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).

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).

Table 2.

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

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

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.