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25 August 2021 The First Survey for Helminths Parasitic in Hybrid and Introduced Giant Salamanders, Genus Andrias (Amphibia: Caudata: Cryptobranchidae) in Kyoto, Japan
Karin Tsuchida, Misako Urabe, Kanto Nishikawa
Author Affiliations +
Abstract

The first survey was conducted for helminth fauna of hybrid giant salamanders (hybrids between Andrias japonicus and other congeneric species), and introduced A. davidianus in Kyoto Prefecture, Japan. Three nematode species, Spiroxys hanzaki, Amphibiocapillaria tritonispunctati and Falcaustra sp., and one trematode species, Liolope copulans, were recovered from their alimentary canals. These results show that hybrid and introduced Andrias species are commonly infected with similar helminth species to those previously reported to infect A. japonicus. We conclude that the spillback of native parasites to introduced A. davidianus has occurred in Kyoto Prefecture. This study is also the first record of Falcaustra species parasitizing Andrias species in Japan.

Introduction

The Japanese giant salamander, Andrias japonicus (Temminck, 1836) (Amphibia: Cryptobranchidae), is endemic to the western and central Japanese Archipelago and listed as both species of special natural monument in Japan and a Near Threatened species on the IUCN Red List (Kaneko and Matsui, 2004; Yoshikawa et al., 2012; Matsui, 2014).

A few surveys have been conducted for the helminths parasitizing A. japonicus. In the early 20th century, Liolope copulans Cohn, 1902 (Trematoda: Liolopidae) and Filaria cingula Linstow, 1902 (Nematoda: Micropleuridae) (now Kamegainema cingulum (Linstow, 1902): Hasegawa et al., 2000) were first found from A. japonicus transported to Europe (Cohn, 1902; Linstow, 1902). Decades later, Yamaguti (1936, 1939, 1941) reported the following species from A. japonicus in Kyoto City, Japan: Diplodiscus japonicus (Yamaguti, 1936) (Trematoda: Diplodiscidae), Pseudoacanthocephalus lucidus (Van Cleave, 1925) (Acanthocephala: Echinorhynchidae) and Megalobatrachonema nipponicum Yamaguti, 1941 (Nematoda: Kathlaniidae). Hasegawa et al. (1998) described Spiroxys hanzaki Hasegawa, Miyata & Doi, 1998 (Nematoda: Gnathostomatidae) recovered from A. japonicus in Hyogo Prefecture, Japan. Hasegawa et al. (2000, 2002) also reported the parasite fauna of A. japonicus in Osaka and Hyogo Prefectures, Japan, as follows: L. copulans, S. hanzaki, K. cingulum, Amphibiocapillaria tritonispunctati (Diesing, 1851) (Nematoda: Trichuridae), Dioctophyme renale (Goeze, 1782) (Nematoda: Dioctophymatidae) and Kathlaniidae gen. sp. (Nematoda). In addition, Physalopteroidea gen. sp. (Nematoda) was recovered but was considered as a pseudoparasite that was accidentally acquired through ingesting parasitized fish (Hasegawa et al., 2002). Tanaka et al. (2016) documented similar parasite species to Hasegawa et al. (2002) in zoo-bred A. japonicus in Hiroshima Prefecture, Japan.

The genetic introgression of Chinese Andrias species into the native population of A. japonicus has been serious in Japan, particularly in Kyoto Prefecture (Yoshikawa, 2011). A recent molecular study concluded that several Andrias species occur in China, including A. davidianus (Blanchard, 1871) and A. sligoi (Boulenger, 1924) (Turvey et al., 2019). These species were introduced to Japan in 1970s, leading to the ongoing hybridization with A. japonicus (Fukumoto et al., 2015).

To fully evaluate the impacts of alien species, it is essential to examine whether parasites are also introduced with novel vertebrate species (e.g., Dunn et al., 2012). Introduced species can increase parasite transmission via spillover or spillback. Spillover occurs when a reservoir host species that was introduced transmits novel parasites to a native species (Hatcher et al., 2012). Alternatively, an introduced species can become a new reservoir for native parasite infection, which can increase infection in native hosts through spillback (Hatcher et al., 2012). In Kyoto Prefecture, the current parasite fauna on Andrias is unclear because parasitological surveys have not been conducted in the almost 80 years since Yamaguti (1936, 1939, 1941). In this study, we documented the current parasite fauna of Andrias, especially of introduced and hybrid individuals, the latter of which is now dominant in the rivers of Kyoto Prefecture. Based on the results, we discuss whether the introduction of Chinese Andrias species affected the parasite fauna of A. japonicus via spillover or spillback.

Materials and Methods

A total of 27 Andrias were euthanized by the injection or immersion in 2-phenoxyethanol (Fig. 1, Tables 1 and 2). All dissections were approved by the Culture Bureau of Kyoto City. Because each Andrias species is difficult to identify by morphology, all collected salamanders were analyzed genetically (Yoshikawa et al., 2012). As a result, we identified 25 “hybrids” between A. japonicus and Chinese Andrias species (species not identified) and two A. davidianus, which is redefined by Turvey et al. (2019) (Nishikawa, unpublished). Parasites were collected from the alimentary canal, liver, lungs and skin of each salamander. All Andrias specimens used for this study were deposited to the Graduate School of Human and Environmental Studies, Kyoto University (KUHE; see Appendix).

Collected nematodes were fixed in 70% ethanol, cleared in undiluted glycerin or mounted in glycerin-gelatin. Some of collected trematodes were fixed in 90% ethanol, and the other were pressed between a cover-slip and glass slide, fixed in alcohol-formolacetic fixative, mordanted in 4% ammonium iron (III) sulfate solution, stained with Heidenhain's iron hematoxylin, differentiated in 4% ammonium iron (III) sulfate solution, dehydrated in 95 and 100% ethanol series, cleared in creosote, replaced in xylene and mounted in Canada balsam. These specimens were observed using a light microscope for morphological study. Liolope copulans, Spiroxys hanzaki and Amphibiocapillaria tritonispunctati were identified based on morphological description in Baba et al. (2011), Hasegawa et al. (1998) and Moravec (1982, 1986), respectively. All measurements are given in micrometre (µm) unless otherwise stated, as range followed by mean±standard deviation in parentheses. All specimens studied were deposited in the Zoological Collection of Kyoto University (catalog no. KUZ Z3908–Z3912).

Fig. 1.

Map of sampling sites for Andrias surveyed for internal parasites in Kyoto City (1, Kamo River; 2, Kurama River; 3, Shizuhara River; 4, Myozin River; 5, Nakatsu River; 6, Katsura River; 7, Teratani River; 8, Takano River and 9, Kiyotaki River).

img-z3-1_109.jpg

Nematodes and trematodes fixed in 90% ethanol were used for genetic study. Genomic DNA was extracted from the specimens using Wizard® SV Genomic DNA Purification System (Promega Corp., Madison, WI). Polymerase chain reaction (PCR) was performed to amplify the internal transcript spacer (ITS) 1 region of S. hanzaki. The PCR was performed using 50 µl PCR reaction mixture containing 5 µl of 10×KOD-Plus-Neo Buffer, 5 µl of dNTPmix (2 mM), 3 µl of MgSO4 (25 mM), 1 µl of KOD-Plus-Neo (TOYOBO Co., Ltd., Osaka, Japan), 1.5 µl of forward primer SSU24HF (5′-AGAGGTGAAATTCG TGGACC-3′) (10 mM) and of reverse primer AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) (10 mM) (Li et al., 2014), and 33 µl of each template. The PCR process was conducted using 2720 Thermal Cycler (Applied Biosystems Inc., Waltham, MA), with thermocycling profile as follows; 30 s at 94°C, 40 cycles of 10 s at 94°C, 30 s at 50°C, 1 min at 72°C, and the final extension for 7 min at 72°C.

To amplify the partial 18S rDNA region of Am. tritonispunctati, PCR was performed in 20 µl PCR reaction mixture containing 13.8 µl of Milli-Q water (MQW), 2 µl of 10×Ex Taq Buffer, 1.6 µl of dNTP mixture, 0.1 µl of Ex Taq (Takara Bio Inc., Shiga, Japan), 1 µl of forward primer NSF4/18 (5′-CTGGTTGATCCTGCCAGT-3′) (10 mM) and of reverse primer SSU18R (5′-TGATCCT TCYGCAGGTTCAC-3′) (10 mM) (Tamaru et al., 2015), and 0.5 µl of each template. Thermocycling profile was as follows: 30 s at 94°C, 40 cycles of 10 s at 94°C, 30 s at 50°C, 1 min at 72°C, and the final extension for 7 min at 72°C.

To amplify the partial 28S rDNA region of Falcaustra sp., PCR was performed in 20 µl PCR reaction mixture containing 7.1 µl of MQW, 10 µl of 2×Gflex PCR Buffer, 0.4 µl of Tks Gflex DNA Polymerase (Takara Bio Inc.), 1 µl of forward primer 28S-F (5′-AGCG GAGGAAAAGAAACTAA-3′) (10 mM) and of reverse primer 28S-R (5′-ATCCGTGTTTC AAGACGGG-3′) (10 mM) (Nadler and Hudspeth, 1998), and 0.5 µl of each template. Thermocycling profile was as follows: 1 min at 94°C, 40 cycles of 10 s at 94°C, 15 s at 50°C, 1 min at 68°C, and the final extension for 7 min at 68°C.

PCR products were visualized on electrophoresis gels with 1 µl Midorigreen Direct (NIPPON Genetics Co., Ltd, Tokyo, Japan) and purified using the Wizard® SV Gel and PCR Clean-up System (Promega Corp.). Sequencing was outsourced to FASMAC Co., Ltd. (Kanagawa, Japan).

Table 1.

Summary for the examined hybrid Andrias and their parasites (TL: Total length shown by mm).

img-z4-2_109.gif

Table 2.

Summary for the examined Andrias davidianus and their parasites (TL: Total length shown by mm).

img-z4-4_109.gif

The quality of returned sequences was checked using the Applied Biosystems Sequence Scanner Software v2.0. All high-quality sequences were aligned using ClustalW implemented in MEGA 7 (Kumar et al., 2016). BLAST searches were performed in GenBank to compare obtained and registered sequences and identify sequences with the lowest E-values and highest similarities.

Results

Morphological study
Nematoda
Family Gnathostomatidae Railliet, 1895
Subfamily Spiroxyinae Baylis & Lane, 1920
Spiroxys hanzaki Hasegawa, Miyata & Doi, 1998

  • Description

  • Male (based on 10 adult specimens): body 13.9–30.0 (21.2±5) mm long and 0.4–0.6 (0.5±0.1) mm wide. Esophagus 2.9–6.6 (4.6±1) mm long and 133–280 (211±54) wide near posterior end. Nerve ring, excretory pore, deirids 534–947 (753±133), 634–1234 (889±176) and 1207–1367 (1309±61), respectively, from anterior extremity. Spicules 760–1234 (1005±124) long and 40–53 (47±5). Tail 227–334 (293±30) long. Female (based on 12 adult specimens): body 21.9–40.2 (29.9±5) mm long and 0.4–0.9 (0.7±0.1) mm wide. Esophagus 2.7–6.5 (4.8±1) mm long and 173–320 (235±44) wide near posterior end. Nerve ring, excretory pore, deirids 667–1121 (845±157), 867–1254 (1005±118) and 1301–1934 (1431±194), respectively, from anterior extremity. Vulva 12.7–24.2 (19.4±4) mm from anterior extremity. Eggs 76–88 (80±4) by 45–76 (58±6) (n=25). Tail 367–667 (519±93) long.

  • Taxonomic summary

  • Host: hybrid Andrias between A. japonicus (Temminck, 1836) and Chinese Andrias species.

  • Infection site: stomach.

  • Stage: adults and third stage larvae.

  • Locality: Kyoto City, Kyoto Prefecture, Japan: Kamo River (35°03′33″ N, 135°45′00″ E) (site 1, Fig. 1), Kurama River (35°06′23″ N, 135°45′52″ E) (site 2, Fig. 1), Katsura River (35°12′19″ N, 135°44′32″ E; 35°14′58″ N, 135°45′56″ E; 35°15′57″ N, 135°44′34″ E) (site 6, Fig. 1), Teratani River (35°13′58″ N, 135°47′35″ E) (site 7, Fig. 1), and Kiyotaki River (35°03′ N, 135°46′ E) (site 9, Fig. 1).

  • Studied specimens: KUZ Z3910.

  • Remarks: general morphology agreed with Hasegawa et al. (1998). This work provides the first measurements of this species parasitizing Andrias spp. in Kyoto.

  • Family Trichuridae (Ransom, 1911)
    Subfamily Capillariinae Railliet, 1915
    Amphibiocapillaria tritonispunctati (Diesing, 1815)

  • Description

  • Female (based on 2 specimens): body 9.2–9.7 mm long and 67–87 wide. Esophagus 203–266 long. Stichocytes and vulva at 3.7–4.2 mm and 4.6–4.9 mm, respectively, from anterior extremity. Nuclei 110–112 in stichosome. Eggs 52–60 (56±3) by 27–30 (30±1) (n=24). Rectum 79–88 long.

  • Taxonomic summary

  • Host: Andrias davidianus (Blanchard, 1871) and hybrid Andrias between A. japonicus and Chinese Andrias species.

  • Infection site: intestine and rectum.

  • Stage: adults.

  • Locality: Kyoto City, Kyoto Prefecture, Japan: Kamo River (35°06′46″ N, 135°43′12″ E), Kurama River (35°06′23″ N, 135°45′52″ E), Nakatsu River (35°06′41″ N, 135°43′27″ E) (site 5, Fig. 1), and Takano River (35°06′03″ N, 135°49′32″ E) (site 8, Fig. 1). Studied specimens: KUZ Z3911.

  • Remarks: general morphology consistent with Moravec (1982, 1986). This work provides the first measurements of this species parasitizing Andrias spp. in Kyoto.

  • Family Kathlaniidae Lane, 1914
    Subfamily Kathlaniinae Lane, 1914
    Falcaustra sp.

  • Description

  • General: body elongate. Three well-developed lips present. Esophagus consisting of three distinct parts; esophageal corpus, short isthmus and esophageal bulb. Tail tapering.

  • Male (based on 10 specimens): body 7.8–12.3 (9.7±1) mm long and 250–434 (334±50) wide in midbody. Lips 27–33 (29±2) by 55–67 (61±4). Pharyngeal part 55–79 (71±8) long and 39–52 (46±5) wide. Esophageal corpus 1.2–1.5 (1.4±0.9) mm long and 67–87 (73±6) wide, short isthmus 100–120 (108±6) long and 73–113 (91±11) wide, esophageal bulb 139–193 (163±17) long and 147–220 (178±21) wide. Nerve ring and excretory pore at 279–349 (318±21) and 1201–1414 (1306±65), respectively, from anterior extremity. Single pseudosucker consisting of 13–15 pairs of muscles, 1.2–2.5 (1.9±0.4) mm from cloaca. Spicules two, elongate, pointed; left spicule 547–727 (614±45) long and 20–40 (31±7) wide, right spicule 600–700 (635±27) long and 21–40 (32±7) wide. Gubernaculum 91–127 (108±10) by 30–47 (37±7). Tail 320–434 (386±32) long.

  • Female (based on 10 specimens): body 9.8–14.0 (11.6±1) mm long and 313–534 (399±62) wide in midbody. Lips 24–36 (31±4) by 36–70 (59±10). Pharyngeal part 58–82 (72±7) long and 24–58 (47±11) wide. Esophageal corpus 1.3–1.9 (1.5±0.2) mm long and 67–87 (78±6) wide, short isthmus 73–120 (100±13) long and 87–127 (103±12) wide, esophageal bulb 147–193 (172±17) long and 173–220 (194±16) wide. Nerve ring and excretory pore at 306–427 (347±41) and 1234–1581 (1393±123), respectively, from anterior extremity. Vulva 6.2–8.8 (7.3±0.8) mm long from anterior extremity. Eggs oval, with a layer, 61–73 (65±3) by 42–55 (48±3) (n=62). Tail 239–1134 (689±227) long.

  • Taxonomic summary

  • Host: Andrias davidianus (Blanchard, 1871) and hybrid Andrias between A. japonicus and Chinese Andrias species.

  • Infection site: intestine and rectum.

  • Stage: adults and larvae.

  • Locality: Kyoto City, Kyoto Prefecture, Japan: Kamo River (35°01′16–52″ N, 135°46′14–17″ E; 35°03′33″ N, 135°45′00″ E; 35°06′46″ N, 135°43′12″ E), Kurama River (35°05′52″ N, 135°45′47″ E; 35°06′23″ N, 135°45′52″ E), Shizuhara River (35°05′52″ N, 135°46′20″ E; 35°06′14″ N, 135°46′51″ E) (site 3, Fig. 1), Myozin River (35°03′27″ N, 135°45′17″ E) (site 4, Fig. 1), Nakatsu River (35°06′41″ N, 135°43′27″ E), Katsura River (35°12′19″ N, 135°44′35″ E; 35°15′57″ N, 135°44′34″ E), Teratani River (35°13′58″ N, 135°47′35″ E), Takano River (35°06′03″ N, 135°49′32″ E), Kiyotaki River (35°03′ N, 135°46′ E).

  • Studied specimens: KUZ Z3912.

  • Remarks: the specimens examined showed morphological features consistent with the genus Falcaustra as defined by Chabaud (2009) in the structure of lips and esophagus. Compared to the native congeneric species previously reported in Japan, Falcaustra sp. differed as follows: (1) single pseudosucker present instead of plural pseudosuckers present in males of F. odaiensis Hasegawa & Nishikawa, 2009, (2) spicules (547–727 long) shorter than those (1.2–1.3 mm long) in F. japonensis (Yamaguti, 1935) (Yamaguti, 1935; Hasegawa and Nishikawa, 2009). Falcaustra sp. also differed from the introduced congeneric species reported in Japan as follows: (1) spicules (547–727 long) longer than those (277–314 long) in F. catesbeianae Walton, 1929, (2) pseudosucker consisting of 13–15 pairs of muscles instead of elongate pseudosucker consisting of 41–44 pairs of muscles in F. wardi (Mackin, 1936) (Baker, 1985; Hasegawa, 2006).

  • Trematoda

  • Family Liolopidae
    Liolope copulans Cohn, 1902

  • Description

  • Adult (based on 9 specimens): body 2.3–3.7 (3.1±0.5) mm by 1.4–1.9 (1.7±0.2) mm. Oral sucker 107–200 (160±26) by 173–247 (210±23). Pharynx 73–113 (95±14) by 80–167 (124±23). Ventral sucker 193–260 (230±24) by 280–340 (307±16). Anterior testis 173–567 (387±117) by 334–494 (417±54), posterior testis 273–614 (391±127) by 287–534 (409±74). Cirrus pouch 400–754 (631±107) by 400–714 (588±91). Seminal vesicle 400–700 (594±94) by 160–300 (223±37). Ovary 187–293 (254±29) by 200–293 (253±30). Eggs 12–26 (19±4) in uterus, 140–147 (145±3) by 73–80 (78±3) (n=38).

  • Taxonomic summary

  • Host: Andrias davidianus (Blanchard, 1871) and hybrid Andrias between A. japonicus and Chinese Andrias species.

  • Infection site: stomach and intestine.

  • Stage: adults.

  • Locality: Kyoto City, Kyoto Prefecture, Japan: Kamo River (35°01′16–52″ N, 135°46′14–17″ E; 35°03′01–33″ N, 135°45′00–29″ E; 35°06′46″ N, 135°43′12″ E), Kurama River (35°05′52″ N, 135°45′47″ E; 35°06′23″ N, 135°45′52″ E), Shizuhara River (35°05′52″ N, 135°46′20″ E; 35°06′14″ N, 135°46′51″ E), Myozin River (35°03′27″ N, 135°45′17″ E), Nakatsu River (35°06′41″ N, 135°43′27″ E), Katsura River (35°12′19″ N, 135°44′32″ E; 35°14′58″ N, 135°45′56″ E; 35°15′57″ N, 135°44′34″ E), Teratani River (35°13′58″ N, 135°47′35″ E), Takano River (35°06′03″ N, 135°49′32″ E), Kiyotaki River (35°03′ N, 135°40′ E).

  • Studied specimens: KUZ Z3908–Z3909. Remarks: general morphology agreed with Baba et al. (2011). This work provides the first measurements of this species parasitizing Andrias spp. in Kyoto.

  • Molecular study

  • The ITS1 region of S. hanzaki was successfully sequenced for 1,551 bp (accession no. LC605542). The BLAST search showed the highest similarity (99%) with a sequence of S. hanzaki from A. japonicus (Japan) (KF530326: Li et al., 2014).

  • The partial 18S rDNA of Am. tritonispunctati was successfully sequenced for 786 bp (accession no. LC605543). The BLAST search showed the highest similarity (94%) with a sequence of Aonchotheca putorii (Rudolphi, 1819) (Nematoda: Trichuridae) (LC052349: Tamaru et al., 2015).

  • The partial 28S rDNA of Falcaustra sp. was successfully sequenced for 596 bp (accession no. LC605539–LC605541). The BLAST search showed the highest similarity (98%) with a sequence of Megalobatrachonema terdentatum (Linstow, 1890) (Nematoda: Kathlaniidae) (MN444706, Chen et al., 2020). The haplotype of larval Falcaustra sp. differed from those of adult Falcaustra sp. by 0.2–0.3% (p-distance). Two haplotypes of adult Falcaustra sp. differed by 0.2% (p-distance).

  • Discussion

    The parasite fauna of Andrias populations in Kyoto Prefecture consisted of Liolope copulans, Spiroxys hanzaki, Amphibiocapillaria tritonispunctati and Falcaustra sp. Liolope copulans and Falcaustra sp. were found in specimens at all of the study sites and were the most abundant species in helminth fauna of Andrias species in Kyoto Prefecture. No parasite species documented by Yamaguti (1936, 1939, 1941) were found in this study.

    Molecular data from the S. hanzaki confirmed the species-level identification of the specimens based on morphology. Molecular studies for Am. tritonispunctati and Falcaustra sp. also supported the subfamily-level identifications based on morphology. Genetic differentiation between the haplotype of larval Falcaustra sp. and those of adults were similar to genetic differentiation between those of two adult nematodes; therefore, we concluded that larval Falcaustra specimens were the same species as adult Falcaustra specimens.

    Spiroxys hanzaki and Am. tritonispunctati are considered native parasites in Japan, because S. hanzaki have been only reported parasitizing A. japonicus in Japan (e.g., Hasegawa et al., 2002). Amphibiocapillaria tritonispunctati is widely distributed over Holarctic region (Moravec, 1986); however, this species has been recorded from multiple different species of Caudata in Japan for many years (Uchida et al., 2019). These facts permit us to regard them as helminths not derived from other countries. Therefore, it was concluded that introduced A. davidianus could act as spillback reservoirs for native parasites in Kyoto Prefecture. It suggests that “enemy release” could not be found in introduced A. davidianus in Kyoto Prefecture, unlike the case demonstrated in Torchin et al. (2003). It is unclear whether such spillback affects the host-parasite relationship between the native populations of A. japonicus and parasites.

    This study is the first record of Falcaustra sp. found in Andrias spp. in Japan. The genus Falcaustra is a cosmopolitan group, and some introduced species of this genus have been reported in Japan (Hasegawa et al., 2006; Oi et al., 2012). Falcaustra sp. morphologically differs from both native and introduced congeneric species reported to parasitize amphibians and reptiles in Japan. Three congeneric species, F. andrias (He, Liu & Ma, 1992), F. fopingensis (He, Liu & Ma, 1992) and F. chengguensis (He, Liu & Ma, 1992), have been once recovered from Chinese Andrias species in China (He et al., 1992). However, these species cannot be compared with Falcaustra sp. due to insufficient morphological study and lacking molecular study. Further taxonomic study is necessary to identify Falcaustra species parasitic in Andrias spp. at species-level.

    Acknowledgments

    We thank Ms. M. Yoshimura (KUHE) for helping dissection of the animals. We thank everyone who participated in the survey of the salamanders around the Kamo River for securing the hybrid salamander used in this study. The procedure in this study followed the guideline of animal experiments in Kyoto University (approval no. 29-A-7). The present study was conducted under the permits issued by the Japan Agency of Cultural Affairs to K. Nishikawa for research in Kyoto City (No. 420) and in Kyoto Prefecture (No. 710).

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    Appendices

    Appendix

    Summary of examined hosts. TL: total length (mm); KUHE: voucher ID of Graduate School of Human and Environmental Studies, Kyoto University; Hybrid: hybrid between Andrias japonicus×Chinese Andrias spp.

    img-z11-3_109.gif
    © 2021 by The Herpetological Society of Japan
    Karin Tsuchida, Misako Urabe, and Kanto Nishikawa "The First Survey for Helminths Parasitic in Hybrid and Introduced Giant Salamanders, Genus Andrias (Amphibia: Caudata: Cryptobranchidae) in Kyoto, Japan," Current Herpetology 40(2), 109-119, (25 August 2021). https://doi.org/10.5358/hsj.40.109
    Accepted: 9 March 2021; Published: 25 August 2021
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