SpCas9) to disrupt genes that are involved in neural crest

formation in X. tropicalis embryos. Among 16 selected gene loci, 15 genes were disrupted

by SpCas9 and the indel mutations were induced with efficiencies in a range up to 65% at the

targeted loci. Furthermore, duplex mutations of pax3 and zic1, and snail1and snail 2 were

induced by SpCas9 and corresponding gRNAs. We also generated segmental deletions and

inversion at the pax3 and snail1 gene loci by using SpCas9 and a pair of gRNAs. Our results

indicate again that the CRISPR/Cas9 system is an effective tool for targeted genome editing

in X. Tropicalis. We found that SpCas9 induced on-target DNA cleavage with high specificity

at most tested gene loci, however cautions needs to be taken as it can induce off-target

cleavages at some loci. The D10A Cas9 carried a mutation that can convert the Cas9

endonuclease into a nicknase. In line with the observation with mammalian cells, D10A

coupled with a pair of gRNA can induce indel mutations in X. tropicalis embryos. We also

showed evidence that D10A nicknase can reduce the off-target mutagenesis rate in X.

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Tropicalis embryos.

Taken together, our study indicated that both TALEN and CRISPR/Cas9 are efficient and

robust tools for genome editing in X. Tropicalis. The gene disruption efficiencies induced by

CRISPR/Cas9 were lower than those by TALENs, but CRISPR/Cas9 system is easier to

handle and good for a large scale screening. The successful applications of TALEN and

CRISPR/Cas9 make the X. tropicalis an attractive genetic model for biomedical research.

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Invited Lecture 3 (15:40 – 16:25, 27th March)

Takuya Nakayama1, Margaret B. Fish1, Marilyn Fisher1, Keisuke Nakajima2, Yoshio Yaoita2, Robert M. Grainger1 1 Department of Biology, University of Virginia, USA, 2 Institute for Amphibian Biology, Hiroshima University, Japan Xenopus tropicalis, a model organism for the new genetics era: from forward to reverse genetics and now to gene targeting

Xenopus has long been a favored model organism for developmental embryology due to its

unique combination of advantageous features including: the ability to acquire large numbers

of fertilized or unfertilized eggs (or oocytes); the availability of relatively simple techniques

for microinjection of mRNA, DNA, or anti-sense morpholino oligonucleotides (MO’s) for

gain-of-function or loss-of-function experiments, respectively; ease of transgenesis allowing

modern molecular developmental biological or biochemical studies; and its suitability for

classical transplantation-type embryology.

Although many early studies were performed using Xenopus laevis, an allotetraploid

species with a long generation time and thus poor suitability for genetics, the recent

development of Xenopus tropicalis as a new model organism with a diploid genome, short

generation time and sequenced genome allows researchers to use genetic tools in frogs.

Forward and reverse genetic approaches have identified developmental mutants and their

responsible genes. However, the number of characterized mutants so far is small and it is

not easy to target mutations in genes of interest at will even by using the method of Targeting

Induced Local Lesions In Genomes (TILLING). While this latter strategy is based on

screening by targeted sequencing of genes of interest, the induced mutations occur at random

and thus the method is still laborious because it requires screening large numbers of

mutagenized animals to find the desired mutations.

Recent technological advances have allowed researchers to perform targeted gene

editing in many organisms. The two major methods that have been used are zinc-finger

nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), both of which

have been successfully applied in Xenopus. Most recently another technology has been

rapidly developed: the CRISPR/Cas (clustered regularly interspaced short palindromic

repeats/CRISPR-associated) system for genome modification, which was originally identified

as part of the naturally occurring bacterial adaptive defense mechanism. This method now has

been successfully applied in major model organisms including Xenopus tropicalis to effect

targeted genome modification, providing an additional tool for Xenopus researchers to

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achieve simple and efficient targeted mutagenesis.

Here we present recent work using several mutants obtained through reverse genetics

using TILLING, TALENs and CRISPRs which show defects in eye development and

illustrate how these mutants are extremely useful in studying important developmental

questions. In addition we highlight several advantages of using X. tropicalis over mouse or

zebrafish in these studies.

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Invited Lecture 4 (16:25 – 17:10, 27th March)

Hajime Ogino Department of Animal Bioscience, Nagahama Institute of Bio-Science and Technology 1266 Tamura, Nagahama, Shiga, JAPAN Evolution of a tissue-specific silencer underlies divergence in the expression of paralogues

Recent analyses of teleost-specific paralogues suggest a role for the differential degeneration

of duplicated enhancers in the evolutionary diversification of paralogue expression. However,

no one has reported evidence for the involvement of innovative cis-regulatory changes. Here

we show that silencer innovation diversified expression of the paralogues, pax2 and pax8, in

Xenopus. pax2 shows multi-tissue expression, as does the ancestral amphioxus orthologue,

pax2/5/8, whereas pax8 expression localizes to a subset of pax2-expressing tissues. We

revealed that, despite their diverged expression, both pax2 and pax8 retain ancestral modes of

cis-activation. pax8 is associated with multiple enhancers, each of which is capable of

directing pax2-like, multi-tissue expression, and one of these pax8 enhancers is conserved in

pax2. However, a silencer within the pax8 proximal promoter suppresses their pleiotropic

enhancer activity outside the pax8-expressing tissues. In contrast, the combination of the pax2

proximal promoter with either the pax8 or pax2 enhancer recapitulates pax2-like expression,

as in the amphioxus pax2/5/8 promoter. We propose that silencer innovation, rather than

enhancer degeneration, was crucial for the divergent expression of paralogues with pleiotropic

enhancers inherited from their common progenitor. Furthermore, silencer innovation might be

an evolutionary escape route for genes whose expression is robustly regulated by redundant

enhancers, such as pax8.

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Invited Lecture 5 (17:20 – 18:05, 27th March)

Masanori Taira and the US-Japan consortium for the Xenopus laevis genome project Department of Biological Science, University of Tokyo, JAPAN The Xenopus laevis genome project: The consequence of allotetraploidization 50 million years ago.

The Xenopus laevis genome sequence has been awaited for a long time by many Xenopus

researchers, not only for using genomic sequence information to perform functional analysis

of genes but also for studying genome evolution after polyploidization. Therefore, one of the

main issues of the X. laevis genome project is to elucidate the structure of its allotetraploid

genome and to evaluate the evolutional conservations of the subgenomes derived from the

hybridization of the parental Xenopus diploid species about 50 million years ago. To clarify

the X. laevis genome structure of 3.1 Gbp, we sequenced genomic DNA isolated from a

homozygous strain, called the J strain, which has been established by Katagiri’s group in

Japan. Shotgun, mate-pair, BAC-end, and fosmid-end sequencing and the following

Meraculous assembly resulted in about 17,000 scaffolds longer than 1 kbp (the longest, 21.6

Mbp; N50, 7.6 Mbp), covering 2.6 Gbp. To map scaffolds to chromosomes, we have carried

out chromosomal FISH (fluorescent in situ hybridization) with BAC clones that reside in

scaffolds longer than 2 Mbp. About 600 BAC clones were mapped to the X. laevis 18

chromosomes, which covered 2.0 Gbp. This analysis confirmed the homoeologous

relationships between chromosomes, which were previously suggested by cDNA FISH (Uno

et al., 2013, Heredity 111:430). These paired homoeologous chromosomes were numbered

according to the corresponding X. tropicalis chromosomes, in which X. tropicalis

chromosomes 9 and 10 are fused in X. laevis, and named chromosome 9/10. From karyotypic

observations, each of the paired chromosomes was distinguished by its length. Based on this,

the chromosomes corresponding to tropicalis chromosome 1 were named 1L and 1S, for the

long and short counterparts, and so on. Using the homoeologous relationships between

chromosomes and scaffolds, we are now able to identify homoeologous gene sets, so-called

"a" and "b" genes, and singleton genes that lost counterparts in their homoeologous regions.

We found that most postulated homoeologous gene sets in the RefSeqs were actually

homoeologous, but a portion of them turned out to be paralogs or even alleles. Conversely,

some of postulated paralogs turned out to be homoeologs.

Scanning the genomic sequence, we identified two types of fossil DNA transposons

which showed a biased distribution in scaffolds, i.e., in a majority of scaffolds, only either of

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the two types, called type A and type B, were present. Remarkably, most scaffolds

possessing type A or type B transposons were mapped along long or short chromosome sets,

respectively. This implies that type A or type B transposons actively propagated within the

parental genomes before allotetraploidization, thus being likely to demarcate the two

subgenomes in X. laevis. Identification of the subgenomes further allows us to investigate

"post-polyploidization" events, such as genome dominance (biased gene expression or loss),

genomic rearrangements, and the subfunctionalization of genes or gene cassettes. We are

now focusing on genes related to developmental regulators (Hox gene clusters etc.), the cell

cycle, signaling pathways, olfactory receptors, sex determination (the W gene), and so on.

Some of our latest findings will be presented in my talk.

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Lecture by IABHU member 1 (9:45 - 10:30, 27th March)

Keisuke Nakajima and Yoshio Yaoita Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima, JAPAN The exploitation of genome editing in Xenopus tropicalis

Gene knockout by homologous recombination in embryonic stem (ES) cells involves the

characterization of altered phenotypes as a means of clarifying the functions of the ablated

genes. Because of the difficulty in establishing ES cell lines from other species, this

technology has only been used in the mouse and the rat genome. The facile gene knockout

technique, which is used to analyze gene function, is desired for use in other animals.

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)

are fusion proteins comprised of a DNA-binding sequence and the nuclease domain from the

restriction enzyme FokI. They bind to target nucleotide sequences and introduce

double-strand breaks, which are repaired by homologous recombination and non-homologous

end-joining.

Because tyrosinase is essential for melanin biosynthesis, ZFN-induced knockout of the

tyrosinase gene should produce the albino phenotype. To generate albino lines of Xenopus

tropicalis, we injected fertilized eggs with mRNAs encoding zinc-finger nucleases (ZFNs)

targeting the coding region of tyrosinase. Surprisingly, vitiligo was observed on the skin of F0

frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes were

disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic

DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase

gene mutations. The data implies that the ZFN-induced tyrosinase gene ablations occurred

randomly over space and time throughout the entire body and that melanocyte precursors

lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X.

tropicalis were obtained by mating the mosaic F0 frogs and were further analyzed.

Multiple TALEN scaffolds have been reported in addition to the basic architecture,

pTAL. A pTAL-encoded TALEN protein contains a nuclear localization signal, an N-terminal

domain (287 amino acids), a target DNA-binding domain composed of many tandem

34-amino-acid repeats, a C-terminal domain (231 amino acids) and the nuclease domain of

FokI. GoldyTALEN was designed to contain a truncated N-terminal domain (158 amino

acids) and C-terminal domain (63 amino acids) upstream and downstream, respectively, of the

DNA binding domain, and has been shown to have greater somatic gene modification activity

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than the pTAL scaffold in zebrafish. Furthermore, a lower toxicity and comparable or higher

somatic mutation activity were reported in zebrafish using TALENs harboring an obligate

heterodimeric FokI nuclease domain than using TALENs containing homodimeric FokI. We

introduced the Sharkey mutation into this TALEN scaffold, as Sharkey is known to enhance

the DNA cleavage activity of FokI in both homodimeric and heterodimeric ZFN architectures.

In the present study, we compared the toxicities and somatic mutation activities of four

TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same

truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and

C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the

truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain.

The strongest phenotype and targeted somatic gene mutation were induced by the injection of

TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate

heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced

toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type

scaffold showed both a high activity of somatic gene modification and high toxicity. The

Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated

somatic mutagenesis.

Gene knockout frogs by injecting TALEN mRNA with a high activity may become

sterile, if the mutated genes are indispensable for fertility. Now, we are exploring the methods

to obtain F0 frogs composed of cells with biallelic mutated genes for phenotype analysis by

improving the procedure of the target gene destruction, and the healthy fertile F0 frogs that

can produce F1 offspring with the homozygous sterile mutation.

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Amphibian Conservation

Invited Lecture 6 (10:20 – 11:05, 28th March)

Miguel Vences Zoological Institute, Division of Evolutionary Biology, Technical University of Braunschweig, GERMANY Challenges to conserve a megadiverse amphibian fauna in Madagascar, one of the poorest countries of the World

Madagascar harbors an extremely rich amphibian fauna, consisting of five endemic

neobatrachian frog clades that independently reached the island and radiated there.

Paradoxically, Madagascar's fauna is at the same time well studied compared to other tropical

regions, with complete phylogenies published and a high proportion of calls and tadpoles

known, and taxonomically incompletely revised, with over 300 candidate species awaiting

description in addition to the currently 285 described species. Madagascar's amphibians are

also remarkable because of their high local species richness (over 100 species in a small area),

high microendemism (large proportion of species restricted to very small ranges) and high

local abundances of individuals, both adults and stream tadpoles. These high abundances

probably reflect the absence of the amphibian chytrid fungus from Madagascar which appears

to be a prime example for a pre-chytrid tropical amphibian fauna. Some of the most important

challenges for the conservation of these animals are (1) including information on the

undescribed candidate species in spatial priority settings for reserve planning, (2) developing

a fast-response strategy or the case of chytrid introduction to the island, including the option

of probiotic treatment of wild specimens, (3) setting in place a long-term monitoring system

at least at some areas of high amphibian richness in Madagascar, and (4) maintaining an

active research schedule on taxonomy, natural history and captive breeding of these frogs,

despite the lack of attractive funding opportunities for such baseline work. Most important,

however, is beyond doubt to ever intensify efforts to reduce the rate of habitat loss which is

mostly caused by small scale slash-and-burn agriculture and which constitutes the most

imminent and by far most important threat to this remarkable amphibian fauna.

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Invited Lecture 7 (11:05 – 11:50, 28th March)

Masafumi Matsui Graduate School of Human and Environmental Studies, Kyoto University, JAPAN Current Status of Japanese Amphibians and Their Conservation

Serious population decline in amphibians was noted and specific surveys were initiated at the

end of the 1970s, when the Japan Ministry of Environment (then Japan Environmental

Agency) compiled a distribution map of some selected amphibian species. In 1986, editorial

planning for the Japanese Red Data Book (JRDB) was begun, and the Book was eventually

published in 1991, in which a total of 19 amphibians (species, subspecies, and local

populations) were included. In 1994, the category of IUCN Red List changed the threatened

category from a qualitative to a quantitative base, and the subsequent revised versions of

JRDB followed that. Additionally, a Red List was newly prepared prior to revising JRDB

since 1997. After publication of the second edition of JRDB in 2000, and the most recent

Red List in 2012, the third version of JRDB will appear this year.

The number of species, subspecies, and local populations of amphibians listed on JRDB

or on the Red List has increased from 19 in 1991 through 22 in 1997 to 36 in 2006, and now

40. Besides the actual increment in endangered status for many taxa, changes in taxonomic

status of some species arising from new data contributed to the augmentation of these

numbers. Newer studies resulted in splitting some species into more than one, and often

narrowed the range of species formerly believed to have wider ranges. Anyhow, it should be

noticed that as many as 61% of known Japanese amphibian fauna is now more or less under

threat.

With the exception of a few species, the RDBs do not list any legal regulation of the

capture of amphibians, or in causing change in their habitat, but nevertheless the publication

of RDB has stimulated the conservation of amphibians. Along with the ratification of

“Biodiversity Act” by the Japanese Government, it is now necessary to assess likely effect on

biodiversity before implementing large-scale environmental changes. In addition to JRDB, all

47 prefectures in Japan have also published their own RDB, and some have already published

revised versions.

There is no information on any recent extinction of any amphibian species in Japan, but

there are many cases of local extirpation of entire populations and in almost all Japanese

amphibian species, populations are declining, although the quantitative details of declines

have not been well documented.

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Some causes of decline have been identified. The first cause is the limited range of

distribution, implying fragile nature of some species with very limited distributional ranges.

The second is habitat requirements, such as special breeding environments necessary for

small salamanders. An endangered frog, Pelophylax porosus brevipodus never moves far

away from still water, and needs lowland marshy areas for its entire life history. This property

is related to the third cause, disjunction of habitat. Mountains and urban areas disrupt

distribution of this species, which inhabits lowland marshy areas. The fourth cause,

destruction of amphibians' habitats has been occurring in Japan for a long time and is the

worst cause of amphibian decline in this country. Development of hilly and lowland areas for

construction of roads and for human inhabitations have resulted in drying of the land and

pollution of the water, which is vital to many species. Also on montane regions, construction

of skiing facilities and golf courses, highways, roads for forestry, and dams, and logging are

all causing deterioration of the environment.

As special examples, modifications of rivers by construction of dams and concrete

enforcement of riverbanks are severely affecting Andrias japonicus, and populations of P.

porosus brevipodus, have been declining because of a decrease of the area devoted to

cultivation of rice, increase in number of human dwellings, construction of roads and of

riverside parks, and water pollution resulting from these activities. For more than 40 years

ago, rice paddies, the main habitat of this species, have been converted to dry fields and to

human habitations. The bottoms and muddy banks of small rivers and ditches were covered

by concrete. Earlier, use of agricultural chemicals on the rice fields adversely affected this

species, but more recently, the complete modification of the structure of the land have

affected frog species formerly inhabiting the rice paddies.

The fifth cause is the introduction of alien species. Along with Lithobates catesbeianus,

introduced long ago and must have already affected the native amphibian fauna, more recent

invader, Rhinella marina, is severely damaging the native fauna of the Ryukyus. Introduced

crayfish, Procambarus clarkii, is one of the predominant predators on tadpoles, and recent

dispersal of the raccoon, Procyon lotor, is increasingly a threat to native fauna, including

amphibians. Fortunately, the chytrid fungus, Batrachochytrium dendrobatidis, proved to

include several haplotypes endemic to Japan and is not so harmful as the introduced one.

Finally, the sixth cause of decline is related to hybridization. Introduced Chinese giant

salamander, A. davidianus, was recently found in the wild in several prefectures. Because this

species occupies an almost similar niche as that of the protected Japanese congener, damage

to the genetic integrity of the latter through hybridization is now a big problem, as is

competition for habitat. The cultivation of rice and other modification of its habitat have

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eliminated the original ecologic segregation between P. p. brevipodus and P. nigromaculatus,

and they now hybridize in nature, resulting in both of them losing their pure genetic identity.

The measures now being taken in Japan are insufficient to meet the above threats to

native amphibians. Andrias japonicus has been designated as a special natural monument, but

the species is still faced with artificial modification of its habitat. Cases of protection by local

governments are also in place for many species, and individual animals cannot be captured

without permission, and land development is restricted in the amphibian habitats. In

Hiroshima Prefecture, groups of volunteer citizens have been trying to restore the numbers of

P. p. brevipodus, collaborating with a zoo. For A. japonicus, artificial breeding nests have

been established and steps have built in constructed at small dams to facilitate upstream

migration in many rivers, although the effect is still minimal. Genetic analysis directed toward

the conservation of this species from hybridization has been made, although these particular

cases are still awaiting assessment.

An urgent problem requiring more attention is the marked decline of common species

in which some local populations are in danger of immediate extirpation. Local populations of

species ranked as Least Concern and not listed on JRDB, like Rana japonica, are also steadily

in decline. This situation is largely related to the changes in the cultivations of rice, due to a

decrease in rice consumption and to aging of farmers. This is a great problem and is not easily

solved, but I am continuously appealing “to consume home-grown rice and protect frogs from

extinction”.

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Invited Lecture 8 (13:50 – 14:35, 28th March)

Kelly Zamudio Goldwin Smith Professor of Ecology and Evolutionary Biology, Cornell University, USA Ecological and Evolutionary dynamics of the amphibian-killing fungus

Our research examines the genetic composition and demography of amphibians experiencing

different stages of chytridiomycosis, an infectious disease caused by the amphibian-killing

fungus Batrachochytrium dendrobatidis (Bd). Chytridiomycosis has caused widespread

declines and extinctions of amphibian populations globally, with high impacts in Central and

South American communities. I will present data on the roles of immunogenetic and

demographic factors in immunity to this infectious disease that is a serious threat to New

World amphibian biodiversity. In my talk I will highlight two research target areas in our

laboratory: (1) identification of the genetic basis of host defense strategies (resistance and

tolerance) and pathogenicity traits, and (2) ecological characterization of shifts in fitness

driving seasonality patterns in host-pathogen interactions. Given the heterogeneity of disease

outcomes and broad host diversity for Bd, genomic information helps characterize the diverse

collection of disease survival strategies for amphibians, which in turn helps build a detailed

map of the components of the innate and adaptive immune system acting in infected hosts in

varying environments. Our results enhance the understanding of the genetic and demographic

factors contributing to this wildlife disease. These are important in conservation efforts and

will allow researchers to take into account long-term maintenance of adaptive evolutionary

potential in conservation research and captive breeding programs, detection of natural

resistance, and management of captive stocks.

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Invited Lecture 9 (14:35 – 15:20, 28th March)

Koichi Goka National Institute for Environmental Studies, JAPAN The Diversity and Endemism of the Chytrid Fungus in Japan

A serious disease of amphibians caused by the chytrid fungus Batrachochytrium

dendrobatidis was first found in Japan in December 2006 in imported pet frogs. This was the

first report of chytridiomycosis in Asia. To inspect the origin and expansion process of the

chytrid fungus in Japan, we surveyed the distribution and genetic variation of the fungus

among captive and wild frog populations. We established a nested PCR assay that uses two

pairs of PCR primers to amplify the internal transcribed spacer (ITS) region of a ribosomal

RNA cassette to detect mild fungal infections from as little as 0.001 pg (1 fg) of B.

dendrobatidis DNA. We collected swab samples from 559 captive amphibians, and more than

5,000 wild collected at field sites from northern to southwestern Japan. We detected

infections in native and exotic species, both in captivity and in the field. Sequencing of PCR

products revealed much haplotype variation of the B. dendrobatidis ITS region. Phylogenetic

analysis for the haplotypes combined with haplotype sequences already detected in other

countries showed that genetic diversity of Bd in Japan was higher than that in other countries.

Furthermore, it was suggested that 3 of the haplotypes detected in Japan were specific to the

Japanese giant salamander (Andrias japonicus) and appeared to have established a commensal

relationship with this native amphibian. The highest genetic diversity of B. dendrobatidis was

found in the sword-tail newt (Cynops ensicauda) endemic to Okinawa Islands and next in the

alien American bullfrog (Rana catesbeiana). From these results, combined with no evidence

of chytridiomycosis occurrence in the Japanese native species, we considered that the chytrid

fungus is native in Japan. To improve chytridiomycosis risk management in the world, we

must monitor and control the amphibian trade between the countries.

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Invited Lecture 10 (15:30 – 16:15, 28th March)

Robert Jehle School of Environment and Life Sciences, University of Salford, UK Conservation genetics in amphibians: integrating spatial and temporal population processes

The emergence of the scientific discipline conservation genetics at the end of the last century

paralleled the general appreciation that the genetic makeup of endangered populations

contributes to their demography, and that genetic markers offer practical tools in wildlife

forensics or for aiding captive breeding programmes. Amphibians represent the most

endangered class of vertebrates, and due to their low dispersal abilities are particularly

deme-structured organisms. Amphibians should therefore offer excellent opportunities for the

application of genetic information to conservation-relevant research. However, conservation

genetics as a scientific discipline largely focuses on charismatic or economically important

species. As a result, amphibians are underrepresented in major scientific journals for

conservation genetic studies when compared to their representative share of species diversity

e.g. among vertebrates. In this talk I aim to highlight current directions and limitations of

conservation genetic studies on amphibians based on examples from own research. A further

aim is to demonstrate how information derived from genetic markers can be used to reveal

both spatial and temporal processes at the level of populations.

The distribution and amount of neutral genetic diversity constitutes the raw material of

evolutionary potential. To this end, detailed measures of local population processes such as

dispersal between demes and genetic drift within demes are vital to achieve conservation

goals. Based on a long-term data set on a European urodele (the crested newt Triturus

cristatus), I describe how genetic data can be used to discern between opposing scenarios of

population connectivity, and how population turnover shapes the maintenance of genetic

variation. I also describe how genetic data could be used to address open questions about the

natural range of T. cristatus, with important consequences for local species management.

When compared to other vertebrates such as birds and mammals, long-term studies on

wild amphibians have revealed rather little about individual fitness measures such as lifetime

reproductive success, and the consequences of such variation on the linkage between

generations. In a second set of examples from own research I argue that DNA-based

parentage analysis to infer population-wide pedigrees has a lot of potential for amphibian

studies. In the case of the South American poison-dart frog Allobates femoralis,

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microsatellite-based gene genealogies demonstrate that reproductive contributions of males

and females to a following generation are high, in line with high levels of effective population

sizes and amount of genetic variation. In the case of a population study on the European

common toad Bufo bufo, I show that pedigree information can be used towards understanding

the genetic causes and consequences of an observed reduction in body size and fecundity that

parallels increasing temperatures through global change.

I finally argue that a main future focus of amphibian conservation genetics lies in the

area of applied conservation management. Although captive breeding programmes for

endangered amphibian species are on the rise, they so far neglected approaches like the

genotype-informed choice of best-suited mating pairs. I describe the development of such a

strategy for the critically endangered central American lemur leaf frog Agalychnis lemur.

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Lecture by IABHU member 2 (9:00 - 9:35, 28th March)

Mohammed Mafizul Islam1, Takeshi Igawa1, Atsushi Kurabayashi1, Ryosuke Kakehashi1, Naoki Satou1, Nozomi Shintani1, Miyuki Tado1, Hirotaka Sugawara1, Takuma Nishitani1, Misato Uchida1, Shohei Oumi2, Seiki Katsuren3, Tamotsu Fujii4, and Masayuki Sumida1 1Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Japan, 2Section of Agriculture and Forest, Amami City Government, Amami, Kagoshima, Japan. 3Biology and Ecology Group, Okinawa Prefectural Institute of Health and Environment, Nanjo, Okinawa, Japan. 4Faculty of Human Culture & Science, Hiroshima Prefectural University, Hiroshima, Japan An ex situ conservation effort for several endangered and near-threaten amphibian species from the Ryukyu Archipelago, Japan: Captive breeding of nine anuran species and cryopreservation of sperm for two of them

Amphibians from most parts the world are in threatened condition compared with other

animals due to their high sensitivity to any environmental change. Due to natural calamities

and other man-made causes their natural habitat and living conditions hampered, thus

amphibian extinction has recently become an international concern. In comparison with other

commercially important animals, the effective maintenance methods for the endangered

amphibians have not been established so far. As a leading facility for amphibian breeding and

maintenance, we conducted captive breeding of endangered amphibian species in the Institute

for Amphibian Biology as an ex situ conservation tool. As the results, we succeeded in

artificial or captive breeding of six Japanese amphibian species (Odorrana ishikawae, O.

splendida, O. amamiensis, Babina holsti, B. subaspera,and Echinotriton andersoni) that are

listed as class B1 endangered species in the IUCN Red List and also designated as a natural

monument in both Okinawa and Kagoshima Prefectures. We have also performed artificial

breeding of three species (Rana ulma, R. kobai and Odorrana supranarina) listed as NT

(Near threatened) in the IUCN Red List. We have induced female frogs for ovulation by

inter-body cavity injection of bullfrog pituitary hormone and after subsequent fertilization

with sperm, the eggs were maintained in adequate temperature. The tadpoles were reared in

incubator or greenhouse fed with boiled spinach and metamorphosed frogs have been fed with

live cricket and reared using the frog rearing facility in Institute for Amphibian Biology

which is a unique system for frogs. In case of Echinotriton andersoni we put the male and

female together and provide them a favorable environment for breeding and collect their

fertilized eggs and maintain them in the laboratory. Due to cannibalistic character of E.

andersoni tadpoles, they were reared individually using small dishes or using net in a big tray

and they were fed with live food like Tubifex worms or tadpoles of Xenopus tropicalis. In

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case of two species (O. splendida and R. ulma), the second-generation offspring were also

obtained in the laboratory via natural mating activities. We have collected the fertilized egg

cluster from the frog rearing box and took care of them, thus we got a good number of

offspring.

We have developed a method of cryopreservation of sperm first time for any Japanese

frog, and it was carried out for an endangered species (O. splendida) and a near-threaten

species (R. ulma). We have conducted these experiments by preserving the sperm in -80°C

deep freezer using the technique used for Xenopus laevis with modifications and we avoided

any use of liquid nitrogen to develop the most cost effective technique. We have confirmed

the capability of frozen sperm not only by checking their motility but also by their ability to

fertilize eggs after thawing. We were able to get a maximum of 80% fertilization rate with 24

hour frozen sperm, and noticed fertilization of eggs by using maximum 40 days frozen sperm.

We also confirmed our success in cryopreservation by observing the normal tadpoles and

metamorphosed frogs obtained from the eggs that were fertilized with frozen sperm.

The knowledge obtained from these studies would be applicable for the international

actions on amphibian conservation.

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Lecture by IABHU member 3 (9:35 - 10:10, 28th March) Takeshi Igawa Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, JAPAN Conservation genetics of endangered amphibians in Ryukyu Archipelago towards sustainable conservation of the biodiversity hotspot of amphibians.

Preservation of genetic variation and the capacity to generate novel variation are crucial

measures for successful long-term conservation of natural populations. Although genetic

variability is often less critical for population persistence than factors like habitat loss or

fragmentation, it can play decisive role in the long term by enabling populations to adapt and

persist in a changing environment. Especially, endemic island species are more immediate

concerned than non-endemic species due to their originally small population size,

distribution area, and possibly less variation of genetic variation.

In Japanese amphibian fauna, remarkable species diversity is seen in the Ryukyu

Archipelago, a fairly long archipelago consisting of approximately 140 subtropical islands

ranging almost 650 km between Kyushu and Taiwan South Western Islands of Japan.

Twenty three native species and subspecies of amphibians are distributed in this region,

reflecting dispersal and divergence via formation of land-bridge and strait during ancient era.

Although such a rich diversity in the Ryukyu, every single species is inevitably island

species and more prone to be extinction. Actually, six frogs (Odorrana ishikawae, Odorrana

splendida, Odorrana utsunomiyaorum, Babina subspera, Babina holist, Limnonectes

namiyei) and a newt (Echinotriton andersoni) are listed in red lists of Japan as Endangered

and Vulnerable, respectively. Although some of these species have been studied for the

genetic relationships between the island populations, population structure within each islands

and genetic variation of each local population, that are essential basic knowledge for

population management for sustainable conservation, are still unknown so far. Therefore, to

clarify fine scale population structure and genetic variation, we firstly conducted population

genetic studies of these endangered frogs (excluding Babina subspera and Limnonectes

namiyei) and a newt in the Rykyu Archipelago by using newly developed microsatellite

markers.

For ishikawa’s frogs, O. ishikawae and O. splendida endemic to the Amami and

northern part of Okinawa Islands, we examined the gene flow, population structure and

demographic histories and also estimated causal factors for shaping population structure by

using environmental niche modeling combined with GIS technics. The resultant population

structures showed the contrasting population structure between two species:

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multiple-population structure in O. splendida and single-population structure of O. ishikawae.

In same manner, we also examined population structure and demography of O. narina

sympatrically or parapatrically distributing with respect to O. ishikawae and obtained same

single-population structure same as O. ishikawae. While, for B. holsti endemic to Okinawa

(also sympatric or parapatric with O. ishikawae) and Tokashiki Isls., somewhat genetically

distinct populations were found in southern limit of distribution area in Okinawa Isl.. Our

results also showed lower genetic variation of these populations and the population in

Tokashiki Isl.. For Anderson’s crocodile newt, E. andersoni endemic to Amami, Uke, Toku,

Okinawa, Sesoko, and Tokashiki Isls., multiple-population structure were found in Amami

and Okinawa Isls., but single-population structure in Toku Isl. The population structure in

both Amami and Okinawa is showed genetic similarities between geographically close

populations, so call “isolation by distance” pattern. Contrary to the previous mitochondrial

study, our Bayesian clustering analyses and estimation of migration rates based on

coalescent theory showed that the shared ancestry of populations and frequent migration

even between populations in adjacent different islands (southern Amami - Uke and southern

Okinawa and Tokashiki). Finally we discuss how each environmental factor affect gene flow

and leads to the formation of such a population structure in each species in association with

their ecological feature. We also mention about rapid and cost-effective microsatellite

development for these specie briefly here.

In addition, we also examined relationship between survivability of tadpoles and

genetic variability to measure the genetic load. By using F1 of O. splendida, artificial

crossing was conducted for three patterns: full-sib (brother-sister) crossing, non-sib crossing,

and crossing between individuals from genetically different populations, respectively. To

eliminate physiological effect on egg condition and environmental effects on water quality

for tadpoles, we divided each egg mass into three and artificially inseminated with the

sperms from different males respectively and these eggs were cultured in same tanks. We

cultured eggs in three different temperatures (18, 20, and 22 °C) and count number of

normally developed individuals until metamorphosis. We conducted the experiments for

three egg masses from F1 females in this time. As the results, effect of higher temperatures

on survivability was severe, showing clear negative relationships between temperature and

survivability. Especially, no individuals were survived in 22 °C. For the relationships

between genetic variety and survivability, positive relationship was observed in 18 °C, but

not obvious in 20 °C. These results suggest that viability of O. splendida populations should

be more threatened when the population size is reduced and naturel water temperatures is

raised by environmental change such as global warming.

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Lecture by IABHU member 4 (16:15 – 16:50, 28th March) Masayuki Sumida1, Mohammed Mafizul Islam1, Takeshi Igawa1, Atsushi Kurabayashi1, Naoki Satou1, Kazuyoshi Ukena2, Shohei Oumi3, Seiki Katsuren4, and Tamotsu Fujii5

1Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima, Japan. 2Section of Life Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashihiroshima, Japan. 3Section of Agriculture and Forest, Amami City Government, Amami, Kagoshima, Japan. 4Biology and Ecology Group, Okinawa Prefectural Institute of Health and Environment, Nanjo, Okinawa, Japan. 5Faculty of Human Culture & Science, Hiroshima Prefectural University, Hiroshima, Japan A case study of fauna conservation in Japan: Current conservation measures of wild populations, captive breeding, genetic diversity, and potential resource materials in the endangered frog species Odorrana ishikawae and O. splendida

Odorrana ishikawae, an anuran endemic to the Okinawa and Amami Islands, has been

described as the most beautiful frog in Japan. However, over-hunting and environmental

destruction over the last several decades have regrettably devastated local populations. As a

result, O. ishikawae is now listed as a class IB endangered species on the IUCN Red List and

is protected by law in both Okinawa and Kagoshima Prefectures. Recently, the Amami

population was considered to be a new and distinct species, O. splendida, and in addition,

large-type specimens were found inhabiting a limited area of the Island and identified as

morphologically distinguishable from the common-type. The present study was performed to

determine fauna conservation of O. ishikawae and O. spendida by examining the following:

(1) the current status of wild populations and maintenance measures in practice; 2) captive

breeding of frogs and testing of farming techniques in the laboratory aimed at preservation

and promotion of natural breeding; (3) evaluation of genetic diversity and population

composition using mitochondrial DNA, allozyme analyses, and microsatellite markers; and

(4) the use of antimicrobial peptide extraction from the skin to identify biologically active

agents with strong antimicrobial effects.

Odorrana ishikawae and O. splendida are both at risk from various factors including

predation by naturalized mongoose species and forest degradation, indicating the need for

maintenance measures of local wild populations. Recovery of various endangered species,

including O. ishikawae and O. splendida, has become apparent following the first mongoose

extermination program (a 10-year plan begun in 2005 on Amami Island), which resulted in a

large decrease in mongoose habitation and population density. The overall goal of the

program is to completely rid Amami Island of mongooses by 2022, with implementation of a

second extermination program running from 2013 through 2022. Ecosystem recovery is the

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ultimate aim and some success is already apparent.

Field-caught male/female pairs of O. ishikawae and O. splendida were bred via artificial

insemination in the 2004, 2006, and 2008 breeding seasons. Although fewer than 50% of the

inseminated eggs achieved metamorphosis, approximately 500, 300, and 250 offspring from

the 3 respective trials are currently being raised in the laboratory. During the 2009–2012

breeding seasons, second-generation offspring were also produced via natural breeding of the

first offspring. These findings appear encouraging with regards the temporary protection of

local populations.

Analysis of mitochondrial ND2 data showed that genetic diversity is relatively low

(1.0%) between the 2 Amami types, but comparatively high (7.5%) between the 2 species.

Allozyme data confirmed the very low (0.016) genetic distance between the Amami types and

comparatively high (0.204–0.234) distance between the species. Based on microsatellite

analysis, O. ishikawae and O. splendida clearly diverged into different genetic clusters, and O.

splendida further diverged into 4 or 5 genetic clusters (including 2 types), while O. ishikawae

formed a single cluster. These results reveal clear genetic diversity between the species, but

only slight genetic divergence between the types. These findings suggest that speciation and

genetic divergence are currently in progress, or alternatively, that speciation occurred in the

past but genetic interaction has resumed.

To examine the presence of biologically active agents with strong antimicrobial effects,

we investigated antimicrobial peptides in the skin of artificially bred frogs. As a result, we

succeeded in identifying 9 novel antimicrobial peptides from the skin of O. splendida. Peptides consisted of 21–46 amino acid residues, classified into 5 antimicrobial peptide

families (esculentin-1, esculentin-2, palustrin-2, brevinin-2, and nigrocin-2). These

antimicrobial peptides showed strong antimicrobial activity and a wide antimicrobial

spectrum against 5 microorganisms (Escherichia coli, Staphylococcus aureus,

methicillin-resistant S. aureus, Bacillus subtilis, and Candida albicans).

Abstracts Poster Presentations

P-01 Artificial Breeding of the Indian Bullfrog (Hoplobatrachus tigerinus): A Case Study on Reproduction and Conservation

*Mohammad Shafiqul Alam1, Md. Mukhlesur Rahman Khan2, Mohammed Mafizul Islam3, Mahmudul Hasan3 and Masayuki Sumida3

1Department of Genetics and Fish Breeding, Faculty of Fisheries, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh, 2Department of Fisheries Biology and Genetics, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh, 3Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Japan

We attempted artificial breeding of the vulnerable Indian Bullfrog, Hoplobatrachus tigerinus, in the laboratory using specimens collected from the field in Bangladesh. A total of 10,332 eggs were obtained from two females and fertilized with the sperm of one male. The developmental stages of H. tigerinus were observed at a water temperature of 23 °C. The results showed that the first cleavage started around 80 min after fertilization. The tail bud was observed at 24 h and hatching at 48 h after fertilization. The tadpoles were fed boiled smashed spinach from day 5 after fertilization. They were then reared for around 2 weeks in Petri dishes in an incubator before being transferred to concrete tanks in a green house. Metamorphosis started around day 42 after fertilization. Of the total eggs, 89% cleaved normally, 70% attained the tail-bud stage, 56% hatched normally, and 46% developed into normal feeding tadpoles. Finally, however, only 10% of the total eggs metamorphosed normally. A strong cannibalistic nature was observed among the tadpoles, which led to a reduced metamorphosis rate. In addition, an unidentified fungal infection and lower water temperatures due to the strong winter also contributed to the reduced rate of metamorphosis. The fecundity of H. tigerinus was excellent, but the mass production of H. tigerinus specimens failed. To ensure mass production and conservation of H. tigerinus in future experiments, we recommend strict regulation of water temperature in all stages preceding metamorphosis.

P-02 Surviving chytridiomycosis: Differential anti-Batrachochytrium dendrobatidis activity inbacterial isolates from three lowland species of Atelopus frogs from Colombia, South America

*Sandra V. Flechas1, Carolina Sarmiento2, Edgar Medina1, Martha Cárdenas1, Silvia Restrepo1, and Adolfo Amézquita1

1Department of Biological Sciences, Universidad de los Andes, Bogotá, Colombia; 2Smithsonian Tropical Research Institute, Republic of Panama

Chytridiomycosis, a disease caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), has been associated with dramatic declines and extinction of amphibian species worldwide. One of the more severely impacted groups of amphibians are the montane harlequin toads of the genus, Atelopus (Anura: Bufonidae) with 80% of species listed as

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critically endangered. In the Neotropics, almost every species of the genus occurring above 1000 m have experienced catastrophic declines. In Colombia nearly all the 33 species of Atelopus have declined sharply, yet lowland species are persisting at 0 – 600 masl. For at least one species, A. elegans from Gorgona Island in the pacific coast of Colombia, we know that the pathogen has been present for at least six years without causing obvious disease or declines. The persistence of lowland Atelopus could be explained by the lower growth of Bd at temperatures above 25ºC. Although, we tested the complementary hypothesis that the toads’ skin microbiota acts as a protective barrier against the pathogen, perhaps delaying or impeding the symptomatic phase of chytridiomycosis. A total of 148 cultivable bacterial morphotypes were isolated, 40 from Atelopus elegans, 85 from Atelopus aff. limosus, and 23 from A. spurrelli. In antagonism assays, we observed anti-Bd activity in 16 of 40 (40%) bacterial morphotypes from A. elegans, 16 of 85 from Atelopus aff. Limosus (19%), and six of 23 (26%) from A. spurrelli. Twenty-six percent (38 strains representing 12 species) of the bacteria inhibited Bd growth and just two of them were shared among the toad species sampled in different localities. Interestingly, the strongest anti-Bd action was measured in bacteria isolated from A. elegans, the only species that tested positive for the pathogen. The cutaneous microbiota is thus revealed as both an exaptation and adaptation against the selective pressure represented by Bd. Our findings reveal enough bacterial strains to eventually develop local probiotic treatments against chytridiomycosis and also shed a light the mechanisms behind the frog-bacteria-pathogen interaction.

P-03 If there’s a silver bullet we can’t find it: options for repatriating chytrid susceptible frogs to the wild are fast evaporating – could genetic modification be our best hope? *Gerry Marantelli1, David Hunter2, Michael McFadden3, Erika Marantelli1, Lee Berger4, Stephanie Versteegen1, Laura Brannelly4

1Amphibian Research Centre, Melbourne, Australia, 2Office of Environment and Heritage, NSW, Australia, 3Taronga Zoo, Sydney, Australia. 4James Cook University, Townsville, Australia.

Emerging Amphibian Chytrid Fungi now occupy many of the earth’s amphibian bearing zones. It seems inevitable that they will eventually occupy all suitable climatic zones. A wide variety of biotic and abiotic variables influence the outcomes of their occupation from seemingly inconsequential to almost immediate extinction. Regardless of the complexities of how it happens, there are two types of amphibians: those that will survive and those who will be driven to extinction by chytrid alone. This clarifies management: we need tools that bring amphibians in the second group into the first group.

Australia has seen twenty plus years of captive assurance colonies and over 16 years of reintroductions, translocations, introductions and chytrid exclusion areas for our most chytrid vulnerable species. We have not established a single sustainable population of a susceptible species in a chytrid occupied area. We have sent thousands of adult frogs and tens of thousands of eggs, tadpoles and juveniles to the frontline in the hope that given time and numbers selection will take its course and resistance emerge – it may yet – but we have seen no measurable demonstration of it. In the meantime ecosystems are changing as a consequence of species absences: prolonged breeding and release programs, while we wait for selection, allow ecosystems to accommodate species’ absence possibly even prohibiting their eventual return. Additionally there may be little or nothing for selection to work on and an expensive drawn out process, limited by their genome, is required for every species. We spent over $200K to grow and infect Corroboree Frogs Pseudophryne corroboree in the hope we

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might extract the more resistant individuals for captive breeding. The result: 4 frogs as likely to have survived at random as by resistance. Meanwhile millions of dollars releasing P.corroboree has resulted in just a handful of wild frogs - taking account of epidemiological processes, survivor lineage and current frog density – this outcome is more likely by chance than resistance.

When species do not have the capacity to evolve can we fix them? Genetic modification (GM) is seen by many as extreme and prohibitively expensive; even inherently undesirable or immoral. But we stand on the precipice after many millions of dollars have been poured into captive assurance, breeding and release to no avail, and the tables are turning: a recent frog management meeting in Australia failed to identify any option other than GM that they believed could be realistically employed against chytrid. We summaries some attempts and expenditures in our failed efforts to recover susceptible species. In hindsight could GM have actually been cheaper and quicker in repatriation of amphibians to the wild? At least it would not be limited by each frogs genome, and once developed may have application across numerous susceptible species. We would like to call on others for assistance and believe there are good candidate species but multidisciplinary teams will be needed to gain traction and funds for such ventures. As for the ethics? Surely GM is less morally reprehensible than ignoring extinction.

P-04 Battle strategies for a chytrid susceptible species: lessons learnt from ten years sending captive bred Spotted Tree Frogs Litoria spenceri to the frontline *Stephanie Versteegen1, *Erika Marantelli1, Gerry Marantelli1, David Hunter2, Matt West3, Glen Johnson4

1Amphibian Research Centre, Melbourne, Australia, 2Office of Environment and Heritage, NSW, Australia, 3Zoos Victoria, Melbourne, Australia. 4Department of Environment and Primary Industries, Victoria, Australia.

The Spotted Tree Frog Litoria spenceri is a montane stream frog that is particularly susceptible to chytridiomycosis. Severe declines and extinctions across its range have left the species restricted to a handful of sites, all with small declining populations. For the past 15 years the Amphibian Research Centre (ARC) has bred and for over ten years released L. spenceri to numerous locations along three streams. These releases spanned juvenile frogs to sexually mature adults, a range of habitat types, altitudes, release densities, climatic variables and threatening processes other than chytrid. They have also been made to modified stream systems, extinction sites and one site that the species is likely never to have occupied. All released frogs have been individually marked and monitored regularly since release.

Despite the presence of the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis at two of our three streams, we have seen some persistence and even breeding of released frogs. We have shown that despite the significant additional cost of rearing frogs to larger sizes or even sexual maturity, the improved survival of released frogs makes this strategy more economical. Unfortunately we are also learning that frog densities below which chytridiomycosis driven extinction occurs are too low to sustain ongoing populations. Our hope that by bridging the initial chytrid outbreaks with captivity and with ongoing releases that the emergence of resistance or equilibrium with chytrid would eventuate now seem optimistic. We are likely dealing with a species with limited potential to evolve to meet this new challenge. Our main hope now is that we can sustain large cheap to operate populations in areas free of chytrid while we continue to pour out large numbers of frogs to the front lines in the hope that they may yet develop their own solution. In the meantime we hope for the development of another method that may enable this frog to persist in the presence of chytrid.

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We have learnt much as each new set of results presented and we have adapted our management accordingly. We review some of our strategy, results and thinking on the last ten years and summarise what we know today about the financial and ecological economics of L. spenceri releases and highlight what we have found to be the most important elements in developing battle strategies for waging war with chytrid.

P-05 Conservation of the Japanese giant salamander Andrias japonicus at Toyosaka-cho, Higashi-Hiroshima, Japan

Norio Shimizu1, Hiromi Yamasaki2, Chika Hashimoto3, Kenta Tsuchioka4, Tetsuo Takamatsu5, Susumu Ueda6, Katsunori Sato7, Kanji Kato8 and *Kazushi Kuwabara9

1,2,3 Hiroshima University Museum, 4,5,6,7,8,9 The japanese giant salamander meeting of Higashi-Hiroshima

The Japanese giant salamander Andrias japonicus, endemic to Japan, is ranked as vulnerable species on Japanese Red List and as near threatened on IUCN Red List. In addition, it is protected as a nation's special natural monument, but recently, the status of it's habitats are serious.

In Higashi-Hiroshima city, it is said that there were several salamander habitats, but now we can confirm the existence of only two habitats in Toyosaka-cho, northeastern area of Higashi-Hiroshima city. In that context, we founded "Higashi-hiroshima giant salamander meeting" in 2011 and then we have surveyed the habitat situations and distribution.

The Japanese giant salamander make their habitat in Mukunashi river and Misasa river. We therefore have mainly surveyed about a 3-killometer stretch of Mukunashi river. This area is located on Kamo-daichi plateau, 380m-420m above sea level, where its forest hill landscape is mixed with rice paddies and houses, like mosaic. The width of the river is comparatively narrow, about 3m-5m. Even though most of the river bank is concreted there, salamanders barely live on limited safe site, for example reed bed or soil bank.

Our surveys did find 28 adults and several hundred larvae of A. japonicus within a three years period (from 2011 to 2014). However, we did not discover any young individuals of total length of 50mm-430mm. This suggests that larval recruitment is not successful. Salamanders are prevented their migration by several weirs of every about 500m distance. We think that the weirs prevent movements of larvae to lower rivers by flowing out larvae to rice paddy.

It is considered that giant salamander living in Mukunashi river at Toyosaka-cho is last population remaining in Kamo-daichi plateau. We think it is very important to maintain our survey in the future and hope to find a way to coexist with the precious animals.

P-06 Understanding the origin to preserve in the future: Conservation of Hylid tree frogs in Mesoamerica using an integrative approach

*Liliana Solano1, 2, Javier Sunyer 3, and Catherine Walton1

1 Faculty of Life Sciences, University of Manchester, United Kingdom, 2 Grupo de Investigación en Evolución y Sistemática Tropical, Programa de Biología, Universidad de Sure, Sincelejo, Colombia, 3 Museo Herpetológico de la UNAN-León (MHUL), Universidad Nacional Autónoma de Nicaragua-León, León, Nicaragua

The amphibian fauna of Mesoamerica is highly diverse yet also highly endangered due in large part to anthropogenic environmental modification, leading to habitat poor quality and ultimately loss. The overall aim of this study was to understand the ecological and historical

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factors leading to the generation of this rich biodiversity, as an effective way of identifying how it may be conserved in the long term. Mitochondrial sequence data was generated from Agalychnis and Dendropsophus tree frog species from Panama, Costa Rica, Nicaragua, Honduras, Belize and Guatemala. The high morphological diversity in Agalychnis callidryas, also corresponds to high genetic diversity in mitochondrial DNA. Analysis of mitochondrial sequence data using the general mixed Yule-Coalescent (GMYC) model identified several divergent clusters indicating the presence of several previously unrecognized species. Comparative phylogeographical analysis of both Agalychnis and Dendropsophus species combined with species palaeo-distribution modeling enabled the identification of dispersal routes through Mesoamerica from the ancestral home of these genera in South America. Moreover, species distribution modeling based on contemporary and predicted future climatic and environmental variables identified sites of conservation importance, which coupled with identified genetic relatedness and connectivity between the frog populations, could contribute to a regional in situ conservation strategy.

P-07 Using advanced molecular techniques in a non-model frog: Lessons for amphibian conservation *Heidi K. Smith-Parker1, John B. Wallingford2, and Michael J. Ryan1

1Department of Integrative Biology, University of Texas-Austin, USA, 2Department of Molecular and Cell Biology, University of Texas-Austin, USA

The túngara frog (Physalaemus pustulosus) serves as model of sexual selection and mate choice in the field of behavioral ecology. However, the molecular and genetic underpinnings of túngara frog behavior are poorly understood. We have begun adapting genetic and molecular techniques employed in model amphibian systems like Xenopus for use in P. pustulosus, allowing us to explore mechanistic questions regarding the development and regulation of morphogenesis in this species. However, our tools and methodologies should be applicable to a wider range of amphibians, including threatened and endangered species. Many of our techniques were adapted by considering ecological traits such as mating and reproductive behaviors, and we have successfully adapted the commonly used methods in Xenopus for in vitro fertilization (IVF) as well as transgenesis. The adapted protocols may be valuable to threatened and endangered amphibians, which are disappearing at alarming rates due to habitat destruction, fragmentation, and diseases such as chytridiomycosis. In many of these cases, the first line of action is to remove threatened animals from the wild and establish a captive population for future reintroduction efforts. However, for most endangered species, general husbandry techniques are lacking, and very little is known about their biology; thus, achieving successful captive matings may be difficult. To assist in culturing such species in captivity, IVF can be used, in conjunction with a gavage procedure in the male to retrieve sperm to ensure offspring production in critical situations. Hence, the use of IVF allows populations to be propagated and provides scientists with increased time to study the life history of the animals before extinction. Beyond population propagation, our widely applicable transgenesis protocol may, in the future, be used to create disease-resistant individuals to help ensure the survival of the species.

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P-08 Fine-scale case study of detecting genetic structure and gene flow restriction of Japanese brown frog caused by recent urbanization. *Soh Kobayashi, Seiya Abe, Rikyu Matsuki Central Research Institute of Electric Power Industry (CRIEPI)

To understand and evaluate connectivity between habitats is one of the most important issues in landscape ecology. There were not so much case studies for amphibians which treat this issue in fine scale of landscape, such as several square kilometers. For efficient evaluation of gene flow in fine scale, landscape genetic approaches will be valid. In the present study, we tested techniques of landscape genetics to evaluate the genetic structure of Japanese brown frog (Rana japonica) in the study site (approximately 3 x 3 kilometers) at Inba area, Chiba prefecture. Japanese brown frog is one of the locally decreasing species, and suitable model animal for such analyses because their breeding sites are considered to form a meta-population. We used D-loop region of mitochondrial DNA (mtDNA) regions as genetic markers for calculating Fst and FCT as genetic distance among populations. Fst was compared with geological distances and locations of breeding sites by using Barrier ver.2.2 and FCT was compared with locations of breeding sites by SAMOVA to detect barriers of gene flow. We took data for three years, and also compared each of them. In the results of mtDNA analyses, two or three genetic groups and barriers of gene flow was found in every year, and those genetic structure would be caused by urbanization in this 50 years which implied from past aerial photographs.

P-09 Microhabitats of Forest Frogs along Molawin Creek, Mt. Makiling Forest Reserve, Philippines Cabreros, Julie Ann1, *de Guzman, Christmas2*, Corales, Valerie3, Molina, Kyla4, Moralina, Melchor Jr.5, Ruiz, Dennis6, Afuang, Leticia7

1-6University of the Philippines Los Baños, College, Laguna, Philippines, 2 MSc Wildlife Studies Candidate, Graduate School, University of the Philippines Los Baños, College, Laguna, Philippines. 7Assistant Professor, Institute of Biological Sciences, University of the Philippines Los Baños, College, Laguna, Philippines

The Mt. Makiling Forest Reserve (MFR) in Los Baños, Laguna Province, Philippines, was established as a reserve in 1910 and as such, the mountain is being protected for research and for the benefit of the species inhabiting the area. The most lucrative inhabitants of Mt. Makiling are the amphibians, especially of the family Ranidae. The objective of this study is to describe different microhabitats of frogs belonging to Family Ranidae in the MFR. Two study sites were selected and three belt-transects (50 x 10) were prepared on each sampling site. Every amphibian found inside each transects were captured by hand, identified, photographed and measured. Microhabitats such as: tree holes, forest floors, spaces between buttress, axils of palms and aroids, tree fern, plant litter and moss accumulation were photographed and recorded. Field observations revealed the microhabitats of are identified and demarcated as follows: (1) water; (2) roots; (3) boulders and clumps of moss; (4) leaf litter; (5) tree stumps and felled logs; (6) tree ferns; and (7) tree bark and buttresses. Seven species of Ranids were apparent during the study in wide-ranging microhabitat inclination. Limonectes macrocephalus is specific in tree bark and buttresses, Rana signata and Rana woodworthi both prefer water, boulders and clumps of moss, and leaf litter but the former also favor microhabitats in roots, tree stumps and felled logs. Occidozyga laevis were present only exclusively in water microhabitat. Platymantis mimulus and Platymantis corrugatus prefer a

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leaf litter microhabitat while Platymantis dorsalis is definite with the tree bark and tree buttresses microhabitat.

P-10 Large scale environmental DNA assessment for Japanese and Chinese giant salamanders in Katsura River, Japan

So Fukumoto1,2, Atushi Ushimaru3 and *Toshifumi Minamoto3

1Facul. of Human Develop., Kobe Univ., Japan; 2Grad. Sch. of Global Env. Stud., Kyoto Univ., Japan; 3Grad. Sch. of Human Develop. and Env., Kobe Univ., Japan

To prevent the decline in endangered endemic species caused by an invasion of exotic species, it is most important to understand correctly the area of distribution in the early stage of invasion when the density of the exotic species is still low, and to cope with it immediately. Recently, a novel examination method using the DNA fragments in the water column (environmental DNA; eDNA) is developed to understand the distribution of target species quickly and sensitively. However, specific detection of closely related species is generally troublesome because related species share similar DNA sequences. Here we developed a specific detection system of Japanese giant salamander (Andrias japonicus) and Chinese giant salamander (A. davidianus) which is known to be introduced to Japanese rivers and produce hybrids with Japanese species. To confirm the species specificity of the detection system developed, DNA was extracted from the shedded skin of both species, and subjected to real-time PCR. As a result, it was possible to specifically detect DNA from each species. Then, four-season surveys were carried out over the entire Katsura River basin, Kyoto Prefecture, Japan, where the invasion of exotic salamanders was reported. The results of the Taqman realtime PCR specific to each species showed that mtDNAs of endemic and exotic species were successfully detected from the river water samples. Overall, DNA detection sites are concentrated in the upstream region, and were consistent with the well-known habitat of the giant salamanders. The distribution of exotic species DNA is generally consistent with the area where exotic and hybrid individuals had been captured. In the near future, with further development of the eDNA methods and combination with the conventional survey method, rare endangered species could be more effectively protected.

P-11 A novel method for detection of the highly endangered cave amphibian Proteus anguinus using environmental DNA and real-time PCR *David Stanković1,2, Špela Gorički3, Magda Năpăruş3,4, Matjaž Kuntner5, Aleš Snoj1, Gregor Aljančič3

1Dep. of Anim. Sci., Biotech. Facul., Univ. of Ljubljana, Slovenia; 2Societas herpetologica slovenica, Slovenia; 3Tular Cave Lab., Society for Cave Biol., Slovenia; 4LASIG, École Polytech. Fédérale de Lausanne, Switzerland; 5Inst. of Biol., Scientific Res. Centre, Slovenian Academy of Sci. and Arts, Slovenia

The Olm, Proteus anguinus Laurenti 1768, is the only cave-dwelling vertebrate of Europe. It inhabits subterranean karst waters of the Dinaric Karst in the North-western Balkan Peninsula. Since the beginning of the research on Proteus, the basic but particularly important question of its exact distribution has been difficult to address. Because only small fragments of its subterranean habitat can be accessed by man, this task seems to be impossible to adequately resolve even today. The current knowledge on the species distribution is incomplete; data are particularly scarce in Bosnia and Herzegovina and partly in Croatia. The olm is recognized as a species in need of strict protection by both the Bern Convention (Annex II) and by the EU

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Habitats Directive (Annexes II* and IV), while it is also protected by the national legislations in most range states: Slovenia, Croatia and Italy. The IUCN Red List of Threatened Species defines this species as vulnerable, and recommends urgent measures to revert its population decline. The extent of its decline, however, cannot be estimated without deeper knowledge of its distribution.

Proteus anguinus presence in inaccessible habitats cannot be ascertained by classical survey methods such as trapping and visual encounters. For this reason, alternative methods are required to improve detectability of subterranean organisms as well as to test for their presence in new potential localities. Environmental DNA (eDNA) collection from water habitats in combination with real-time PCR approach has already been shown as suitable for monitoring surface dwelling vertebrates. The aim of the study was to develop a novel method for detection of P. anguinus DNA in cave/spring water samples.

We developed a SYBR chemistry-based assay with two sets of specific primers to amplify short mitochondrial DNA sequences in the 16S rDNA gene (153 bp) and in the control region (106 bp). The specificity of the primers was tested on trout, crested newt and human DNA. In the majority of cases there was no DNA amplification; if amplicons were present, however, they could easily be separated from amplified DNA of P. anguinus by the shape of the melting curve. The eDNA filtration coupled with real-time PCR amplification was tested in controlled conditions of Proteus husbandry in the Tular Cave Laboratory (Slovenia), followed by testing in nature.

The minimum detection limit of the method is 1 animal per 128,000 L (128 m3) of water. Utilizing this method, the species presence was confirmed at all test sites: the springs at Vir pri Stični and Mahniči and the cave Kompoljska jama (all in Slovenia).

With real-time PCR-based method of eDNA detection we significantly improved the efficiency of detection of a cave-dwelling organism and reduced the possibility of contamination as post-PCR analysis can be cancelled out. Furthermore, the DNA-based method to detect Proteus from water samples coupled with local spatial data will provide a vulnerability map of Proteus, which will help visualize zones most threatened by human impacts. Our real-time PCR approach is the most time-efficient method currently known for detection of the cave-dwelling Proteus anguinus.

P-12 Genetic variation of three different populations of Indian Bull frog, Hoplobatrachus tigerinus for conservation

*Md. Rakeb-Ul Islam Department of Fisheries and Marine Science, Noakhali Science and Technology University, Bangladesh

Bangladesh is a small riverine country in Southeast Asia with rich biodiversity. The Indian bullfrog, Hoplobatrachus tigerinus, is widely distributed in South and South-East Asian countries (Husain and Rahman 1978). H. tigerinus plays a significant role in maintaining the natural balance in the ecosystem. It also plays an important role in controlling the various agricultural pests because of its omnivorous feeding habit (mainly insectivorous). According to survey conducted by IUCN Bangladesh (2000), H. tigerinus was fairly common in Bangladesh and had a wide distribution mainly in irrigated, cultivated fields that create a new habitat for feeding, sheltering and breeding. The indiscriminate catch of frogs during breeding season is a common phenomenon in Bangladesh. But now-a-days the availability of frogs is going to be vulnerable due to different anthropological effects and environmental changes. If this situation is going on, once frog population will vanish from Bangladesh and it will disturb the biological balance and consequently lead to the ecological disaster. In these

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circumstances, it has been seriously thought to save the frogs from being extinction. So, it is urgently needed to know their genetic variation of different natural sources of frog population. Therefore, the genetic variation in Indian bullfrog was investigated using allozyme electrophoresis technique. Frog samples were collected from three different regions of Bangladesh namely Rangpur, Khulna and Mymensingh. The genetic variations were analysed with five enzymes in CA 6.1 buffer system. Four polymorphic loci (Mdh-1*, Est-1*, Gpi-1* and Pgm*) were interpretable in muscle with starch gel electrophoresis. Among the 5 presumptive loci, the mean proportion of polymorphic loci was observed 80%, 80% and 60% in Rangpur, Khulna and Mymensingh populations respectively. The highest mean number of allele per locus and mean proportion of heterozygous loci per individual were observed in the Rangpur population. The average observed heterozygosity (Ho) was 0.163 and expected heterozygosity (He) was 0.469. In pair-wise analysis, comparatively higher Nm value (5.507) was estimated between the Rangpur and Khulna populations corresponding lower level of FST value (0.043). The dendrogram showed that the Mymensingh population separated from Rangpur and Khulna populations by a genetic distance of 0.177 whereas the Khulna population is different from the Rangpur population by the genetic distance of 0.052. The findings suggested that the considerable genetic variation is maintained among the natural H. tigerinus populations.

P-13 A new species of the Fejervarya limnocharis complex from Japan (Anura, Dicroglossidae) *Djong Hon Tjong1,4, Masafumi Matsui2, Mitsuru Kuramoto3, Midori Nishioka1, and Masayuki Sumida1 1Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima, Japan, 2Graduate School of Human and Environmental Studies, Kyoto University, Sakyo-ku, Kyoto, Japan, 33-6-15, Hikarigaoka, Munakata, Fukuoka, Japan, 4Department of Biology, Faculty of Science, Andalas University, Padang 25136, West Sumatra, Indonesia

Asian dicroglossid frogs related to Fejervarya limnocharis Gravenhorst are wide-ranging and notoriously difficult to classify taxonomically. Recent extensive studies in this genus proved the presence of two distinct genetic groups, i.e. South Asian and Southeast Asian groups. The Southeast Asian group includes East Asian taxa and systematic studies of this group date back to the finding of two distinct populations occurring sympatrically on Java, the type locality of F. limnocharis. The cryptic Javanese population genetically distinct from F. limnocharis was later described as F. iskandari Veith, Kosuch, Ohler and Dubois. The population from Japan Mainland substantially differs from F. limnocharis. This study is aimed to provide a description of the unnamed population from Japan Mainland so as to facilitate future studies of the surrounding populations. A total of 116 preserved specimens of the F. limnocharis complex from East and Southeast Asia were examined. In order to assess morphometric differences among the population samples, we took 14 body measurements to the nearest 0.1 mm with dial calipers, or with a stereoscopic binocular microscope.

We described a new species, Fejervarya kawamurai sp. nov., of dicroglossid frog of the Fejervarya limnocharis complex from western Honshu, Japan Mainland. Holotype: IABHU - F2184, an adult male collected from paddy field around 2 km from Hiroshima University, Higashihiroshima-shi, Hiroshima Prefecture, western Honshu, Japan (340 23’ N, 1320 42’ E, alt. 200 m a.s.l) on 30 May 2006 by H. T. Djong and M. Sumida. Diagnosis: A small-sized species of the F. limnocharis complex, with adult SVL (36.8 – 48.7 mm in females, 30.7 – 41.8 mm in males). This new species is genetically closer to F. sakishimensis than to F. limnocharis, but is differentiated from F. sakishimensis by smaller tympanum, head, forelimb,

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hindlimb, foot, and tibia lengths, all relative to SVL. It differs from F. multistriata by relatively shorter forelimb, hindlimb, foot, and tibia. From F. limnocharis and F. iskandari, it is differentiated by relatively smaller forelimb, hindlimb, foot, and tibia lengths. Etymology: We dedicate the species name “kawamurai“ to the late Dr. Toshijiro Kawamura, Emeritus Professor of the Institute for Amphibian Biology of Hiroshima University, who was one of the outstanding Japanese herpetologists and the pioneer founder to establish the Institute.

P-14 Evolution of the Sado-frog, a newly described species, in the small Japanese Island

Ikuo Miura1, *Mitsuaki Ogata2, Kunio Sekiya3 and Hiromi Ohtani1

1Hiroshima Univ., 2Preservation and Res. Cent., The City of Yokohama, 3Niigata Univ., Japan

In September 7th of 2012, we have described a new frog species, Rugosa sussura, which was discovered in Sado Island, Japan (Sekiya et al., 2012 in Zootaxa).

The frog is a sister species of Rana rugosa. The two species are definitely discriminated from each other in morphology, and are genetically differentiated based on the mitochondrial DNA. Particularly, the two species, which are allopatrically distributed in the same island, are reproductively isolated from each other: the mating call of R. sussura is unique, and the inter-specific reciprocal hybrids are almost all males with extremely low fertility or quite few females with degenerated ovaries.

Interestingly, the new species is just one endemic species among vertebrates that have ever been identified in Sado Island. The phylogenetic origin of the new species is also curious, because it shows the highest genetic affinity to the northeastern Japan population of R. rugosa, which is located over the high mountain ranges in mainland Honshu, but lower affinity to its northwestern population that is geographically closest to. The phylogenetic history constructed on the basis of previous studies and the evolutionary reason why it is just one endemic vertebrate in the island will be introduced and discussed.

P-15 Major groups of Hynobius nebulosus in Western Japan

Hiroshi Okawa Hiroshima-Kokusai Univ.,Japan

Hynobius nebulosus in western Japan have a significant regional variation; until now they have been organized into more than 10 types based on region. This paper attempts to re-organize these into 3 major groups by morphological characteristics: a yellow line on the top and bottom of the tail ,the type of the hind limb toe type, and spawning habit.

There are three main groups of Hynobius nebulosus in western Japan . They are, first, the Japan Sea Type, distributed across Kyushu, Yamaguchi Prefecture, Shimane Prefecture, Tottori Prefecture, second , the Setouchi Type distributed from the Seto Inland Sea to Fukuyama ,the Middle-Southern region of Okayama, the Southern region of Hyogo Prefecture and Kagawa Prefecture, and third, the Highland type , distributed within the highlands area sandwiched between the other two Major types.

The subgroups Abu Type, Iko type, Aki Type and Iwami Type become easier to organize if thought of as groups that became isolated from other nearby groups then specialized.

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It is believed that the differences between these 3 major groups may be related to how the Hynobius nebulosus migrated from the mainland to the Japanese archipelago and how their populations then distributed across Japan.

P-16 Cytonuclear Discordance and Historical Demography of Two Brown Frogs, Rana tagoi and R. sakuraii (Amphibia: Ranidae) *Koshiro Eto and Masafumi Matsui Graduate School of Human and Environmental Studies, Kyoto University

Closely related species sometimes exhibit paraphyletic or polyphyletic relationships. Such phylogenetic patterns help to clarify their evolutionary history, but it is often difficult to identify the cause of the discordance between phenotypes and genotypes. Two phenotypically similar brown frogs Rana tagoi and R. sakuraii show a polyphyletic pattern in mitochondrial (mt-) genealogy, and this pattern is thought to have arisen from incomplete lineage sorting (ILS) or past gene introgression. To assess their evolutionary history, we estimated phylogenetic relationship and dated the divergence time based on mtDNA, and conducted population assignment and isolation-with-migration (IM) analyses using multiple nuclear (n-) genes. Rana tagoi was split into two major mt-clades, Clades A and B. Rana sakuraii formed two lineages (A-2 and A-3) within one of two R. tagoi-clades, and moreover, one R. sakuraii-lineage (A-2) contained samples assigned to R. tagoi. Lineages A-2 and A-3 were estimated to have separated from the other lineages ca. 2.8–2.6 MYA, followed by the splitting of two lineages (ca. 2.1–1.9 MYA) and intra-lineage divergence (ca. 1.4–0.9 MYA). In contrast to the results of the mtDNA analyses, R. sakuraii tended to form a unique subcluster in the population assignment analysis based on n-DNA, although the species was included in one of the two major clusters of R. tagoi. From the IM analysis, gene flow between species was estimated to be limited, and they separated ca. 1.2–1.1 MYA. These results suspect that polyphyly of the two species occurred through recent speciation of R. sakuraii from R. tagoi, resulting in ILS at the species-level. The resultant population assignment suggests that R. tagoi includes several cryptic taxa, some of which are genetically isolated from each other despite their sympatric distribution.

P-17 Evolutionary relationships and postmating isolation among Fejervarya species from Lesser Sunda, Indonesia and other Asian countries revealed by mtDNA Cyt b gene sequences and crossing experiments *Mahmudul Hasan1, Nia Kurniawan2, Aris Soewondo2, Mohammed Mafizul Islam1, Takeshi Igawa1, Wilmientje Marlene Mesang Nalley3, Masafumi Matsui4, and Masayuki Sumida1

1Inst. for Amphibian Biol., Hiroshima Univ., Japan; 2Dept. of Biol., Facul. of Math. and Sci., Brawijawa Univ., Indonesia; 3Facul. of Anim. Husb., Univ. of Nusa Cendana, Indonesia; 4Grad. Sch. of Human and Environ. Stud., Kyoto Univ., Japan

In order to elucidate the evolutionary relationships and the degree of postmating isolation among Fejervarya species from Indonesia (Lesser Sunda), Bangladesh, China, and Japan, molecular phylogenetic analyses and crossing experiments were carried out using eighteen frogs of eight populations of six Fejervarya species from these countries. The phylogenetic analyses using mtDNA Cyt b gene sequences demonstrated that F. iskandari formed a sister clade with F. verruculosa from Lesser Sunda, Indonesia with 8.1% sequence divergence of

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Cyt b gene. Fejervarya multistriata from China made a clade with Thailand, Malaysia and Indonesian (Topotype) populations of F. limnocharis which showed sister relationships to F. kawamurai from Japan with 8.9% sequence divergence of Cyt b gene. Fejervarya sp. small type from Bangladesh formed a clade with the other South Asian members of Fejervarya group and made a sister clade with Southeast-Asian Fejervarya group having 23.7% sequence divergence of Cyt b gene. The crossing experiments revealed that the reciprocal hybrids among F. iskandari, F. verruculosa, and F. sp. large type, and those between F. multistriata and F. kawamurai were viable through metamorphosis, but those between F. iskandari group and F. limnocharis group were completely or partially inviable at the tadpole stage, and those between Southeast-Asian and South-Asian Fejervarya groups were completely inviable at the embryonic stage. The matured reciprocal hybrids between F. iskandari and F. verruculosa from Indonesia, Lesser Sunda showed some degree of abnormality in spermatogenesis. This study showed that the degree of postmating isolation reflects the molecular phylogenetic relationships, and that the two species, F. iskandari and F. verruculosa, from Indonesia (Lesser Sunda) genetically diverge, and are reproductively isolated by abnormal spermatogenesis.

P-18 Development and characterization of 27 new microsatellite markers of Indian Bullfrog, Hoplobatrachus tigerinus (Daudin, 1802) and its congeneric species

*Nasrin Sultana1, Takeshi Igawa1, Masafumi Nozawa1,2, Mohammed Mafizul Islam1, Md. Mukhlesur Rahman Khan3, Mahmudul Hasan1, Mohammad Shafiqul Alam4 and Masayuki Sumida1

1Inst. for Amphibian Biol., Hiroshima Univ., Japan; 1Center for Information Biol., National Inst. of Genetics, Mishima, Shizuoka, Japan; 2Dept. of Genetics, SOKENDAI, Hayama, Kanagawa, Japan 3Bangladesh Agricultural Univ., Mymensingh, Bangladesh; 4Bangabandhu Sheikh Mujibur Rahman Agricultural Univ., Gazipur, Bangladesh.

Indian Bullfrog, Hoplobatrachus tigerinus belonging to the family Dicroglossidae, is a common large sized frog species found throughout South Asia. As it is mostly found in adjacent canals and ditches of agricultural paddy fields and riparian and emergent vegetation, they are considered excellent bio-indicators of ecosystem due to their sensitivity to environmental change. Actually, it is predicted that populations might be declined in the near future because of habitat loss due to infrastructure development, water pollution from industrial plants and agricultural pesticides, and hunting for consumption by local peoples. This frog is also exploited as a model organism in genetic, ecological, embryological and physiological research and frequently sacrificed by biology students. However, the basic genetic information is limited only for the mitochondrial genealogy. In the present study, to clarify the genetic population structure and population dynamics of this species for the further study, we therefore developed and characterized microsatellite loci for this frog. For rapid and cost-effective development, we obtained genomic data by using Ion Torrent PGM™ and designed 71 primer sets for candidate loci. As the result of screening of polymorphic loci for individuals of H. tigerinus and its congeneric species, 27 loci were isolated as highly polymorphic microsatellite loci. The number of alleles per locus and expected heterozygosity ranged from 6 to 31 and 0 to 0.938 respectively. No significant deviation of linkage disequilibrium was observed across all loci and 5 of the 27 loci showed a significant deviation from Hardy-Weinberg equilibrium in some populations after Bonferroni correction. Finally, these polymorphic microsatellite markers provide a powerful tool to study population genetics across varying scales from inter-population to inter-individual.

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P-19 Inter- and intra-island divergence in Odorrana ishikawae (Anura, Ranidae) of the Ryukyu Archipelago of Japan, with description of a new species Mitsuru Kuramoto3, Naoki Satou1, Shohei Oumi2, Atsushi Kurabayashi1, and Masayuki Sumida1

1Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Hiroshima, Japan, 2Section of Agriculture and Forest, Amami City Government, Amami, Kagoshima, Japan, 3 3-6-15 Hikarigaoka, Munakata, Fukuoka, Japan

The endangered frog, Odorrana ishikawae (Anura, Ranidae), is a species endemic to the Amami and Okinawa Islands of the Ryukyu Archipelago, Japan. Vicariance of these islands has been considered to occur middle or upper Pleistocene. Our morphometric analyses revealed obvious differences between the Amami and Okinawa populations. Two distinct morphotypes were also recognized from the Amami population (Amami common and Amami large types). Furthermore, the Amami and Okinawa populations could be distinguished clearly by coloration and dorsal tuberculation. Based on 16S rRNA gene data, the Okinawa and Amami populations were phylogenetically separated but the genetic divergence (1.44 – 2.16%) was lower than the value suggested as species threshold in anurans (> 3% in 16S). Individuals of the Amami common and large types were nested within a single clade. Artificial hybridization experiments revealed normal hybrid viability between the two Amami types, with one exception. By contrast, between Okinawa females and two Amami type males, complete hybrid inviability was observed at early embryonic stages in the hybrids contrary to expectations from their low divergence in 16S. The reciprocal hybrids between two Amami type females and Okinawa males were viable, but spermatogenesis in the hybrid males showed some degree of abnormality. These results strongly indicate specific separation of the Amami population from the Okinawa population of O. ishikawae. Thus, we describe the Amami population as a new species, Odorrana splendida sp. nov., which is readily distinguishable from O. ishikawae by smaller ruggedly edged dorsal spots and an immaculate ventral surface.

P-20 New insights into evolutionary history among Asian pond frog species *Shohei Komaki1, Takeshi Igawa1, Koji Tojo2, Atsushi Kurabayashi1, Si-Min Lin3, Mi-Sook Min4, Masayuki Sumida1

1Inst. Amphibian Biol., Hiroshima Univ., Japan; 2Dept. Biol., Shinshu Univ., Japan; 3Dept., Life Sci., National Taiwan Normal Univ., Taiwan; 4Res., Inst., Seoul National Univ., Korea

Asian pond frogs are middle sized anurans dwelling rice fields and swamps, found from western china to Japanese Archipelago, consisting of four species and a subspecies (Pelophylax nigromaculatus, P. plancyi, P. fukienensis, P. porosus porosus and P. p. brevipoda). Phylogenetic relationships of this species complex were often studied based on mitochondrial DNA (mtDNA) previously. In more recent, study combining nuclear DNA (ncDNA) and mtDNA analyses revealed rampant historical introgressions among these species, suggesting their complex evolutionary history which was not expected in previous studies. But uncertainty has still remained in the phylogenetic relationships and processes of introgression so far. We therefore conducted phylogeographic analyses based on multiple genes (mitochondrial cytb gene and nuclear six genes) combining historical distribution modeling to clarify their true evolutionary history including introgressions among the ancestral lineages.

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As the result, our phylogenetic tree based on ncDNA suggested the sister relationships between P. plancyi and P. fukienensis and between P. nigromaculatus and P. porosus, contrasting to the previous studies. While phylogenetic tree based on mtDNA showed consistent results with the previous studies. On the basis of comparison of these results, we hypothesized following historical introgression events among and within ancestral lineages of P. nigromaculatus and P. plancyi: (1) rampant directional mitochondrial introgression had occurred from ancestor of P. nigromaculatus into that of P. plancyi and genuine mitochondrial genome of P. plancyi had been replaced by that of P. nigromaculatus; (2) ancestral lineage of Japanese P. nigromaculatus were created through the mitochondrial introgression from ancestral lineages of Chinese to Korean P. nigromaculatus; (3) ancestral lineage of Russian P. nigromaculatus were created through same pattern of introgression with (2) in more recent period. For hypotheses (1) and (2), historical distribution modeling of P. nigromaculatus and P. plancyi during the glacial period supported these scenarios, suggesting extreme shrinkage of distribution range of P. plancyi and continuous distribution of P. nigromaculatus around land-bridges from Yellow sea to Kyushu area of Japan. Because the periods of (1) and (2) are differed based on the relaxed molecular clock, these events may have been occurred in different glacial periods. For hypothesis (3), a previous study and our mismatch distribution analyses supported this by suggesting sudden expansion of Chinese lineage from central China to far eastern Russia. Taken together, our findings revealed complex evolutionary history and recurrent introgressions of Asian pond frogs associated with population dynamics during glacial and interglacial periods.

P-21 Genetic population structure of an endangered frog species, Babina holsti *Ryosuke Kakehashi, Takeshi Igawa and Masayuki Sumida Inst. for Amphibian Biol., Hiroshima Univ., Japan

The Holst’s frog (Babina holsti) is a ranid frog endemic to the Okinawa Islands, Japan, and has been designated as natural assets in Okinawa prefecture. This species display unique morphological and ecological characteristics which are evolutionary interesting, such as possessing a pseudothumb in the forelimb and creating a nest for oviposition. However, due to their small original distribution range, recent habitat loss by deforestation, this species is in danger of declining and has been listed as class B1 endangered species in the IUCN Red List of Threatened Species. In this study, we performed genetic analyses using 12 polymorphic loci and mtDNA sequences to investigate genetic diversity, genetic structure and demographic history. The resultant NJ tree based on mtDNA sequences indicated that the Tokashiki Island population (TOK) was largely differentiated from mainland populations. The number of haplotypes of TOK was one haplotype only. In the mainland, the number of haplotypes of southern two populations (KIJ and TAK) was lower than other populations. Furthermore, over the 80% of individuals in southern populations showed the specific haplotypes to each population. The results of Bayesian clustering approach based on microsatellite loci indicated Holst’s frog species divided into three genetically distinct populations (TOK, KIJ and others).The expected heterozygosity was the lowest in TOK, followed by KIJ. This result was consistent with the mtDNA analysis. Furthermore, significant bottlenecks were detected in these two populations. We found that the pairwise FST based on both markers were correlated with geographic distances between mainland populations. However, the pairwise FST values calculated from mtDNA sequences between KIJ and the marginal populations were deviated from the regression line. In conclusion, our results suggested that TOK and KIJ populations were genetically differentiated from other populations, and the sizes of both populations were

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declining. Our results also showed KIJ population may be isolated by ecological factors, while almost mainland populations were isolated by geographical distances.

P-22 Effect of low temperature on metamorphosis of the salamander Hynobius retardatus in the field and under laboratory conditions

*Takuma Midori1, Ryoga Kato1, Tomohiro Ichikawa1, Naoyuki Ushiro1, Takuya Sato1, Tomoaki Kuwahara2, and Naoko Yamashiki1

1Lab. of Develop. Biol., Rakuno Gakuen Univ., Ebetsu, Japan; 2Kon Photography & Research, Sapporo, Japan

In Hynobius retardatus, Hokkaido salamander called Ezo Sansho-uo in Japanese, larvae of some populations in cold mountainous ponds do not metamorphose within the year and become aged, overwintered larvae. In Sapporo, overwintered larvae were found in ponds at high elevations and were also recently found in a pond located at a low elevation in Sapporo. We studied larval histories in populations bred in two nearby ponds (A pond and B pond) for comparison. Both ponds are in the same riverine system and the distance between ponds is approximately 250m. A pond was artificially constructed dam across a brook in the 1960’s. In A pond, temperature and level of the water are maintained almost constant by abundant spring water throughout the year, and overwintered larvae and larvae under the age of 1 year were found in this pond. In B pond, water temperature rises and level drops during summer, and only larvae under the age of 1 year were found. Egg sacs of salamander are generally seen during April at both ponds. On June 1, 2013, the mean (±SE) body lengths of larvae captured at the two ponds were almost the same (A: 23.58 ± 0.51 mm, B: 23.49 ± 0.72 mm). In B pond, almost all of the larvae metamorphosed and moved to land around July, while almost all of the larvae in A pond developed slowly and showed no sign of metamorphosis. From June 1 to July 6 in 2013, the mean water temperature in B pond was 13.8℃, while that in A pond was 10.6℃. In larval populations in A pond, there were some differences in the timing of completion of metamorphosis between 2012 and 2013. Larvae that had hatched in the previous year moved to land during a two-month periods (June 1 to August 6) in 2013, a one-month period (June 9 to June 25) in 2012. Larvae that hatched in the same year move to land in the period from August 20 to September 20 in 2012, but we did not find any larvae on land in 2013. In A pond, the mean water temperatures during a four month period (May 11 to September 20) were almost the same in 2012 (10.79℃) and 2013 (10.75℃), but the mean water temperatures during the period from April 25 to May 14 were 8.7℃ in 2013 and 11.0℃ in 2012. We reared some larvae collected from the field at 7℃ and 10℃ under laboratory conditions. When the larvae were maintained at 10℃, all of the larvae underwent metamorphosis within 140 days from hatching. However, when they were kept at 7℃, metamorphosis was not completed even after 210 days from hatching. These results indicate that a 2-3℃ difference in low water temperature causes large differences in the process of the larvae development, especially in metamorphosis of the salamander.

P-23 Frog fauna around Imori Pond

*Naoki Sato and Yoshikazu Takegoshi Kaiyo-high school and Miyokoukogen Visitor center, Niigata, Japan

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Imori Pond is a small pond with a 500-m circumference located in Ikenotaira Onsen of Myoko Kogen within Joshinetsu-kogen National Park in Niigata prefecture, Japan. The pond is inhabited by many newts as suggested by its name Imori, which means “newts” in Japanese. It is surrounded by hydrophytes and white birch trees, and is famous for Lysichiton camtschatcensis flowers, which are in full blossom from mid-April to early May. Frog fauna differs among surrounding ponds and swamps. Six frog species, two in Rhacophoridae, two in Ranidae, one in Bufonidae, and one in Hylidae, were found in two ponds (Ponds A and B) between 2012 and 2013. The diversity of the frog fauna was greater in Pond A than in Pond B, although both ponds were suitable frog habitats. Rhacophorus arboreus lays eggs in the pond, and many frogs can be seen in the breeding season (around June). We will continue investigating the distribution and calls of the resident frogs and any new frogs species we discover. This study was carried out to introduce the kind of the frogs distributed around Imori Pond.

P-24 Radiocesium accumulation of frog species in forest floors 2.5 years after the Fukushima Nuclear Power Plant accident

*Teruhiko Takahara1, Satoru Endo2, Momo Takada1, Yurika Oba1, Wim Ikbal Nursal1, Toshihiro Yamada1, and Toshinori Okuda1

1Grad. Sch. of Integrated Arts and Sci., Hiroshima Univ., Japan; 2Grad. Sch. of Eng., Hiroshima Univ., Japan

The radioactive contamination (134Cs and 137Cs) level of abiotic environment (e.g. soils, water) has been surveyed well, particularly in forest vicinity area of the Fukushima Nuclear Power Plant (FNPP), while the contamination level in small vertebrates is a lacking of information.

Anuran frogs are the key components of the food webs in the forest ecosystems, and play major roles as higher consumers predating small insects, while they serve as food sources for higher hierarchies (e.g. snakes or birds). Therefore, the radiocesium accumulation in frogs is a key issue for understanding how the radiocesium transfers from lower to higher trophic levels in forest ecosystems. In these respects, we investigated the concentration of 134Cs and 137Cs of frogs captured in forest floors about 2.5 years after the FNPP accident.

On 31 July to 6 August 2013, 69 frogs (including Rana japonica, R. ornativentris, R. tagoi tagoi, and Bufo japonicas formosus) were collected in the range of 22.6–48.7 km (e.g., Soma City, Minami-Soma City, and Iitate Village) from the FNPP. The frog samples were dried and powdered. Then, concentration of 134Cs and 137Cs in the frog samples (Bq kg–1 dry) was individually measured by a gamma spectrometer with a well-type germanium detector.

The air radiation dose rates decreased as a function of distance from the FNPP. And, the radiocesium concentration of the frogs was positively correlated to the degrees of the air radiation dose rates. Radiocesium concentration of individual frogs was also positively correlated to their mass, but exhibited a large variation among individuals. This suggests that differences of the diet menu within the local sampling sites result in the large variations of individual-based contamination. The monitoring of the contamination levels of potential food sources for frogs is necessary for elucidating the processes and mechanisms of radiocesium transition through the food webs of forest ecosystems. To our knowledge, this study is the first report that evaluated the detailed radiocesium contamination levels of the vertebrate, such as frogs, after the FNPP accident.

By DNA sequencing, we will identify species and sexes of these frog samples. The age of the frog samples will be assessed by skeletochronological analysis using phalange obtained from the samples. We will clarify species-, sex- and age-related variation of the accumulated radiocesium concentration of the frogs.

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P-25 Effects of female calls on male behavior during the breeding season in frogs, Rana porosa brevipoda and Rana nigromaculata *Makoto Itoh Grad. Sch. of Sci., Kyoto Univ., Japan

Generally, male frogs give advertisement calls whereas female frogs emit only a soft simple release call. In a very few species, however, female frogs also give a courtship call. It is, therefore, expected that the species with female courtship call have some evolutionary consequences to emit such a call. Two relative frogs, Rana porosa brevipoda and Rana nigromaculata inhabit paddy fields in Japan. These species hybridize in the laboratory but F1 hybrid males are sterile. Therefore, there are strong effects of reproductive interference and it seems difficult for them to occupy the same habitat. However, they are sympatric and synchronously breeding in nature in some areas in Japan. There might be some mechanisms of reproductive isolation between these species. In the present study, I observed their behavior in meeting the other species. I used frogs of both species captured during the breeding season for experiments. They were large enough to breed. I placed one male and one female together in a test tank, and then observed their behavior with an infrared video camera and recorded their calls with an IC-recorder. The test tank of 110 x 80 x 40 cm was filled with water in 20 cm depth and a few bricks were placed as a land in it. There were total 4 patterns in the experiments. Intraspecific or interspecific pairs were examined in both species. Twenty experiments were tested for each pattern. An individual frog was used in two patterns of experiments. Frog behavior was recorded for 3 hours but the first 1 hour was not used for analyses. These experiments were conducted at night. I classified their behavior into 5 patterns and counted them. Then, I made sonograms of their calls. I found that female frogs of both species often emitted calls. The sonograms of both female calls were similar. These calls were emitted to male Rana nigromaculata more frequently than to male Rana porosa brevipoda. Females responded to the movement of male Rana nigromaculata and emitted calls to the male in both species. The female calls of both species had effects to freeze male Rana nigromaculata. These results suggest that the call of female Rana porosa brevipoda and Rana nigromaculata has a negative effect on male Rana nigromaculata breeding and approaching. Thus, it also suggests that a female Rana porosa brevipoda call is effective to avoid approach of male Rana nigromaculata. It is probable that female calls function as a mechanism for avoiding reproductive interference.

P-26 Eco-evo-devo study of phenotypic plasticity in the Hokkaido salamander (Hynobius retardatus) *Masatoshi Matsunami1, 2, Osamu Kishida3, Jun Kitano4, Masafumi Nozawa5, Takeshi Igawa6, Hirofumi Michimae7, Toru Miura2, Kinya Nishimura1

1Grad. Sch. of Fisheries Sci., 2Grad. Sch. of Environ. Sci., 3FSC, Hokkaido Univ., Japan; 4Ecological Genet. Lab., 5Lab. for DNA data analysis, Natl. Inst. of Genet., Japan; 6Inst. for Amphibian Biol., Hiroshima Univ., Japan; 7Sch. of Pharm. Sci., Kitasato Univ., Japan

The Hokkaido salamander (Hynobius retardatus) inhabits the Hokkaido island of Japan archipelago. Larvae of the Hokkaido salamander exhibit two distinct morphs, "attack morphs" and "defense morphs", as a result of inducible phenotypic response to their prey and predators, respectively. The existence of prey, Rana pirica tadpoles, leads to the induction of attack morphs, which have broad heads likely suitable for swallowing tadpoles. The existence of predators, dragonfly nymphs (Aeshna nigroflava), induces them to become defense morphs,

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which have enlarged external gills and high tails suitable for hiding and avoiding the predatory attacks, respectively. The expression of these morphs is regulated by ecological interaction and the induction rates of attack morph show large variation depending on their regional populations. Although the Hokkaido salamander is an ideal model organism of eco-evo-devo study due to these features, molecular mechanisms and genetic backgrounds underlying this phenotypic plasticity have yet to be elucidated. To reveal the landscape of molecular mechanisms and genetic variations underlying expression of both plastic morphs, we conducted two experimental approaches, 1) de novo transcriptome analysis and 2) development of microsatellite markers.

1) We exposed salamander larvae to either predators or prey to induce the different morphs. Morph induction was completed about 7 days after the start of exposure. RNAs were extracted from 4 tissues (brain, head, external gill and tail) and 3 time points (0 hour, 12 hours, and 7 days after the exposure onset) and sequenced. Obtained reads and contigs of treatment samples were compared to identify differentially expressed genes. Finally, 32 contigs were identified as differentially expressed genes. These are candidate responsible genes of expression of phenotypic plasticity.

2) We sequenced salamander genomic DNA and obtained 2,975,641 reads (mean: 201 bp). These reads included 806 microsatellite sequences and 111 of 806 loci were capable of primer design. From these candidate microsatellite marker sequences, we designed 48 primers to identify useful markers, which will help investigation of the genetic diversity and population structure.

P-27 Molecular cytogenetic studies on the process of genomic and chromosomal evolution in X. laevis after WGD and the origin and evolution of sex chromosomes in anuran species *Yoshinobu Uno1, Chizuko Nishida2, Chiyo Takagi3, Takeshi Igawa4, Naoto Ueno3, 5, Masayuki Sumida4 and Yoichi Matsuda1

1Lab. Anim. Genet., Grad. Sch. Bioagr. Sci., Nagoya Univ., 2Dep. Biol. Sci., Fac. Sci., Hokkaido Univ., 3Div. Morphogen., Natl. Inst. Basic Biol., 4Inst. Amphibian Biol., Grad. Sch. Sci., Hiroshima Univ., 5Sch. Life Sci., Grad. Univ. Adv. Studies (Sokendai)

A draft assembly of the first genome sequence in amphibians was generated in the Western clawed frog (Xenopus (Silurana) tropicalis, Pipidae, Anura) (2n = 20). However, there is still no detailed information on genome and chromosome structures for the other anuran species. Comparative gene mapping among many anuran species enables us to understand the process of karyotype evolution in anurans. Here we show the latest data of our research on the process of genomic and chromosomal reorganization in an allotetraploid species, the African clawed frog (Xenopus laevis, Pipidae, Anura) (2n = 36), after whole-genome duplication (WGD) and the origins and evolution of sex chromosomes in anurans. We constructed a high-resolution cytogenetic map of 140 functional genes for X. tropicalis using FISH. Then, to examine the process of genomic and chromosomal reorganization in X. laevis after WGD, we performed comparative gene mapping for X. laevis using 60 cDNA clones of genes that covered entire regions of 10 pairs of X. tropicalis chromosomes. Hybridization signals on two pairs of homoeologous chromosomes were detected for 50 of 60 (83%) genes, and genetic linkages were highly conserved between the two species and also between homoeologous chromosome pairs of X. laevis. Furthermore, we identified the sex-linked genes of X. laevis, the Japanese wrinkled frog (Rana rugosa, Ranidae, Anura) (2n = 26) and the endemic Japanese bell-ring frog (Buergeria buergeri, Rhacophoridae, Anura) (2n = 26) by comparative gene mapping.

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Genetic linkages of sex chromosomes were different among X. laevis, X. tropicalis and R. rugosa. By contrast, in B. buergeri, the homology with the X. tropicalis sex chromosomes was found for the sex chromosomes and a pair of autosomes. Comparative chromosome mapping between X. tropicalis and X. laevis revealed that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in X. laevis lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD. Comparative mapping among X. laevis, X. tropicalis, R. rugosa and B. buergeri suggested the diversity of sex chromosomal origins of anuran species and the possibility that X. tropicalis and B. buergeri have the same origin of sex chromosomes.

P-28 Species diversity of hynobiid salamanders of the genus Onychodactylus from Japan *Natsuhiko Yoshikawa and Masafumi Matsui Grad. Sch. of Human and Environ. Stud., Kyoto Univ., Japan

Clawed salamander, the genus Onychodactylus, is a group of hynobiid salamanders endemic to northeast Asia, especially surrounding area of Japan Sea. This genus has unique characters among hynobiids, such as large premaxillary fontanelle, lack of lungs, black acute claws on digits of larvae and breeding adults, significant sexual dimorphism, larval morphology that is highly adapted to torrents. The species diversity of this genus was long underestimated, only two species were recognized: O. japonicus (Houttuyn, 1782) from Honshu and Shikoku Islands of Japan and O. fischeri (Boulenger, 1886) from Korean Peninsula, northeastern China and Russian Far East. However, a series of recent molecular phylogenetic studies uncovered that this genus comprises multiple cryptic species.

Of the two species traditionally recognized, O. japonicus (sensu lato), long considered as a wide-ranging common species in the mountain regions of Honshu and Shikoku Islands, was divided into four species by descriptions of three new species done in the past two years: (1) O. nipponoborealis Kuro-o et al., 2012, which is widely distributed in northern half of Tohoku District, northern Honshu. (2) O. tsukubaensis Yoshikawa et Matsui, 2013 endemic to Tsukuba Mountains of Kanto District, eastern Honshu. (3) O. kinneburi Yoshikawa et al., 2013 from Shikoku and several localities of western Honshu. By this series of taxonomic revisions, O. japonicus (sensu stricto) was delimited to southwestern half of Honshu (corresponding to SW-Honshu group in the previous study). Besides, some more unnamed species remained to be described. These newly described species are morphologically similar and not easy to distinguish from each other, that is why species diversity of this group has been overlooked.

Molecular phylogenetic analysis and divergence time estimates revealed that diversification of Japanese Onychodactylus species started in the Upper Late Miocene. The speciation and distributional patterns of this species complex might have been strongly associated with recent climate oscillation, as well as geohistorical events, such as volcanic activity and mountain formations, that occurred in the process of formation of the Japanese archipelago.

As menthioned above, O. japonicus (sensu lato) was long considered as a wide-ranging common species, and thus this species was tended to be disregarded in conservation actions. For example, only O. japonicus (sensu lato) was not listed in the Japanese Red Data Book among Japanese salamanders. However, the series of taxonomic revisions separated the wide-ranging species into several species of restricted distributional ranges. Unfortunately,

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most of these species are facing to threats of population declines, and urgent conservation actions on these species are needed.

P-29 Functional analysis of the sex-linked gene SOX3 for female determination in the frog

*Ikuo Miura1, Mitsuaki Ogata2, Yoshinori Hasegawa3 and Hiromi Ohtani1

1Hiroshima Univ., 2Preservation and Res. Cent., The City of Yokohama, 3Kazusa DNA Res. Inst., Japan

The sex chromosome of Japanese frog Rana rugosa (northwestern population) is morphologically differentiated as ZZ♂/ZW♀type.

In the undifferentiated gonads of tadpoles, the autosomal genes of CYP17, CYP19 and FOXL2 show a sexually dimorphic expression. And, prior to them, the sex-linked gene SOX3 begins to be highly expressed in ZW female gonads. SOX3 is well known as the ancestral gene of SRY, a male determining gene of eutherians, and in fact, its miss-expression could determine maleness in place of SRY in mouse. However, the expression pattern shown in the frog suggests a female determining function, which is completely opposite to the potential function of eutherian SOX3.

Then, we have carried out functional analysis of SOX3 for female determination in the frog. For driving the gain-of-function in ZZ males, a construct of W-SOX3 (allele from W chromosome) under the upstream promoter region was introduced into fertilized eggs using meganuclease technique. On the other hand, for driving the loss-of-function in ZW females, the translation was inhibited using SOX3 antisense-morpholino injection into fertilized eggs, or the genomic SOX3 was disrupted using TALEN gene knockout technique. We will report the results.

P-30 Gene dose, compensation, and anurans *John H. Malone Institute of Systems Genomics, Department of Molecular and Cell Biology, University of Connecticut

How do genomes respond to changes in gene dose? Reducing or increasing DNA dose by altering copy number can lead to changes in gene expression levels that may percolate across gene expression networks to alter phenotypes. Interestingly, dosage compensation that offsets copy number change is often observed so that the effects of copy number changes are dampened or removed. Previous work has shown that dosage compensation, or the lack thereof, is a gene-specific response, largely mediated by interactions with the rest of the transcriptome. Frog sex chromosomes offer an ideal system for advancing our understanding of genome evolution and function because of the variety of sex determination systems in the group, the diversity of sex chromosome maturation states, and the ease of experimental manipulation during early development. Initial sequencing of the African Bullfrog (Pyxicephalus adspersus) provides the identification of sex-linked sequences for comparative and functional analysis of dosage compensation and other mechanisms of genome dose tolerance. Investigating how the frog genome responds to changes in gene dose will provide important insights into the evolution of sex chromosomes and dosage compensation systems.

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P-31 Transient receptor potential channel vanilloid 4 (TRPV4) functions as the hypotonic osmoreceptor in the Japanese tree frog, Hyla japonica *Sho Maejima1,2, Hiroyuki Kaiya3, Norifumi Konno2, Minoru Uchiyama2

1Grad. Sch. of Integrated Arts and Sciences, Hiroshima Univ., Japan; 2Grad. Sch. of Science and Engineering, Univ. of Toyama, Japan; 3National Cerebral and Cardiovascular Center Research Institute, Japan.

Transient receptor potential (TRP) is the non-selective cation channel family and is also well known as a sensor molecule for various noxious stimuli such as heat, acid, plasma membrane stretch and so on. TRP superfamily is divided into at least six subfamilies; TRPA, TRPC, TRPM, TRPML, TRPP and TRPV. Recently, in the rodents and C. elegans, it was reported that TRPV1 and TRPV4, members of TRP vanilloid subfamily, were activated in response to hypertonic and hypotonic stimuli, respectively. These results suggest that TRPV might function as an “osmoreceptor”. However, the molecular entity of osmoreceptors in vertebrates is unclear yet. In the present study, we investigated whether TRPV4 functioned as an osmoreceptor in the anuran amphibians. Anurans adapt in various environments such as the desert and the brackish water area, therefore they might have superior osmotic sensors. First, we cloned and characterized cDNA of frog TRPV4 from the Japanese tree frog, Hyla japonica, by molecular biological technique. Putative primary structure of frog TRPV4 showed several properties such as six transmembrane domains, TRP box and TRP domain which were structural characteristics of TRP channels. Although TRPV4 mRNAs were expressed ubiquitously, high expression was observed in the kidney which is the osmoregulatory organ. In addition, mRNA expression of TRPV4 changed in response to hypo-osmotic treatment in the brain (especially midbrain) and the kidney. This means that frog TRPV4 may be responsive to hypotonic stimulation. Next, we transfected cloned frog trpv4 gene into Chinese hamster ovary (CHO) cells, and established culture cell lines expressing frog TRPV4 protein (frog TRPV4-CHO). Moreover, we examined response to osmotic variations of frog TRPV4-CHO with intracellular calcium concentration change by fluorescent imaging plate reader. Rat TRPV4 was used as the positive control. Intracellular calcium concentrations of frog TRPV4-CHO and rat TRPV4-CHO were increased by 4α-PDD, TRPV4 specific agonist, in a dose-response manner, and these effects were prevented by pre-treatment of TRPV4 specific antagonist, RN-1734. Both frog TRPV4-CHO and rat TRPV4-CHO did not respond to hyper-osmotic or low temperature stimuli. Interestingly, intracellular calcium concentration of frog TRPV4-CHO was increased only in response to hypo-osmotic stimulation whereas rat TRPV4-CHO responded to both high temperature and hypotonic stimuli. This might be caused by the difference between the homothermal animals and the poikilothermic animals. These findings suggest that TRPV4 channel functions as a hypotonic-specific sensor in the anuran amphibians.

P-32 Discovery and phylogenetic distribution of a short interspersed nuclear element (SINE) subgroup in neobatrachian frogs *Atsushi Kurabayashi1, Nobuaki Furuno1, Masayuki Sumida1, Hideaki Mizuno2, Kazuhiko Ohshima3, Miguel Vences4

1 Inst. for Amphibian Biol., Hiroshima Univ., Japan, 2 Kamakura City, Kanagawa Pref., Japan, 3 Dept. of Computer Biosci., Nagahama Inst. of Bio-Sci. and Thech., Japan, 4 Zool. and Evol. Biol., Tech. Univ. of Braunschweig, Germany

SINEs (Short Interspersed Nuclear Elements), a type of retrotransposons, can be found in many eukaryotic genomes. The SINEs are generally short (<500 bp) DNA sequences and

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represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase III into tRNA and 5S and 7SL ribosomal RNA. They do not encode a functional reverse transcriptase and rely on other mobile elements for transposition (specifically reverse transcriptases encoded in Long Interspred Nuclear Elements: LINEs). Because SINEs have a unique property –once a SINE gets copied into a new genomic position, it is never cutout (ie, copy-past type increase)–, they have been regarded as strong phylogenetic makers to support the monophyly of the taxa sharing the same SINE loci (known as SINE method). In fact, the SINE makers have been used with success in resolving some phylogenetic problems (such as fish, reptile, and mammal taxa) that were difficult to be solved by common “sequence based” phylogenetic approaches. In amphibians, SINEs have been found only from one frog species Xenopus tropicalis (family Pipidae), a member of basal frog group, archaeobatrachia so far; and “real” SINEs have not been reported from neobatrachian frog genomes. The suborder Neobatrachia is phylogenetically nested anuran taxon containing over 95% frog species and phylogenetic relationships of many neobatrachian taxa are still pending. In this study with the final purpose of resolving the problematic neobatrachian phylogenies by using the SINE method, we first tried to find SINEs from the genomes of three neobatrachian species, Breviceps adspersus (family Breviciptidae), Mantella betsileo (family Mantellidae), and Rana japonica (family Ranidae). We partially sequenced these genomes with 454-NGS. From the NGS data (53, 47, and 22 Mbp, respectively), we discovered SINE2-1XT (one of six SINEs known in X. tropical genome) homologues in all three genomes surveyed here. The estimated abundance ratio of the SINE2-1XT homologues in each genome varies among the taxa (0.04% in X. tropicalis to 1.84% in M. betsileo), suggesting that the SINE method is easely applicable for some neobatrachian taxa (ie, high abundance taxon, Mantellidae). In the Rana and Boophis genomes, we found unique SINE2-1 subtypes with modified 3’ sequence (in R. japonica) and with apploximately 70 bp repetitive sequences (in M. betsileo). Southern blot analysis indicates that SINE2-1 homologues are shared all 22 amphibian taxa surveyed here including caecilian and caudata species, suggesting an ancient origin (common ancestry of amphibians) and long-term survival of this SINE. Southern blot analyses also suggested that the SINE2-1 subtypes found in Rana and Boophis have occurred in limited neobatrachian lineages (genus Rana and part of natatanurans, respectively).

P-33 Molecular domestication of transposons in Xenopus

*Akira Hikosaka, Seigo Konishi, Sarina Uno, Akira Kawahara Graduate School of Integrated Arts and Sciences, Hiroshima University, Japan

A transposable element (TE) or a transposon is a DNA sequence that can transpose and proliferate in a host genome and is normally viewed as a parasitic genetic element. Its mobility and proliferative nature have a potential harmful effect on its host; it can cause deleterious mutations and unusual chromosomal recombination between TE copies. Therefore, in general, the mobility of a TE is not conserved by purifying selection. One way for a TE to survive in its host is by molecular domestication i.e., co-option of a TE or its component by its host as a useful genetic element, such as a novel protein-coding gene or a cis-regulatory element. For example, we have reported a domesticated transposon, Kobuta, in Xenopus, which originated from a piggyBac superfamily transposon Uribo but ceased to transpose and has been co-opted by the host. The Kobuta transposase gene exists as a single copy, and its conservation between X. tropicalis, X. borealis, and X. laevis indicates that purifying selection has conserved the Kobuta gene.

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A predominant Miniature Inverted-repeat Transposable Element (MITE) family, T2-MITE, is found in Xenopus species and certain fish. We systematically searched the genome of X. tropicalis for T2-MITEs and classified them into 16 major subfamilies. Among them, T2-A1 and T2-C were found to be the predominant subfamilies, each consisting of more than 1,000 copies and comprising the currently active elements. Their presence in both X. tropicalis and X. laevis suggests that they may have originated approximately 65 million years ago prior to the deep branching of these 2 species. It is highly unlikely that the mobility of these MITEs has been conserved for such a long period without purifying selection. An intriguing hypothesis is that the mobility of the subfamily has been conserved by purifying selection because their mobility has been beneficial for X. tropicalis.

T2-MITE is a non-autonomous TE that does not encode the transposase gene. Hellsten et al. (2010) reported a new DNA transposon family, Kolobok, from the X. tropicalis genome; the transposases (Kol-Tpases) of this family could be candidate proteins that mediate (or meditated) transposition of T2-MITE subfamilies. If certain Kol-Tpases mobilize the T2-MITE subfamilies, it stands to reason that these transposases should also have been conserved for the same period of time as the T2-MITE subfamilies. We researched and characterized multiple Kol-Tpase genes in the genome of X. tropicalis and found 26 Kol-Tpase subtypes. The significant conservation of the Kol-Tpase genes between two distant lineages of X. tropicalis (Nigerian and Asashima) and expression of their mRNA during embryogenesis suggests that the Kol-Tpases (or at least some of them) are domesticated by X. tropicalis to make a contribution to the host and are thus conserved by purifying selection.

P-34 Inbreeding coefficient and genetic relationship of seven strains of Xenopus tropicalis inferred from genome wide genotyping of 54 microsatellite loci

*Ai Watanabe1, Takeshi Igawa1, Akihiko Kashiwagi1, Atsushi Suzuki1, Atsushi Kurabayashi1, Tamotsu Fujii2, Masayuki Sumida1

1Inst. for Amphibian Biol., Hiroshima Univ., Japan; 2Dept. Hlth. Sci. Fac. Hum. Cult. Sci., Pref. Univ. Hiroshima

Western clawed frog, Xenopus tropicalis is a small, fully aquatic frog, found in the West African rainforest belt. It is a diploid relative of another amphibian experimental X. laevis, a frog that has traditionally used in the field of developmental biology. Some of superior characteristics of X. tropicalis to X. laevis are smaller body size, shorter generation time, and smaller diploid genome (2n = 20), together with whole-genome sequence data.

At Institute for Amphibian Biology, Hiroshima University (IABHU), multiple strains of X. tropicalis has been exploited, proliferated, maintained, and supplied to researchers as the National BioResource Project since 2003. However, despite of these advantages, inbreeding coefficient and genetic relationships among multiple strains of X. tropicalis has not been fully understood because of short history as experimental animal. To clarify these uncertainties, we therefore investigated heterozygosities and genetic relationships of multiple lines maintained in IABHU and the other abroad institutes by genotyping multiple microsatellites that locate widely in the genome and sequencing a mitochondrial gene.

We conducted genome-wide genotyping of 54 microsatellite loci for every 10 individuals of four inbreeding lines from IABHU (Nigerian A, Nigerian H, Golden, Ivory Coast) and three non-inbreeding lines called as ‘Nigerian’ provided from the other institutes. We also sequenced 2047 bp of D-loop region of mitochondrial genome for every single individual of these lines.

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As the result of genotyping, expected heterozygosities (HE)of the four inbreeding lines from IABHU were less than the other three Nigerian lines from abroad institutes, ranging 0.086 – 0.284 and 0.456 – 0.542, respectively. Of these IABHU lines, Nigerian H line was the lowest in HE (0.086), which followed by Golden line (0.140). Comparison of nucleotide sequence of D-loop region of mt-genome highlighted genetic divergence between Ivory Coast and Nigerian (including Golden) lines with 4.29 % of nucleotide substitution, while no substitution were observed within all Nigerian lines. Phylogenetic trees based on FST, DA distance, and proportion of shared alleles consistently showed clear divergence between Ivory Coast and Nigerian lines. Our trees also showed fine genetic relationships within Nigerian lines. These findings should be useful information for further genetic studies on X. tropicalis.

P-35 International Xenopus Resource Network and Educational Activities at Institute for Amphibian Biology *Atsushi Suzuki, Akihiko Kashiwagi, Keioko Kashiwagi, Hideki Hanada, Kimiko Takebayashi-Suzuki, Ichiro Tazawa, Nobuaki Furuno, Atsushi Kurabayashi, Satomi Kobayashi, Junko Takenaka, Yuuna Tamaki, Takeshi Igawa, Takeshi Uto, Chiyo Nanba, Ai Watanabe, Hitoshi Yoshida, Aki Shimada, and Masayuki Sumida Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashihiroshima, JAPAN

African clawed frogs (Xenopus laevis and tropicalis) have been essential for biomedical researches and educational resources. Institute for Amphibian Biology at Hiroshima University plays important roles to make easily available genetic and live frog resources to the international Xenopus research community. We are currently working closely with Xenopus recourses centers in the US and UK by organizing an international Xenopus resource network. We are not only providing a variety of resources but also technical assistance for researchers through workshops and a web-based discussion forum in the National Bio-Resource Project (NBRP). In addition, we also provide educational hands-on training opportunities and a series of lectures for high school students/teachers and for students in domestic as well as foreign universities. In collaboration with the Xenopus laevis genome consortium of Japan and European Xenopus Resource Centre in the UK, we will distribute Xenopus laevis genomic library clones to researches around the world. Our effort will strongly enhance the advancement of amphibian research and highlight Xenopus as an essential model organism.

P-36 National BioResource Project (NBRP) Xenopus (Silurana) tropicalis: Standard High Quality Inbred Strains for Biological Research *Akihiko Kashiwagi1, Keiko Kashiwagi1, Hideki Hanada1, Atsushi Suzuki1, Kimiko Takebayashi1, Atsushi Kurabayashi1, Ken-ichi Suzuki2, Nobuaki Furuno1, Ichiro Tazawa1, Keisuke Nakajuma1, Takashi Yamamoto2 and Masayuki Sumida1

1Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima, Japan, 2Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima, Japan

Since the Age of Enlightenment in 17th- and 18th-century Europe, amphibians have long been important experimental animals because of a variety of their compelling characteristics. In 1771, Luigi Galvani caused muscular contraction in dead frog legs by applying electricity,

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and termed this vital force in muscle tissue “animal electricity”. Wilhelm Roux (1888) began his study of experimental embryology using frog eggs. Since then investigations using amphibians have contributed to the progress of developmental biology, cell biology and other life sciences. In 1935, Hans Spemann was awarded the Nobel Prize in Physiology or Medicine for his work on the organizer effect in embryonic newt development.

The African clawed frog Xenopus leavis, which is native to Sub-Saharan Africa, has been used in many biomedical investigations for more than half a century. John B. Gurdon won the 2012 Nobel Prize in Physiology or Medicine for his discovery that mature X. laevis cells can be reprogrammed and become pluripotent. On the other hand, the closely related species Xenopus (Silurana) tropicalis, distributed in the lowland rain forest of West Africa from Senegal to Nigeria, was adopted for research in the early 1980s. This species should prove to be very important as an experimental animal in the postgenomic era for the following reasons: (1) X. tropicalis greatly simplifies genetic studies and easily establishes stable transgenic and knockout lines because of its small diploid genome, as well as its rapid development and relatively short generation time. A targeted gene mutagenesis technology may be employed utilizing engineered transcription activator-like effector nucleases (TALENS) in order to produce knockout frogs. In contrast to this, X. laevis has several shortcomings, including a larger allotetraploid genome, and a relatively long lifecycle. (2) The X. tropicalis’ genome sequence was determined in 2010, showing a high correspondence with mammalian genomic sequences.

Hiroshima University Institute for Amphibian Biology has been designated as the sole supplier for X. tropicalis to researchers for the five years from 2012 to 2016 under the sponsorship of the Ministry of Education, Culture, Sports, Science and Technology.

Discrepancies in experimental results using a variety of different frogs is probably due to different genetic backgrounds when environmental conditions are held constant. It is necessary therefore to use high quality inbred strains for obtaining accurate and consistent information in biological studies. Gynogenetic reproduction is very effective in producing inbred strains in amphibians. We would like to produce gynogenetic diploid X. tropicalis within a few years. We are also planning to develop a method for cryopreserving germ cells in order to avoid having to house a large number of males and females.

A metamorphosis assay using X. tropicalis is a valuable method for examining chemically induced thyroid hormone dysfunction. Using this method, BPA and UV filters were found to suppress T3-induced and spontaneous metamorphosis.

The purpose of the present poster presentation is to introduce the merits and usefulness of X. tropicalis as material in biological experiments.

P-37 Application of CRISPR/Cas system to Xenopus laevis *Minoru Watanabe Institute of Socio-Arts and Sciences, The University of Tokushima, Japan

CRISPR/Cas is an artificial nuclease that makes it possible to edit genomes of organisms. This system has several advantages over the other artificial nuclease systems: zinc-finger nucleases (ZENs) and transcription activator-like effector nucleases (TALENs). While ZENs and TALENs use proteins to recognize target DNA sequences, CRISPR/Cas system uses RNAs (sgRNAs) to recognize them, which makes construction of sgRNA expression vector quite easy. Off-target effects of CRISPR/Cas system have been reported hard to occur. In near future, this system would become the primary choice to edit genomes of many organisms.

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Xenopus laevis has been one of the favorite animals in variety of research fields. Genetic approaches, however, have not been as applied as other model organisms because of its long reproduction span and allo-tetoraploid nature. Recently, targeted mutagenesis of tyrosinase (tyr) gene of Xenopus laevis by TALEN has been reported, showing the possibility of genome manipulation of this animal. Here, we report the application of CRISPR/Cas system to Xenopus laevis. When we injected Cas9 mRNA together with sgRNA against tyr gene into embryos, the embryos developed with mosaic pigmentation pattern in retinal pigment epitheliums and melanophores. Mutations of tyr locus were confirmed by Cel-I assay and sequencing. This result shows that CRISPR/Cas system would be applicable to Xenopus laeivs and extends the possibility of Xenopus genetic researches.

P-38 Sex reversibility in amphibians and its applications: sex-hormone-inducible sex reversal in Rana rugosa, sex determination in Silurana tropicalis, and model amphibians as environmental indicators

*Minoru Takase Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Japan

Both the continuity of the life since appearances of organisms more than three billion years ago and the diversity of living organisms are considered to depend primarily on characteristics of germ cells. There are four major biological events regarding germ cells during life cycles of amphibians as follows: (1) appearances of germ cells during the pre-hatch embryo life, (2) sex differentiation of germ cells during the tadpole life, (3) production of mature germ cells through spermatogenesis and oogenesis, during the adult life, and (4) functions of germ cells to produce totipotent fertilized eggs for making the next generation. With these backgrounds, I have investigated differentiation, development and functions of germ cells using amphibians. In this symposium, I will present studies on sex-hormone-inducible sex reversal. Many studies have supplied many pieces of evidence that sex steroid hormones induce sex reversal in many amphibians, where androgens induce generally female-to-male sex reversal and estrogens generally male-to-female sex reversal. Sex steroid hormone induces one direction of sex reversal, the male-to-female type or the female-to-male type, in most of amphibians. But, both directions of sex reversal are induced in the Hiroshima population of Rana rugosa by sex steroid hormones. The sex-hormone-inducible sex reversal is very useful phenomenon for investigating sex differentiation of germ cells among four major biological events described above. I have investigated so far effects of sex hormones on gonad differentiation and gene expressions including sex hormone receptor genes during sex reversal, using Rana rugosa, and expect in future to be able to identify triggers to differentiate primordial germ cells into male-type germ cells, such as spermatogonia, or female-type germ cells, such as oogonia. Sex reversibility in amphibians could be applied to identification of sex-determination systems of amphibians and development of environmental indicators. Furthermore, I will present in this symposium studies on a possible sex determination system in Silurana tropicalis, a ZZ/ZW type or a XX/XY type, and impacts of environmental chemicals on gonad differentiation, such as formation of testis-ova, in R. rugosa and Pelophylax nigromaculata.

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P-39 Bipolar spindle formation during meiosis I required the proper localization cyclin B2 in Xenopus oocytes Yoshitome, S1., Prigent, C2., Hashimoto, E3. and *Furuno, N.4

1Iwaki Meisei Univ., 2Univ. Rennes 1, 3Tottori Univ., 4Hiroshima Uiv.

A Maturation/M-phase promoting factor (MPF), which consists of cyclin B and Cdc2 kinase, regulates multiple aspects of M-phase (nuclear envelop breakdown, chromosome condensation and spindle formation). Cyclin B is a regulatory subunit of MPF and B type cyclin has several subtypes in many species. Cyclins B1 and B2 are subtypes of cyclin B, and they are also highly conserved in many vertebrate species. Cyclin B1 is essential for mitosis, whereas cyclin B2 is regarded as dispensable. However, the overexpression of the cyclin B2 N-terminal fragment containing the cytoplasmic retention signal (CRS), but not cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes and embryos.

Here we show that endogenous cyclin B2 was localized in and around the germinal vesicle. The perinuclear localization of cyclin B2 was perturbed by the overexpression of its N-terminal fragment with the CRS domain, which resulted in a spindle defect. This spindle defect was rescued by the overexpression of bipolar kinesin Eg5, which is located at the perinuclear region in the proximity of endogenous cyclin B2. Moreover, the overexpression of either murine or zebrafish cyclin B2 N-terminal fragments with their CRS domains, but not their cyclin B1's, affected spindle formation in Xenopus oocytes. These results suggest that the proper localization of cyclin B2 is essential for bipolar spindle formation in Xenopus oocytes, and it is possible that vertebrate cyclin B2 has a common role for spindle formation.

P-40 Effects of Spermine on Neural Morphogenesis in Xenopus laevis Tailbud Embryos *Hitoshi Yoshida1, Takeshi Kondo1, Kimiko Takebayashi-Suzuki2, Atsushi Suzuki2, and Koichiro Shiokawa1

1Sch. of Science and Engineering., Teikyo Univ., Japan; 2Inst. for Amphibian Biol., Hiroshima Univ., Japan

Spermine is the most largest molecule among major polyamines in eukaryotic cells and has function that up-regulates and down-regulates N-methyl-D-aspertate receptor (NMDAR) in neural cells depending on the dosage. In Xenopus embryogenesis, levels of spermine and mRNA for S-adenosyl-l-methyonine decarboxylase (SAMDC) and its enzyme activities are respectively low. However, SAMDC mRNA increase at 20 hours after fertilization, and its enzyme activities is elevated at 40 hours after fertilization. Although we previously found that spermine bend the body axis in Xenopus tadpoles, the mechanism of this effect has not been clarified and little is known about the function of spermine in Xenopus tadpoles. We examined here the effect of spermine on Xenopus embryos. Tailbud stage embryos cultured in 10 % Steinberg’s solution from Nieuwkoop-Faber (NF) stage 2 to NF stage 42 which contained 1 mM Spermine induced abnormal axial structure. In head part of these embryos brain atrophy was observed, but there was little change in the spinal cord. When Xenopus tadpoles was treated with spermine, NCAM expression in the brain that is essential for neural development in Xenopus laevis embryos became lower as compared with untreated embryos. These results suggest that spermine inhibits elongation of axis, and inhibits also morphogenesis of neural tissue, especially in the brain of Xenopus tadpoles.

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P-41 Functional analyses of a novel Insulin-like factor

Yoshikazu Haramoto1, *Shuji Takahashi2, Yasuko Onuma1, Yuzuru Ito1, Makoto Asashima1

1.Research Center for Stem Cell Engineering (SCRC), National Institute of Advanced Industrial Science and Technology (AIST), Japan. 2. Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Japan

Insulin family members have significant roles as bioactive substances. Insulin controls blood glucose levels, and IGF influences growth, differentiation, and survival of cells. In Xenopus, IGF signaling is known to be required for anterior neural development. We cloned a novel insulin-like factor (insl), which is expressed from gastrula to tailbud stage. The amino acid sequence of Insl is the most similar to insulin, but Insl has no proteolytic cleavage site, which is necessary to release C peptide. The protein structure of Insl corresponds to pro-insulin or mature IGF. Over-expression of insl mRNA enlarged anterior structure and, depletion of Insl by antisense MO causes morphological defects in head development and reduces the expression of anterior neural genes. These results showed that Insl is required for anterior neural development. We demonstrate here that Insl modulates components of Wnt signals and inhibit its signalling via a unique mechanism.

P-42 Establishment of vertebrate body plan via coordinated regulation of dorsal-ventral and anterior-posterior patterning during early Xenopus embryogenesis

*Kimiko Takebayashi-Suzuki, Hidenori Konishi and Atsushi Suzuki Inst. for Amphibian Biol., Hiroshima Univ. Grad. School of Sci., Japan

The formation of the dorsal–ventral (DV) and anterior–posterior (AP) axes, fundamental to the body plan of animals, is regulated by several regulatory pathways including the TGF-β, FGF, and Wnt families. In order to ensure the establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated during early development. However, the molecular mechanisms responsible for these interactions remain unclear. Here, we report the molecular mechanisms of neural induction and patterning regulated by Biz, a novel candidate protein involved in the coordinative regulation of the DV and AP body axes. Overexpression of Biz resulted in inhibition of BMP signaling and activation of Wnt signaling, thereby promoted neural induction and posterior neural development in isolated ectodermal explants. Knockdown of Biz resulted in repression of neural gene expression. Interestingly, we found that Biz functionally interacted with forkhead transcription factor FoxB1, another important factor in the coordinative regulation of DV and AP patterning. Our results suggest that Biz plays essential roles in the establishment of the body plan by modulating BMP and Wnt signaling during early development.

P-43 Induction of homeotic limbs by retinoid treatment on the amputated tails of Japanese anurans *Ichiro Tazawa and Yoshio Yaoita Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Japan

Two decades ago, the first homeotic transformation of the vertebrates was reported. After tail-amputated Indian anuran tadpoles were treated with retinyl palmitate, hindlimb-like

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organs appeared on the amputation site instead of tail regenerates. This phenomenon has been expected to be a key to understand the pattern formation of the vertebrates. It has been reproduced so far only by using some species of anurans in India and Europe, but not by using popular experimental model animals such as Xenopus laevis and Rana catesbeiana (Lithobates catesbeianus). This situation would make it difficult for researchers to analyze this phenomenon especially in the areas where such species are not available. It would be a reason why molecular analyses of it have not been performed. Therefore, we tried to induce the homeotic limbs using X. laevis and several species of Japanese Rana. The Xenopus tadpoles did not generated ectopic limbs, whereas we successfully induced the homeotic transformation in the Rana species including R. japonica and R. ornativentris, the common frogs in Japan. The ectopic limbs expressed adult type genes of myosin and epidermal keratin. In these limbs, tbx4, a hindlimb marker gene, was expressed much more strongly than tbx5, a forelimb marker gene. These expression patterns showed that the induced ectopic limbs expressed the same genes that hindlimbs do.

P-44 Translucent frogs created through crossbreeding, and their inheritance and dermal chromatophore structure Masayuki Sumida1, Mohammed Mafizul Islam1, Takeshi Igawa1, Atsushi Kurabayashi1, Yukari Furukawa2, Naomi Sano2, Tamotsu Fujii2, Norio Yoshizaki3, and *Midori Nishioka1

1Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashihiroshima, Japan, 2Faculty of Human Culture & Science, Hiroshima Prefectural University, Hiroshima, Japan, 3Faculty of Applied Biological Science, Gifu University, Yanagido, Gifu, Japan

The skin of frogs is generally covered with dermal chromatophore units consisting of xanthophores, iridophores, and melanophores, so the internal organs cannot be seen through the skin. Although some small fish are translucent, no translucent tetrapods have been reported. We have succeeded for the first time in producing translucent frogs from two natural color mutants of the Japanese brown frog Rana japonica, whose dorsal surface is usually ochre or brown, enabling their organs, blood vessels, and eggs to be observed through the skin without performing dissection. Two kinds of recessive color mutants (black-eyed and gray-eyed) in this species are known to lack the normal chromatophores (iridophores and melanophores, respectively), which causes the frogs to be pale or albino. In order to produce translucent frogs from these two color mutants and to clarify the mode of inheritance of the translucent frogs, we crossed the two color mutant frogs through artificial insemination, and the F2 offspring led to frogs whose skin is translucent throughout their life. Crossing gray-eyed females with black-eyed males produced F1 offspring that were all wild-type with two heterozygous recessive color mutant loci. Crossing wild-type F1 females with wild-type F1 males produced F2 offspring that were wild-type (53.4%), gray-eyed (21.4%), black-eyed (19.3%), and translucent (5.9%). Crossing gray-eyed F2 females and wild-type F1 males produced F3 offspring that were wild-type (40.6%), gray-eyed (38.7%), black-eyed (11.2%), and translucent (9.5%). Crossing translucent F2 females and translucent F2 males produced F3 offspring that were all translucent, but the survival rate was very low. From the abovementioned results, it was found that crossing gray-eyed F2 females with wild-type F1 males was the most effective means of producing translucent frogs. We also observed the dermal chromatophores in the skin of wild-type, black-eyed, gray-eyed, and translucent frogs under an electron microscope to examine the dermal microstructure of these frogs. As a result, three kinds of dermal chromatophores—xanthophores, iridophores, and melanophores—were observed in a dermal layer of the wild-type frogs. The skin of gray-eyed frogs contained

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xanthophores and iridophores but no melanophores, and many abnormal round reflecting platelets were observed in the iridophores. The skin of black-eyed frogs contained xanthophores and melanophores but no iridophores, and immature pterinosomes or carotenoid vesicles were observed in the xanthophores. In the skin of the translucent frogs, the number of chromatophores was low, and immature xanthophore-like cells containing melanosome-like structures were observed.

P-45 The effect of pituitary hormone and ovaprim in frog ovulation and the difference of food effect to Fejervarya cancrivora tadpoles growth

Ardyah Ramadhina I.P1., Agung Pramana W.M.1, *Nia Kurniawan1

1Biology Department, Mathematics and Natural Sciences Faculty, Brawijaya University, Malang

Fejervarya cancrivora is an amphibian which its habitat is strongly influenced by the conditions of the habitat and the change of human activities. One of the efforts to maintain the biodiversity is using artificial reproduction technologies by pituitary hormones and ovaprim injection to induce gonadal maturation. The purpose of this study was to determine the effect of pituitary hormones (Hip) and ovaprim (Ova) on gonadal maturation and fertilization success in Fejervarya cancrivora, and to understand the effect of different feeding on the growth of tadpoles. Injections were performed on early mature gonads female frogs with a total dose of 250 µl, using intraperitonial injection. After spawned and fertilized, the eggs reared until hatched and given with two different feed treatments such as, feed boiled spinach (P1) and boiled spinach with boiled egg yolk (P2). Results showed the treatment pituitary can lying 632 eggs cells (100%) and only 65.67% hatched, treatment ovaprim lying 108 egg cells (100%) and only 75.92% of eggs can hatched. The results of P1 and P2 treatment of pituitary and ovaprim injection that there were no length difference (p>0.05) but it has weight difference (p<0.05) during its metamorphosis. Percentage of tadpoles that successfully metamorphosed by pituitary treatment is 41.12% and ovaprim treatment is 31.67 %.

P-46 Ghrelin stimulates food intake in bullfrog larvae Sunsuke Shimizu1, Hiroyuki Kaiya2, *Kouhei Matsuda1

1Lab. of Regul. Biol., Grad. Sch. of Sci. and Eng., Univ. of Toyama; 2Dept. of Biochem., National Cerebral and Cardiovascular Center Res. Inst., Suita, Japan

Ghrelin is a potent orexigenic peptide implicated in appetite regulation in rodents. However, except for teleost fish, the involvement of ghrelin in the regulation of feeding in non-mammalian vertebrates has not been well studied. Anuran amphibian larvae feed and grow during the pre- and prometamorphic stages, but, thereafter they stop feeding as the metamorphic climax approaches. Therefore, orexigenic factors seem to play important roles in growing larvae. In the present study, we examined the effect of intraperitoneal (IP) or intracerebroventricular (ICV) administration of synthetic bullfrog ghrelin (n-octanoylated 28-amino acid form) on food intake in larvae at the prometamorphic stages. Cumulative food intake was significantly increased by IP (8 and 16 pmol/g body weight (BW)) or ICV (0.5 and 1 pmol/g BW) administration of ghrelin during a 15-min observation period. The orexigenic action of ghrelin at 8 pmol/g BW (IP) or at 0.5 pmol/g BW (ICV) was blocked by treatment with a growth hormone secretagogue-receptor antagonist, [D-Lys3]GHRP-6 at 80 pmol/g BW (IP) or at 5 pmol/g BW (ICV). We then investigated the effect of feeding status on expression

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levels of the ghrelin transcript in the hypothalamus and gastrointestinal tract. Ghrelin mRNA levels in both were decreased 15 and 60 min after feeding. These results indicate that ghrelin acts as an orexigenic factor in bullfrog larvae.

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List of Participants ◯ indicates expected attendants at the social gathering. Presentation No. follows speaker’s name.

I: Invited Lecture; A: Lecture by IABHU Member; P: Poster Presentation.

◯ Alam, Md. Shafiqul P-01 (Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh)

◯ Amaya, Enrique I-01 (University of Manchester, UK)

◯ de Guzman, Christmas P-09 (University of the Philippines, Philippines)

◯ Eto, Koshiro P-16 (Kyoto University, Japan)

◯ Flechas, Sandra Victoria P-02 (Universidad de los Andes, Colombia)

◯ Furuno, Nobuaki P-39 (Hiroshima University, Japan)

◯ Goka, Koichi I-10 (National Institute for Environmental Studies, Japan)

◯ Hasan, Mahmudul P-17 (Hiroshima University, Japan)

◯ Hikosaka, Akira P-33 (Hiroshima University, Japan)

◯ Igawa, Takeshi A-03 (Hiroshima University, Japan)

◯ Islam, Mohammed Mafizul A-02 (Hiroshima University, Japan)

◯ Islam, Md. Rakeb-Ul P-12 (Noakhali Science and Technology University, Bangladesh)

◯ Itoh, Makoto P-25 (Kyoto University, Japan)

◯ Jehle, Robert I-09 (University of Salford, UK)

◯ Kakehashi, Ryosuke P-21 (Hiroshima University, Japan)

◯ Kashiwagi, Akihiko P-36 (Hiroshima University, Japan) Kashiwagi, Keiko (Hiroshima University, Japan)

◯ Kobayashi, Soh P-08 (Central Research Institute of Electric Power Industry, Japan)

◯ Komaki, Shohei P-20 (Hiroshima University, Japan)

◯ Kurabayashi, Atsushi P-32 (Hiroshima University, Japan)

Kuramoto, Mitsuru P-19 (Japan)

◯ Kurniawan, Nia P-45 (Brawijaya University, Indonesia) Kuwabara, Kazushi P-05 (The Japanese Giant Salamander Meeting of Higashi-Hiroshima, Japan)

◯ Maejima, Sho P-31 (Hiroshima University, Japan)

◯ Malone, John H. P-30 (University of Connecticut, USA)

◯ Marantelli, Erika P-04 (Amphibian Research Centre, Melbourne, Australia)

◯ Marantelli, Gerry P-03 (Amphibian Research Centre, Melbourne, Australia)

◯ Matsuda, Kouhei P-46 (University of Toyama, Japan)

◯ Matsui, Masafumi I-07 (Kyoto University, Japan)

◯ Matsunami, Masatoshi P-26 (Hokkaido University, Japan)

Midori, Takuma P-22 (Rakuno Gakuen Univirsity, Japan) ◯ Minamoto, Toshifumi P-10 (Kobe University, Japan)

◯ Miura, Ikuo P-29 (Hiroshima University, Japan)

◯ Nakajima, Keisuke A-01 (Hiroshima University, Japan)

◯ Nakayama, Takuya I-03 (University of Virginia, USA)

Nasrin, Sultana P-18 (Hiroshima University, Japan) Nishioka, Midori P-44 (Hiroshima University, Japan)

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Ogata, Mitsuaki P-14 (Preservation and Res. Cent., The City of Yokohama, Japan)

◯ Ogino, Hajime I-04 (Nagahama Institute of Bio-Science and Technology, Japan)

◯ Okawa, Hiroshi P-15 (Hiroshima-Kokusai University, Japan)

◯ Sato, Naoki P-23 (Kaiyo-high school and Miyokoukogen Visitor center, Japan)

◯ Shinozuka, Masaru (Japan)

◯ Smith-Parker, Heidi K. P-07 (University of Texas-Austin, USA)

Solano, Liliana P-06 (University of Manchester, Colombia) ◯ Stanković, David P-11 (University of Ljubljana, Slovenia)

◯ Sumida, Masayuki A-04 (Hiroshima University, Japan)

◯ Suzuki, Atsushi P-35 (Hiroshima University, Japan)

◯ Taira, Masanori I-05 (University of Tokyo, Japan)

◯ Takahara, Teruhiko P-24 (Hiroshima University, Japan)

◯ Takahashi, Shuji P-41 (Hiroshima University, Japan) Takase, Minoru P-38 (Hiroshima University, Japan)

◯ Takebayashi-Suzuki, Kimiko P-42 (Hiroshima University, Japan)

◯ Tazawa, Ichiro P-43 (Hiroshima University, Japan) Tjong, Djong Hon P-13 (Andalas University, Indonesia)

◯ Uno, Yoshinobu P-27 (Nagoya University, Japan)

◯ Vences, Miguel I-06 (Technical University of Braunschweig, Germany)

◯ Versteegen, Stephanie P-04 (Amphibian Research Centre, Melbourne, Australia)

◯ Watanabe, Ai P-34 (Hiroshima University, Japan)

◯ Watanabe, Minoru P-37 (The University of Tokushima, Japan) Yamashiki, Naoko (Rakuno Gakuen University, Japan)

◯ Yaoita, Yoshio (Hiroshima University, Japan)

Yoshida, Hitoshi P-40 (Teikyo University, Japan) ◯ Yoshikawa, Natsuhiko P-28 (Kyoto University, Japan)

◯ Yoshimura, Masako (Japan)

◯ Zamudio, Kelly I-08 (Cornell University, USA)

◯ Zhao, Hui I-02 (The Chinese University of Hong Kong, China)

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Access

To the Higashi Hiroshima Campus From Hiroshima Airport Please take the Geiyo Bus to Shiraichi Station and transfer to the train bound for Saijo, Hiroshima and Iwakuni and get off at Saijo Station. It takes approximately 15 minutes to Shiraichi Station by bus and a further 10 minutes to Saijo Station by train.

From Higashi Hiroshima Station There is a very limited bus service to the Higashi Hiroshima Campus from Higashi Hiroshima Station. We recommend that you take a taxi to the Higashi Hiroshima Campus or organize for someone to pick you up. If there is no taxi waiting at Higashi Hiroshima Station, please call Saijo Taxi on 082-423-2525 or Higashi Hiroshima Taxi on 082-423-2121.

From Saijo Station There are two bus companies that provide a bus service between Saijo Station and the Higashi Hiroshima Campus. Please refer to the Geiyo Bus website1 for an updated timetable (in Japanese). Buses depart from bus stop 3 in front of Saijo Station. It will take approximately 15 minutes. Please refer to the campus map2 to determine which bus stop to get off at when you reach the Higashi Hiroshima Campus.

From other international airports than Hiroshima Airport Please access to the guiding web page3 of Hiroshima University for details. 1http://www.geiyo.co.jp/Unyu/daigakuH25-3.htm 2http://www.hiroshima-u.ac.jp/add_html/access/en/saijyo1.html 3http://www.hiroshima-u.ac.jp/en/top/access/

To the Higashi Hiroshima Campus

From Hiroshima AirportPlease take the Geiyo Bus to Shiraichi Station and transfer to the train bound for Saijo, Hiroshima and Iwakuni and get off at Saijo Station. It takes approximately 15 minutes to Shiraichi Station by bus and a further 10 minutes to Saijo Station by train.

From Higashi Hiroshima StationThere is a very limited bus service to the Higashi Hiroshima Campus from Higashi Hiroshima Station. We recommend that you take a taxi to the Higashi Hiroshima Campus or organize for someone to pick you up. If there is no taxi waiting at Higashi Hiroshima Station, please call Saijo Taxi on 082-423-2525 or Higashi Hiroshima Taxi on 082-423-2121.

From Saijo StationThere are two bus companies that provide a bus service between Saijo Station and the Higashi Hiroshima Campus. Please refer to the Geiyo Bus website for an updated timetable (in Japanese). Buses depart from bus stop 3 in front of Saijo Station. It will take approximately 15 minutes. Please refer to the campus map to determine which bus stop to get off at when you reach the Higashi Hiroshima Campus.

From other international airports than Hiroshima AirportPlease access to the guiding web page of Hiroshima University (URL: http://www.hiroshima-u.ac.jp/en/top/access/) for details.

Japan

Access

To the Higashi Hiroshima Campus

From Hiroshima AirportPlease take the Geiyo Bus to Shiraichi Station and transfer to the train bound for Saijo, Hiroshima and Iwakuni and get off at Saijo Station. It takes approximately 15 minutes to Shiraichi Station by bus and a further 10 minutes to Saijo Station by train.

From Higashi Hiroshima StationThere is a very limited bus service to the Higashi Hiroshima Campus from Higashi Hiroshima Station. We recommend that you take a taxi to the Higashi Hiroshima Campus or organize for someone to pick you up. If there is no taxi waiting at Higashi Hiroshima Station, please call Saijo Taxi on 082-423-2525 or Higashi Hiroshima Taxi on 082-423-2121.

From Saijo StationThere are two bus companies that provide a bus service between Saijo Station and the Higashi Hiroshima Campus. Please refer to the Geiyo Bus website for an updated timetable (in Japanese). Buses depart from bus stop 3 in front of Saijo Station. It will take approximately 15 minutes. Please refer to the campus map to determine which bus stop to get off at when you reach the Higashi Hiroshima Campus.

From other international airports than Hiroshima AirportPlease access to the guiding web page of Hiroshima University (URL: http://www.hiroshima-u.ac.jp/en/top/access/) for details.

Japan

Access

Hiroshima Prefecture