Introduction
Nematodes are among the most common parasites of anurans, and many species exploit different developmental stages of their hosts as intermediate or definitive hosts (Baker Reference Baker1984, Reference Baker1987; Chen et al. Reference Chen, Zhang, Feng and Li2020a, Reference Chen, Zhang, Sinsch, Scheid, Balczun and Li2020b, Reference Chen, Gu, Ni and Li2021; González and Hamann Reference González and Hamann2008; González et al. Reference González, Hamann and Duré2021; Gu et al. Reference Gu, Mu, Chen and Li2025; Ni et al. Reference Ni, Barton, Chen and Li2022, Reference Ni, Barton and Li2023; Zeng et al. Reference Zeng, Chen, Ni, Kang and Li2024a, Reference Zeng, Chen, Xu and Li2024b). Tadpoles are frequently infected with oxyuroid nematodes of the genus Gyrinicola Yamaguti, Reference Yamaguti1938 (Adamson Reference Adamson1981a, Reference Adamsonb, Reference Adamsonc, Reference Adamsond; Kehr and Hamann Reference Kehr and Hamann2003; Sata and Nakano Reference Sata and Nakano2020; Walker et al. Reference Walker, Bolek, Zieman, Neubig and Jiménez2024, Reference Walker, Aguilar-Aguilar, León-Règagnon and Jiménez2025), whereas cosmocercoid and rhabdiasid nematodes commonly occur in adult frogs and toads (Alcantara et al. Reference Alcantara, Ebert, Müller, Úngari, Ferreira-Silva, Emmerich, Santos, O’Dwyer and da Silva2022; Chen et al. Reference Chen, Zhang, Feng and Li2020a, Reference Chen, Zhang, Sinsch, Scheid, Balczun and Li2020b, Reference Chen, Gu, Ni and Li2021; González et al. Reference González, Hamann and Duré2021; Kirillova and Kirillov Reference Kirillova and Kirillov2015; Kirillova and Kirillov Reference Kirillova and Kirillov2021; Ni et al. Reference Ni, Barton, Chen and Li2022, Reference Ni, Barton and Li2023; Zeng et al. Reference Zeng, Chen, Ni, Kang and Li2024a, Reference Zeng, Chen, Xu and Li2024b).
The Indian rice frog Fejervarya kawamurai Tjong, Matsui, Kuramoto, Nishioka & Sumida, 2011 (Amphibia: Dicroglossidae) is a common and widespread species inhabiting paddy fields in Japan (Nakamura Reference Nakamura2024; Noha et al. Reference Noha, Toyoda, Takai and Shimada2018), which breeds mainly in rice paddies and other shallow freshwater habitats during spring and summer. Following an aquatic larval stage lasting approximately one to two months, tadpoles undergo metamorphosis and become terrestrial juveniles. Adults overwinter in soil and reach sexual maturity by the following breeding season.
Although F. kawamurai is one of the most common frogs inhabiting rice paddies in Japan, information on its helminth fauna remains limited. To date, Gyrinicola japonicus has been reported from tadpoles of this host, whereas members of the Cosmocercidae have been recorded primarily from post-metamorphic individuals (Abe et al. Reference Abe, Matsumoto and Matsuda2026; Sata and Nakano Reference Sata and Nakano2020; Yamaguti Reference Yamaguti1938). Recent morphological and molecular studies have further suggested that F. kawamurai may harbour previously unrecognized diversity of cosmocercid nematodes in Japan, highlighting the need for integrative taxonomic investigations (Abe et al. Reference Abe, Matsumoto and Matsuda2026). Despite these findings, comprehensive information on helminth assemblages across different developmental stages of F. kawamurai is still lacking. In addition, because amphibians experience a major ecological transition from an aquatic larval stage to a predominantly terrestrial post-metamorphic stage, surveys restricted to adult hosts may overlook important components of parasite diversity. Therefore, examining both tadpoles and adult frogs is essential for understanding the diversity and host associations of helminths infecting F. kawamurai.
In the present study, tadpoles and post-metamorphic individuals of F. kawamurai were examined for nematode parasites using an integrative taxonomic approach combining morphology, scanning electron microscopy, and molecular analyses. We redescribe G. japonicus based on new morphological observations and additional molecular markers, and also describe a new species of Cosmocerca from adult frogs.
Materials and methods
Parasite collection
A total of 60 specimens of F. kawamurai, including 22 tadpoles, 15 juveniles, and 23 adults, were collected from an experimental paddy field at Chubu University (35°16′18.2″N, 137°01′02.7″E), Japan, between June 2020 and February 2023. Individuals of F. kawamurai were captured by hand and transported alive to the laboratory, where they were kept in water and dissected as soon as possible after collection, usually within a few days. Collection dates and dissection dates for all host specimens are provided in Supplementary Data S1. Prior to dissection, all host specimens were confirmed to be alive to ensure accurate assessment of parasite prevalence, intensity, and abundance, because host death may alter parasite condition, abundance, or location within the host. Species identification was determined following Matsui (Reference Matsui2016). Tadpoles were defined as pre-metamorphic individuals retaining a tail, irrespective of the degree of limb development. Total length (from snout to tail tip) was recorded for all tadpoles. Post-metamorphic frogs were identified by the complete absence of the tail. Following dissection, post-metamorphic individuals were classified as juveniles or adults based on gonadal development. Individuals lacking mature reproductive organs were regarded as juveniles, whereas those possessing identifiable testes or ovaries were regarded as adults. Sex was determined from the gonads at dissection. Snout–vent length (SVL) was recorded for all post-metamorphic frogs. Detailed information for each host specimen is provided in Supplementary Data S1.
Adult (mature) and juvenile (immature) frogs were deeply anaesthetized with isoflurane vapour and subsequently euthanized before dissection. The body-cavity was opened longitudinally, and the alimentary tract was removed under a stereomicroscope (SMZ800, Nikon, Japan) and placed in a Syracuse watch glass containing ×0.7 phosphate-buffered saline (PBS). The alimentary tract was then opened longitudinally with fine tweezers and examined for parasitic nematodes. Parasitic nematodes were collected using a thin-wire picker and transferred to a new Syracuse watch glass containing ×0.7 PBS. Tadpoles were anaesthetized on ice until complete loss of responsiveness and subsequently dissected. Detailed information on sampling date, host developmental stage, host sex, and nematode infections (including developmental stage, sex, and abundance) is provided in Supplementary Data S1.
Research involving amphibians is not subject to review by the institutional animal ethics committee of Chubu University under the current university regulations. Therefore, no ethics approval number was required for this study. Nevertheless, all animals were handled and anaesthetized in accordance with accepted ethical practices for amphibian research, and every effort was made to minimize animal suffering during handling and dissection.
Morphological observation
For light microscopy, nematode specimens were transferred to anhydrous glycerin via slow evaporation, following Seinhorst (Reference Seinhorst1959) and mounted in the same medium. The edges of the coverslips were sealed with wax rings to prepare permanent slides. Morphological measurements are given in micrometres except where indicated. Photomicrographs were taken using a Nikon digital camera mounted on a Nikon optical microscope (Nikon ECLIPSE Ni-U, Nikon Corporation, Tokyo, Japan) following the procedures of Gu et al. (Reference Gu, Mu, Chen and Li2025). For scanning electron microscopy (SEM), specimens were post-fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 6.0) overnight, followed by a one-hour treatment in 2% osmium tetroxide. The nematode specimens were then dehydrated through an ethanol series, treated with hexamethyldisilazane, and air-dried in a fume hood according to Morffe et al. (Reference Morffe, García, Véliz, Hasegawa and Carreno2022). Dried specimens were mounted on double-sided aluminium tape, sputter-coated with gold using an E-1030 sputter coater (Hitachi, Japan), and observed using a JSM-6510LA scanning electron microscope (JEOL, Japan) at an accelerating voltage of 15 kV. All nematode specimens were deposited in the Nematological Collection of Hasegawa Laboratory (NCHL), Chubu University, Japan and in the College of Life Sciences, Hebei Normal University (HBNU), Hebei Province, China.
Molecular procedures
Genomic DNA was extracted from each nematode specimen using either the Qiagen DNeasy Blood and Tissue Kit (Qiagen, USA) or the Column Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China), following the manufacturers’ instructions. Whole nematode specimens were used for DNA extraction. Voucher specimens were not retained from sequenced individuals, but representative specimens from the same host population were deposited in the NCHL and the HBNU. The nuclear 28S rDNA (D1D2 and D2D3 regions) and internal transcribed spacer (ITS) data, and the mitochondrial cox1 gene, were amplified by PCR using the primers provided in Table 1 and under the cycling conditions described in previous studies (Li et al. Reference Li, Lü, Nadler, Gibson, Zhang, Chen, Zhao and Guo2018; Ni et al. Reference Ni, Barton, Chen and Li2022; Ozawa and Hasegawa Reference Ozawa and Hasegawa2018). PCR products were sequenced in both directions.
Primer set used to amplify each gene marker in this study
Table 1. Long description
The table is organized into four columns: Gene, Primers, Sequence (5 prime to 3 prime), and Reference.
* Gene I T S 1: Includes primers S S 1 (G T T T C C G T A G G T G A A C C T G C G) and S S 2 R (A G T G C T C A A T G T G T C T G C A A). Reference: Zhu et al. 2000.
* Gene I T S 2: Includes primers N C 1 3 (A T C G A T G A A G A A C G C A G C) and N C 2 (T T A G T T T C T T T T C C T C C G C T). Reference: Zhu et al. 2000.
* Gene 28 S r R N A L S U: Divided into two primer pairs. Pair one: 28 S-F (A G C G G A G G A A A A G A A A C T A A) and 28 S-R (A T C C G T G T T T C A A G A C G G G), Reference: Nadler and Hudspeth 1998. Pair two: D 2 a (A C A A G T A C C G T G A G G G A A A G T T G) and D 3 b (T C G G A A G G A A C C A G C T A C T A), Reference: Nunn 1992.
* Gene cox 1: Includes primers C O I F (T T T T T T G G T C A T C C T G A G G T T T A T) and C O I R (A C A T A A T G A A A A T G A C T A A C A A C). Reference: Lazarova et al. 2006.
Although a single clear band was obtained using the above primer sets, direct sequencing of the ITS region of Gyrinicola japonicus was problematic. Therefore, ITS amplicons were cloned using the pCR®-Blunt II-TOPO® vector (Thermo Fisher Scientific, USA) prior to sequencing. All sequences were aligned using ClustalW2. The DNA sequence data generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov).
ASAP and BI analyses
Species delimitation was performed using the Assemble Species by Automatic Partitioning (ASAP) algorithm (Puillandre et al. Reference Puillandre, Brouillet and Achaz2021; https://bioinfo.mnhn.fr/abi/public/asap/). ASAP analyses for Cosmocerca spp. and Gyrinicola spp. were conducted on the ASAP online server under the Kimura two-parameter (K80) model using 28S, ITS, and cox1 sequence datasets. Bayesian inference (BI) was performed to assess interspecific evolutionary relationships among Cosmocerca spp. and Gyrinicola spp. Analyses were performed in MrBayes 3.2.7 with two parallel runs of 1,000,000 generations under the following optimal substitution models: for Cosmocerca spp., HKY + F + G4 model for 28S, SYM + G4 model for ITS, and HKY + F + I + G4 model for cox1; for Gyrinicola spp., HKY + F model for 28S, and HKY + F + G4 model for both ITS and cox1. Cosmocercoides qingtianensis Chen, Zhang, Nakao & Li, 2018 (Cosmocercoidea: Cosmocercidae) and Enterobius vermicularis (Linnaeus, 1758) (Oxyuroidea: Oxyuridae) were chosen as outgroups for the ASAP and BI analyses, respectively.
For ASAP analyses, the partition with the lowest ASAP score was regarded as the primary species-delimitation hypothesis. Species delimitation followed an integrative taxonomic framework in which species were recognized based on the congruence of diagnostic morphological characters, phylogenetic distinctiveness recovered from Bayesian inference analyses of independent nuclear and mitochondrial markers, and support from ASAP species-delimitation analyses. No single line of evidence was considered sufficient on its own; instead, species boundaries were inferred from the concordance of morphological and molecular evidence.
Results
Parasitic nematode prevalence, mean intensity, and mean abundance
Two morphologically and molecularly distinct nematode species were recovered from F. kawamurai: the oxyuroid nematode Gyrinicola japonicus from tadpoles and the cosmocercid nematode Cosmocerca kasugaiensis n. sp. from post-metamorphic individuals. Among the 22 tadpoles examined, 19 individuals were infected with G. japonicus (prevalence = 86.4%, mean intensity = 3.9 worms per infected host, mean abundance = 3.4 worms per examined host). No other helminths were detected in tadpoles.
Among the 38 post-metamorphic individuals (15 juveniles and 23 adults), 37 individuals were infected with C. kasugaiensis n. sp. (prevalence = 97%, mean intensity = 9.2 worms per infected host, mean abundance = 9.0 worms per examined host). In addition to C. kasugaiensis n. sp., a small number of unidentified helminths were occasionally observed. Because the available material was insufficient for reliable identification, these specimens were not considered further in the present study. The detailed infection data for each host specimen is provided in Supplementary Data S1.
Taxonomy
Cosmocerca kasugaiensis n. sp. (Figures 1–3)
Photomicrographs of Cosmocerca kasugaiensis n. sp. collected from Fejervarya kawamurai (Anura: Dicroglossidae) in Japan: (A) entire body of female (excretory pore arrowed), lateral view; (B) anterior region of female (nerve-ring arrowed), lateral view; (C) vulval region (vulva arrowed), lateral view; (D) well-developed, embryonated eggs (arrowed) in uterus; (E) posterior end of male (precloacal plectanes arrowed), lateral view; (F) entire body of male (spicule arrowed), lateral view; (G) posterior end of female, lateral view.
Figure 1. Long description
A collection of seven light microscopy photos labeled A through G.
Panel A shows the entire body of a female nematode curved in a C-shape. An arrow at the anterior end points to the excretory pore. A vertical scale bar indicates 500 micrometers.
Panel B provides a close-up of the anterior region of a female. An arrow points to the nerve-ring located around the esophagus. A vertical scale bar indicates 100 micrometers.
Panel C displays the vulval region of a female. A horizontal arrow on the left points to the vulva opening. A vertical scale bar indicates 100 micrometers.
Panel D shows the uterus containing several well-developed embryonated eggs, indicated by two diagonal arrows. A vertical scale bar indicates 100 micrometers.
Panel E depicts the posterior end of a male, which is sharply curved. Six horizontal arrows point to a series of precloacal plectanes along the ventral surface. A vertical scale bar indicates 50 micrometers.
Panel F shows the entire body of a male nematode. An arrow at the posterior end points to the spicule. A horizontal scale bar indicates 100 micrometers.
Panel G shows the posterior end of a female, tapering to a pointed tail. A vertical scale bar indicates 100 micrometers.
Line drawings of Cosmocerca kasugaiensis n. sp. collected from Fejervarya kawamurai (Anura: Dicroglossidae) in Japan: (A, B) anterior region of female (excretory pore arrowed), lateral view; (C) anterior region of male (excretory pore arrowed), lateral view; (D, E) non-embryonated and embryonated eggs; (F) vulval region, lateral view; (G) tail of female, lateral view; (H) posterior end of male (precloacal plectanes and spicule arrowed), lateral view; (I) posterior end of male (gubernaculum arrowed), ventral view.
Figure 2. Long description
A series of nine line drawings labeled A through I.
Panel A and B: Lateral views of the female anterior region. A long, striated esophagus leads to a bulbous posterior end. An arrow points to the excretory pore on the ventral side. Scale bar is 50 micrometers in A and 100 micrometers in B.
Panel C: Lateral view of the male anterior region, similar in structure to the female, with an arrow pointing to the excretory pore. Scale bar is 50 micrometers.
Panel D and E: Eggs. D shows a non-embryonated egg with a granular interior. E shows an embryonated egg containing a coiled larva. Scale bars are 50 micrometers and 100 micrometers respectively.
Panel F: Lateral view of the female vulval region showing the tubular vaginal duct opening through the cuticle. Scale bar is 100 micrometers.
Panel G: Lateral view of the female tail, tapering to a fine point with internal reproductive structures visible. Scale bar is 100 micrometers.
Panel H: Lateral view of the male posterior end. Five precloacal plectanes are marked with arrowheads along the ventral surface. An arrow points to the curved spicule near the cloaca. Scale bar is 100 micrometers.
Panel I: Ventral view of the male posterior end showing the distribution of caudal papillae in rows. An arrow points to the gubernaculum located near the spicules. Scale bar is 50 micrometers.
Scanning electron micrographs of Cosmocerca kasugaiensis n. sp. collected from Fejervarya kawamurai (Anura: Dicroglossidae) in Japan: (A) cephalic end of female (cephalic papillae arrowed), apical view; (B) cephalic end of male (amphids arrowed), apical view; (C) posterior end of male (somatic papillae and lateral ala arrowed), ventro-lateral view; (D) posterior end of male, lateral view; (E) tail of female (somatic papillae arrowed), lateral view; (F) enlarged image of somatic papilla; (G) entire body of female (vulva arrowed), ventral view; (H) enlarged image of vulva. Abbreviations: d, dorsal lip; s, subventral lip.
Figure 3. Long description
An eight-panel S E M series.
Panel A. Apical view of a female cephalic end. Three lips surround the mouth opening. Arrows point to cephalic papillae on the dorsal lip labeled d and subventral lips labeled s. Scale bar is 10 micrometers.
Panel B. Apical view of a male cephalic end. Arrows point to the amphids located laterally. Scale bar is 5 micrometers.
Panel C. Ventro-lateral view of a male posterior end. Arrows indicate a series of somatic papillae and a prominent lateral ala. Scale bar is 50 micrometers.
Panel D. Lateral view of a male posterior end showing a tightly coiled tail. Multiple arrows point to a dense row of ventral somatic papillae. Scale bar is 10 micrometers.
Panel E. Lateral view of a female tail tapering to a point. Arrows indicate two somatic papillae on the striated cuticle. Scale bar is 50 micrometers.
Panel F. High-magnification view of a single somatic papilla showing a circular, raised base with a central pore. Scale bar is 1 micrometer.
Panel G. Ventral view of the entire female body. An arrow points to the vulva located in the mid-body region. Scale bar is 500 micrometers.
Panel H. Enlarged view of the vulva from panel G, showing a transverse slit-like opening in the striated cuticle. Scale bar is 50 micrometers.
General. Small-sized, whitish nematodes. Body cylindrical, with maximum width at about mid-body (Figures 1A and F). Cuticle bears fine transverse striations. Somatic papillae present in both sexes (Figure 3F). Excretory pore usually situated just anterior to oesophageal bulb in male, and at level of or just posterior to oesophageal bulb in female (Figures 1A, B, 2A–C). Oral aperture simple, somewhat triangular, surrounded by 3 small lips, each with narrow inner flanges (Figures 3A and B). Dorsal lip with one pair of large double cephalic papillae; subventral lips each with one single and one double cephalic papilla plus amphid (Figures 3A and B). Ring of 8–9 papillae present at base of cephalic extremity (Figures 3A and B). Oesophagus muscular, divided into short anterior pharynx, cylindrical corpus, slightly narrow isthmus, and posterior oesophageal bulb with valves (Figures 1A, B, 2A–C). Nerve-ring is located at about 2/5 of oesophageal length. Tail of both sexes conical, with filamentous tip (Figures 1A, E, G, 2G, H, I and 3E).
Male (based on 11 mature specimens). Body 0.75–1.99 (1.51) mm long; maximum width 114–162 (125). Entire oesophagus 180–315 (270) long (including posterior bulb), representing 15.9–24.0 (18.5) % of body length; pharynx + corpus + isthmus 152–279 (228) long; size of posterior bulb 34–58 (49.9) × 33–56 (45.7). Nerve-ring 64–143 (101) and excretory pore 104–332 (237) from cephalic extremity. Lateral alae well developed, extending between some distance from base of cephalic extremity and level of third or fourth pair of precloacal plectanes (Figures 3C and D). Posterior end of body distinctly ventrally curved. Spicules equal in length, 73–108 (96.5) long; distal end pointed, representing 5.44–9.72 (6.68) % of body length (Figures 1E, F, 3H and I). Gubernaculum small, well sclerotized, 34–75 (61.3) long (Figure 2I). Total of 5 pairs of subventral precloacal plectanes present, each surrounded by incomplete circle of tubercles (central papilla surrounded by about 1/4 circle of tubercles) (Figures 1E, 2H, I, 3C and D). Paracloacal and postcloacal rosettes absent. Some pairs of small, simple, subventral papillae (indistinguishable from somatic papillae) located in postcloacal region (Figures 1E, 2H, I, 3C and D). Tail 101–222 (169) long, representing 9.40–13.5 (11.4) % of body length.
Female (based on 10 mature specimens). Body 2.37–4.07 (3.25) mm long; maximum width 150–310 (240). Entire oesophagus 299–362 (338) long (including posterior oesophageal bulb), representing 8.40–14.1 (10.6) % of body length; pharynx + corpus + isthmus 242–315 (274) long; size of posterior bulb 65–97 (81.5) × 72–108 (90). Nerve-ring 120–175 (151) and excretory pore 251–400 (335) from cephalic extremity. Lateral alae well developed, extending from some distance posterior to cephalic extremity to base of tail tip (Figure 3E). Vulva a transverse slit; vulval lips not distinctly protruded (Figures 1C, D, 2F, 3G and H), 1.10–1.90 (1.50) mm from cephalic extremity, representing 39.9–52.1 (46.2) % of body length. Eggs oval, thin-walled, with smooth surface, embryonated or non-embryonated, 43–118 (83) × 38–80 (57) (n = 20) (Figures 1C, D, 2D and E). Tail 310–409 (362) long, representing 7.85–14.8 (11.3) % of body length.
Type-host: Indian rice frog Fejervarya kawamurai (Amphibia: Dicroglossidae).
Type-locality: Experimental paddy field at Chubu University, Kasugai, Aichi, Japan.
Site in host: Large intestine of the juvenile and adult frogs.
Type-specimens : Holotype: male (HBNU-N-A20260501NL); paratypes: 3 males and 10 females (HBNU-N-A20260502NL), deposited in 80% alcohol in the College of Life Sciences, Hebei Normal University, China; paratypes: 9 females (NCHL B109–117) and 8 males (NCHL B101–108) mounted in permanent slides, deposited in the Nematological Collection of Hasegawa Laboratory, Chubu University, Japan.
Etymology: The specific name refers to the type-locality (Kasugai City, Japan).
Representative DNA sequences: Representative nuclear and mitochondrial sequences were deposited in the GenBank database under the accession numbers: PP627202–PP627204, OQ878512–OQ878514, OQ878493–OQ878499 for 28S; PP627552–PP627554, OQ877238, OQ877239 for ITS; PP627207, OQ466536–OQ466539 for cox1.
ZooBank registration: To comply with the regulations set out in Article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:3BDA5808-DDAA-4077-934F-335F02E2F24D. The LSID for the new name Cosmocerca kasugaiensis is urn:lsid:zoobank.org:act:595828DA-A4B0-46AF-A613-8C27CF17E4B7.
Remarks: The genus Cosmocerca Diesing, 1861 currently comprises more than 40 nominal species parasitizing amphibians worldwide and represents one of the most speciose genera within the family Cosmocercidae (Alcantara et al. Reference Alcantara, Ebert, Müller, Úngari, Ferreira-Silva, Emmerich, Santos, O’Dwyer and da Silva2022; Baker and Green Reference Baker and Green1988; Bursey et al. Reference Bursey, Goldberg, Siler and Brown2015; Chen et al. Reference Chen, Zhang, Feng and Li2020a; Harnoster et al. Reference Harnoster, du Preez and Svitin2022; Ni et al. Reference Ni, Barton, Chen and Li2020; Sou et al. Reference Sou, Sow and Nandi2018). Among its congeners, C. kasugaiensis n. sp. is characterized by the combination of well-developed lateral alae, two equal spicules distinctly longer than the gubernaculum, five pairs of precloacal plectanes surrounded by incomplete circles of tubercles, and tails lacking spines in both sexes. This combination of characters is shared with a limited number of congeners, including C. japonica, C. simile, C. ornata, C. microhylae, C. kalesari, C. banyulensis, C. daly, C. goroensis, C. makhadoensis, C. monicae, C. novaeguineae, and C. paraguayensis (Chabaud and Campana-Rouget Reference Chabaud and Campana-Rouget1955; Chen et al. Reference Chen, Zhang, Feng and Li2020a; Harnoster et al. Reference Harnoster, du Preez and Svitin2022; Rizvi et al. Reference Rizvi, Bursey and Bhutia2011; Sou and Nandi Reference Sou and Nandi2015; Svitin et al. Reference Svitin, Kuzmin, Harnoster, Nel and du Preez2023). Cosmocerca kasugaiensis n. sp. can be differentiated from C. simile by its relatively longer spicules in the male (representing 5.44–9.72% of body length in the former vs 3.28–4.10% in the latter), and a smaller body length and shorter tail in the female (body 2.37–4.07 mm long, tail 0.31–0.38 mm long in the new species vs body 4.65–6.21 mm long, tail 0.59–0.84 mm long in C. simile) (Chen et al. Reference Chen, Zhang, Feng and Li2020a). The new species can easily be distinguished from C. daly, C. goroensis, C. japonica, C. ornata, C. kalesari, C. makhadoensis, C. monicae, C. novaeguineae, and C. paraguayensis by having spicules distinctly longer than the gubernaculum (vs spicules shorter than or nearly as long as the gubernaculum). Cosmocerca kasugaiensis n. sp. also differs from C. microhylae by the lateral alae ending at the level of the third or fourth pairs of precloacal plectanes in the male (vs ending at the base of the caudal filament), relatively shorter spicules (representing 5.44–9.72% of body length vs 13.5–14.1% in C. microhylae) and the presence of somatic papillae in female (vs absent in C. microhylae) (Sou and Nandi Reference Sou and Nandi2015). Cosmocerca banyulensis is clearly different from the new species, as it has a specialized cuticular hood.
ASAP and BI analyses of Cosmocerca spp. (Figures 4, 5)
Bayesian inference of Cosmocerca spp. based on different nuclear and mitochondrial genetic markers. Abbreviations: 28S, large ribosomal subunit RNA; ITS, internal transcribed spacer; cox1, cytochrome c oxidase subunit I; OG, outgroup. Bayesian posterior probability values ≥0.80 are shown in the phylogenetic trees. Sequences generated in the present study are shown in blue.
Figure 4. Long description
A three-panel phylogenetic diagram. Each panel displays a rooted tree with Bayesian posterior probability values at the nodes and a scale bar at the bottom left.
* The first panel, labeled 2 8 S, shows a large clade of Cosmocerca kasugaiensis n. sp. in blue at the top, followed by Cosmocerca simile in pink, Cosmocerca goroensis in brown, Cosmocerca ornata in maroon, and Cosmocerca longicauda in light blue. The outgroup O G Cosmocercoides qingtianensis is at the base.
* The second panel, labeled I T S, shows a similar branching order. Cosmocerca kasugaiensis n. sp. (blue) is at the top, followed by Cosmocerca japonica (green), Cosmocerca simile (pink), Cosmocerca commutata and Cosmocerca ornata (red/maroon), Cosmocerca goroensis (brown), Cosmocerca kalesari (orange), and Cosmocerca longicauda (light blue). The outgroup O G Cosmocercoides qingtianensis is at the bottom.
* The third panel, labeled c o x 1, displays Cosmocerca kasugaiensis n. sp. (blue) at the top, followed by Cosmocerca japonica (green), Cosmocerca simile (pink), Cosmocerca ornata (maroon), Cosmocerca albopunctata (light blue), Cosmocerca podicipinus (purple), and Cosmocerca parva (red). The outgroup O G Cosmocercoides qingtianensis is at the bottom.
In all trees, the blue text indicates new sequences from the current study. Node support values range from 0.80 to 1.0.
Assemble Species by Automatic Partitioning (ASAP) analyses of Cosmocerca spp. based on different nuclear and mitochondrial genetic markers. Abbreviations: 28S, large ribosomal subunit RNA; ITS, internal transcribed spacer; cox1, cytochrome c oxidase subunit I; OG, outgroup. Nb groups indicate the number of species-level groups recognized by ASAP, whereas Score represents the ASAP score for each partition (lower scores indicate stronger support). Numbers within coloured bars denote species groups inferred under each partition. Coloured symbols on the nodes indicate support from different molecular datasets. The asterisk indicates the partition with the lowest ASAP score. Sequences generated in the present study are shown in bold.
Figure 5. Long description
The figure consists of three horizontal panels labeled 28 S, I T S, and cox 1 from top to bottom. Each panel contains three main components.
1. Species List: On the far left of each panel is a list of Cosmocerca species and outgroups with their accession numbers. Bold text indicates new sequences.
2. A S A P Partitions: To the right of the names are vertical bars representing different partitions. Above each bar, Nb groups indicates the number of species clusters and Score indicates the A S A P score.
* In the 28 S panel, there are 7 partitions ranging from 23 groups (score 7.5) to 2 groups (score 2.0). The partition with 6 groups (score 2.5) is marked with an asterisk.
* In the I T S panel, there are 10 partitions ranging from 11 groups (score 8.5) to 3 groups (score 8.0). The partition with 9 groups (score 2.5) is marked with an asterisk.
* In the cox 1 panel, there are 10 partitions ranging from 14 groups (score 8.5) to 3 groups (score 8.5). The partition with 8 groups (score 2.0) is marked with an asterisk.
Colored blocks within these bars are numbered to show how species are grouped together as the number of groups decreases from left to right.
3. Phylogenetic Trees: On the far right of each panel is a cladogram. Nodes are marked with colored circles (red, yellow, orange, or grey) representing different levels of molecular support. The trees show the branching relationships between the species listed on the left, with horizontal lines often broken by double forward slashes to indicate shortened branch lengths.
ASAP and BI analyses of Cosmocerca spp., based on the nuclear 28S, ITS, and mitochondrial cox1 data, all supported C. kasugaiensis n. sp. as representing a distinct species in relation to its congeners (Figures 4, 5). BI results based on the ITS and cox1 data both showed that the new species was sister to C. japonica + C. simile in the genus Cosmocerca (Figure 4). However, BI analysis of 28S data did not include the species C. japonica, due to inaccessibility of genetic data, which suggested a close affinity between C. kasugaiensis n. sp. and C. simile. The optimal ASAP partitions consistently recovered C. kasugaiensis n. sp. as an independent species-level unit (Figure 5). This result was congruent with its morphological characters and with the phylogenetic relationships inferred from the 28S, ITS, and cox1 dataset.
Gyrinicola japonicus Yamaguti, Reference Yamaguti1938 (emend.) (Figures 6, 7 and Table 2)
Photomicrographs of Gyrinicola japonicus collected from tadpoles of Fejervarya kawamurai (Anura: Dicroglossidae) in Japan: (A) entire body of female (excretory pore arrowed), lateral view; (B) anterior region of female, lateral view; (C) vulval region (vulva arrowed), lateral view; (D) posterior end of female, lateral view; (E) thick-shelled egg; (F) thin-shelled egg; (G) anterior end of male, lateral view; (H) posterior end of male (caudal papillae arrowed), lateral view; (I) entire body of male (spicule arrowed), lateral view.
Figure 6. Long description
A collection of nine photomicrographs labeled A through I.
Panel A shows the entire elongated body of a female nematode in lateral view with a scale bar of 500 micrometers. An arrow points to the excretory pore near the anterior end.
Panel B provides a close-up of the female anterior region with a 100 micrometer scale bar.
Panel C focuses on the vulval region where an arrow indicates the vulva opening.
Panel D displays the tapered posterior end of the female with a 100 micrometer scale bar.
Panel E shows a thick-shelled egg with a distinct outer layer and internal embryo, scale bar 50 micrometers.
Panel F shows a thin-shelled egg with a more granular interior, scale bar 50 micrometers.
Panel G depicts the blunt anterior end of a male nematode, scale bar 50 micrometers.
Panel H shows the curved posterior end of a male where an arrow points to the caudal papillae, scale bar 50 micrometers.
Panel I shows the entire body of a male nematode in lateral view with a scale bar of 100 micrometers. An arrow points to the internal spicule near the tail.
Scanning electron micrographs of Gyrinicola japonicus collected from tadpoles of F. kawamurai (Anura: Dicroglossidae) and Zhangixalus arboreus (Anura: Rhacophoridae) in Japan: (A) cephalic end of female (two prominent horn-like amphids arrowed), apical view; (B) cephalic end of male (six labial lobes arrowed), apical view; (C) entire body of male (lateral ala arrowed), lateral view; (D) tail of male (one pair of precloacal and one pair of postcloacal papillae located on genital cone indicated with black arrows, ventral-median postcloacal double papillae indicated with white arrow), ventral view; (E) tail of female, subventral view; (F) thick-shelled eggs.
Figure 7. Long description
A collection of six S E M micrographs labeled A through F.
* Panel A: Apical view of a female cephalic end. Two black arrows point to prominent horn-like amphids on either side of the central oral opening. Scale bar is 10 micrometers.
* Panel B: Apical view of a male cephalic end. Six black arrows point to labial lobes surrounding the mouth. Scale bar is 5 micrometers.
* Panel C: Lateral view of the entire male body, showing a curved, tapered shape with fine horizontal striations. A black arrow points to a longitudinal lateral ala running down the side. Scale bar is 50 micrometers.
* Panel D: Ventral view of a male tail. Four black arrows indicate two pairs of papillae on the genital cone, while a white arrow points to a ventral-median postcloacal double papilla. Scale bar is 10 micrometers.
* Panel E: Subventral view of a female tail, showing a long, smooth, pointed terminus. Scale bar is 50 micrometers.
* Panel F: Close-up of several thick-shelled, oval-shaped eggs with smooth surfaces. Scale bar is 10 micrometers.
Morphometric comparison of Gyrinicola japonicus (Oxyuridomorpha: Gyrinicolidae) in different studies. Measurements are given in micrometres (μm) unless otherwise stated
Table 2. Long description
The table compares morphometric data for G y r i n i c o l a japonicus. Measurements are in micrometres unless specified as millimetres (m m).
* Length of body (B L) (m m): Present study (Male: 0.62–0.88, Female: 1.51–3.66); Yamaguti 1938 (Male: 0.80–0.87, Female: 2.00–3.70); Sata and Nakano 2020 (Male: 0.76–1.07, Female: 2.03–4.39).
* Width of body (W L): Present study (Male: 57–81, Female: 140–280); Yamaguti 1938 (Male: 50–60, Female: 150–300); Sata and Nakano 2020 (Male: 78, Female: 194–329).
* Length of entire oesophagus (O L): Present study (Male: 101–170, Female: 290–443); Yamaguti 1938 (Male: 142–152, Female: 300–460); Sata and Nakano 2020 (Male: 119–157, Female: 295–431).
* Size of oesophageal bulb: Present study (Male: 26–44 by 31–42, Female: 68–105 by 82–132); Yamaguti 1938 (Male: 28–32, Female: 90–126); Sata and Nakano 2020 (Male: 35 by 37, Female: 55–78 by 80–107).
* O L / B L percentage: Present study (Male: 16.4–19.6, Female: 10.8–20.6); Yamaguti 1938 (Male: 17.5–17.8, Female: 12.4–15.0); Sata and Nakano 2020 (Male: 14.1–15.6, Female: 11.2–15.6).
* Distance of nerve-ring from cephalic end: Present study (Male: 50–80, Female: 91–164); Yamaguti 1938 (Male: 60–63, Female: 100–165); Sata and Nakano 2020 (Male: 56–89, Female: 89–130).
* Distance of excretory pore from cephalic end: Present study (Male: 188–273, Female: 386–736); Yamaguti 1938 (Male: 242–252, Female: 570–920); Sata and Nakano 2020 (Male: 273, Female: 458–895).
* Length of tail (T L): Present study (Male: 90–143, Female: 179–270); Yamaguti 1938 (Male: 80, Female: 200–310); Sata and Nakano 2020 (Male: 99–135, Female: 210–273).
* T L / B L percentage: Present study (Male: 12.4–16.4, Female: 6.21–12.4); Sata and Nakano 2020 (Male: 11.5–13.5, Female: 7.30–11.2).
* Spicule length (S L): Present study (Male: 31–40); Yamaguti 1938 (Male: 42–44); Sata and Nakano 2020 (Male: 37).
* Vulva from cephalic end (V C E) (m m): Present study (Female: 0.80–2.00); Sata and Nakano 2020 (Female: 1.14–1.84).
* V C E / B L percentage: Present study (Female: 47.8–60.6); Yamaguti 1938 (Female: 42.9–47.6); Sata and Nakano 2020 (Female: 53.5–67.3).
* Size of eggs: Present study (Female: 75–108 by 30–58); Yamaguti 1938 (Female: 87–126 by 30–54); Sata and Nakano 2020 (Female: 70–102 by 36–52).
* Host: Present study (Fejervarya kawamurai); Yamaguti 1938 (Hoplobatrachus chinensis); Sata and Nakano 2020 (Zhangixalus arboreus).
* Locality: All studies conducted in Japan.
Small, whitish nematodes. Body cylindrical, with maximum width at about anterior 2/5 of body (Figures 6A and I). Cuticle with fine transverse striations. Somatic papillae absent in both sexes. Sexual dimorphism distinct in cephalic structure (Figures 7A and B). Lateral alae present in male, extending from some distance posterior to base of cephalic extremity to about level of anterior region of genital cone (Figure 7C); lateral alae absent in female. Excretory pore located posterior to oesophageal bulb in both sexes. Oesophagus muscular, divided into indistinct anterior pharynx, cylindrical corpus, slightly narrow isthmus, and posterior oesophageal bulb with valves (Figures 6A, B, G and I). Nerve-ring located at about 2/5 of oesophageal length (Figure 6B). Genital cone well developed in male. Caudal papillae in male sessile, including one pair precloacal, one pair immediately postcloacal, one pair postcloacal, ventromedial and fused, and one pair located at about middle of tail (Figure 7D). Single spicule and sclerotized V-shaped gubernaculum present (Figure 6H). Vulva a transverse slit in female, without protruded vulval lips. Uterus didelphic; one branch containing thin-shelled eggs at various stages of development; other branch containing thick-shelled eggs at early stage of cleavage (Figures 6E and F, 7F). Tail of both sexes conical, with pointed tip (Figures 6D, H, I, 7D and E). Measurements and morphometric comparisons are provided in Table 2.
Host of present specimens: Fejervarya kawamurai (Amphibia: Dicroglossidae).
Locality of present specimens: Experimental paddy field at Chubu University, Kasugai, Aichi, Japan.
Site in host: Large intestine of tadpole of F. kawamurai.
Voucher specimens : 3 males (NCHL B119–121), 10 females (NCHL B123–132), deposited in the Nematological Collection of Hasegawa Laboratory, Chubu University, Japan; 1 male (HBNU-N-2023M025L), 13 females (HBNU-N-2023M026L), deposited in the College of Life Sciences, Hebei Normal University, China.
Representative DNA sequences: Representative nuclear and mitochondrial sequences were deposited in the GenBank database under the accession numbers: PP627555, PP627556, OQ878561–OQ878565 for 28S; PP590615 for ITS; PP549858–PP549860 for cox1.
ASAP and BI analyses of Gyrinicola spp. (Figures 8, 9)
Assemble Species by Automatic Partitioning (ASAP) analyses of Gyrinicola spp. based on different nuclear and mitochondrial genetic markers. Abbreviations: 28S, large ribosomal subunit RNA; ITS, internal transcribed spacer; cox1, cytochrome c oxidase subunit I; OG, outgroup. Nb groups indicate the number of species-level groups recognized by ASAP, whereas Score represents the ASAP score for each partition (lower scores indicate stronger support). Numbers within coloured bars denote species groups inferred under each partition. Coloured symbols on the nodes indicate support from different molecular datasets. The asterisk indicates the partition with the lowest ASAP score. Sequences generated in the present study are shown in bold.
Figure 8. Long description
The figure consists of three horizontal panels labeled 2 8 S, I T S, and c o x 1. Each panel contains a list of species sequences on the left, vertical colored bars representing A S A P partitions in the center, and a cladogram on the right.
Top Panel (2 8 S): Lists 17 sequences including O G Enterobius vermicularis and various Gyrinicola species. Four vertical partition bars are shown. The first bar has 16 groups (Score 4.0) with individual colors for most sequences. The fourth bar, marked with an asterisk, shows 3 groups (Score 1.5) where Gyrinicola japonicus sequences are grouped in a large green block labeled 10. The tree on the right features a red node and a dark brown node indicating high support.
Middle Panel (I T S): Lists 11 sequences. Seven partition bars are shown. The fourth bar (asterisk) shows 5 groups (Score 1.5). In this partition, Gyrinicola japonicus is group 10 (green), Gyrinicola batrachiensis is group 2 (light green), and Gyrinicola gulabrevioris is group 3 (orange). The tree shows nodes marked with yellow, red, and grey symbols.
Bottom Panel (c o x 1): Lists 10 sequences. Eight partition bars are shown. The fifth bar (asterisk) shows 5 groups (Score 1.5). Gyrinicola japonicus sequences are grouped in purple (labeled 3), while Gyrinicola batrachiensis and Gyrinicola gulabrevioris are grouped in red (labeled 2) and green (labeled 3) respectively. The tree on the right contains multiple yellow and grey node symbols.
Across all panels, sequences in bold indicate new data from the current study. The A S A P scores generally decrease from left to right as the number of groups is consolidated.
Bayesian inference of Gyrinicola spp. based on different nuclear and mitochondrial genetic markers. Abbreviations: 28S, large ribosomal subunit RNA; ITS, internal transcribed spacer; cox1, cytochrome c oxidase subunit I; OG, outgroup. Bayesian posterior probability values ≥0.80 are shown in the phylogenetic trees. Sequences generated in the present study are shown in blue.
Figure 9. Long description
The figure consists of three horizontal phylogenetic trees arranged side-by-side.
1. The leftmost tree is labeled 28 S. It features a large upper clade of Gyrinicola japonicus sequences, with the top seven entries in blue (O Q 8 7 8 5 6 1 through P P 6 2 7 5 5 6) and the bottom three in light blue. Below this is a clade containing Gyrinicola gulabrevioris in red and Gyrinicola batrachiensis in purple. The outgroup at the bottom is Enterobius vermicularis L C 4 1 6 0 6 9. A scale bar of 0.03 is at the bottom left.
2. The middle tree is labeled I T S. It shows Gyrinicola japonicus P P 5 9 0 6 1 5 in blue at the top. Below it is a clade of Gyrinicola batrachiensis in purple, followed by a clade of Gyrinicola dehradunensis in green. Further down is a clade of Gyrinicola gulabrevioris in red. The outgroup at the bottom is Enterobius vermicularis H Q 6 4 6 1 6 4. A scale bar of 0.04 is at the bottom left.
3. The rightmost tree is labeled cox 1. The top clade contains three Gyrinicola japonicus sequences in blue (P P 5 4 9 8 5 8 through P P 5 4 9 8 6 0). Below this is a clade of Gyrinicola batrachiensis in purple. The next clade down contains Gyrinicola gulabrevioris in red, followed by a single entry for Gyrinicola moohsia in light blue. The outgroup at the bottom is Enterobius vermicularis A B 9 7 1 6 7 0. A scale bar of 0.09 is at the bottom left.
All trees include Bayesian posterior probability values at the nodes, such as 1, 0.999, or 0.875, and use double forward slashes on long branches to indicate truncation.
The ASAP and BI analyses of Gyrinicola spp., based on the nuclear 28S, ITS, and mitochondrial cox1 sequence data, all confirm that G. japonicus represents a species distinct from its congeners (Figures 8, 9). The BI results, based on the 28S, ITS, and cox1 data, all show that G. japonicus is sister to the other species of Gyrinicola. The optimal ASAP partitions (indicated by an asterisk in Figure 8) consistently recovered G. japonicus as a distinct species, in agreement with morphological observations and phylogenetic analyses.
Discussion
The molecular identification of cosmocercid nematodes is still in its infancy due to the limited genetic data available for this group. Molecular analysis showed no intraspecific nucleotide variation in the 28S, ITS, and cox1 regions among the different individuals of C. kasugaiensis n. sp. ASAP and BI analyses, based on different genetic data, all indicated that C. kasugaiensis n. sp. represented a distinct species, which is in accord with the morphological observations. Because all specimens were collected from a single locality, the observed genetic homogeneity was not unexpected, particularly for the relatively conserved 28S marker. Nevertheless, no intraspecific variation was detected even in the more rapidly evolving ITS and mitochondrial cox1 regions, supporting the conclusion that the sequenced specimens belong to a single species.
However, molecular data are currently available for only a subset of the known species of Cosmocerca, including only some of the species that are morphologically similar to C. kasugaiensis n. sp. Consequently, additional sequence data from presently unsampled congeners will be required to fully evaluate genetic diversity and evolutionary relationships within the genus. Although differences in body size may sometimes reflect variation associated with host species, environmental conditions, or reproductive maturity, the recognition of C. kasugaiensis n. sp. is supported by a unique combination of diagnostic morphological characters together with independent molecular evidence. In particular, the new species was consistently recovered as a distinct lineage in the ITS and cox1 phylogenies and was independently supported by ASAP species-delimitation analyses. Therefore, the observed morphological differences are unlikely to represent intraspecific variation alone, and the taxonomic status of the new species is supported by an integrative taxonomic framework combining morphological and molecular evidence.
The emendation of the species name from Gyrinicola japonica to Gyrinicola japonicus follows the provisions of the International Code of Zoological Nomenclature (ICZN). According to Article 30.1.4.2 of the Code, generic names ending in the suffix ‘-cola’ are to be treated as masculine, regardless of their linguistic derivation. As the specific epithet japonica is an adjectival species-group name, it must agree in gender with the generic name and is therefore correctly rendered as japonicus. In contrast, the generic name Cosmocerca is treated as feminine; consequently, the species named Cosmocerca japonica remains grammatically correct and does not require modification.
The genus Gyrinicola is a relatively small group of tadpole-parasitic nematodes, containing five valid species according to the most recent taxonomic reviews (Planade et al. Reference Planade, Bain, Lena and Joly2008; Sata and Nakano Reference Sata and Nakano2020). However, molecular data remain available for only a few species within the genus, most notably G. japonicus and G. batrachiensis, limiting our understanding of species boundaries and evolutionary relationships within the group. The present study provides the first ITS and cox1 sequences for G. japonicus, thereby substantially expanding the molecular resources available for the genus. Yamaguti (Reference Yamaguti1938) erected the genus Gyrinicola, and designated G. japonica Yamaguti, Reference Yamaguti1938 (here emended to G. japonicus), from tadpoles of Rana rugulosa (now Hoplobatrachus chinensis) (Anura: Dicroglossidae) in Japan, as the type-species of this genus.
Sata and Nakano (Reference Sata and Nakano2020) redescribed G. japonicus based on specimens collected from Rhacophorus arboreus (now Zhangixalus arboreus) (Anura: Rhacophoridae) also in Japan. The morphology and morphometrics of our specimens agree well with the previous studies in most aspects, including the body size, the morphology of the lips, the position of the nerve-ring and excretory pore, the morphology and length of the oesophagus, spicule and tail, the position of the vulva, and the morphology and size of the eggs (Figures 4–5). It is also worth mentioning that the present specimens, collected from F. kawamurai (Amphibia: Dicroglossidae) at Aichi, Japan, belong to the same family as the type-host and were collected in a region neighbouring the type-locality (Kyoto, Japan). Sata and Nakano (Reference Sata and Nakano2020) also reported the 28S sequence (LC489226) of G. japonicus, and pairwise comparison with the 28S sequences of our specimens showed no genetic variation, which is also indicative of conspecificity. This study also revealed the morphology of the cephalic extremity of the male and the egg using SEM for this species for the first time. Our SEM observations have shown the presence of additional ventral double papillae between the pair of postcloacal papillae on the genital cone of the male, which has not been mentioned in previous studies (Sata and Nakano Reference Sata and Nakano2020; Yamaguti Reference Yamaguti1938). We also report, for the first time, ITS and cox1 data for G. japonicus, which should prove to be helpful for the molecular identification and population genetics of this species in the future.
Amphibians undergo a major ecological transition during development, shifting from an aquatic larval stage to a predominantly terrestrial or semi-aquatic post-metamorphic stage. Consequently, individuals may encounter different parasite assemblages throughout their lifetime. In the present study, tadpoles of F. kawamurai were infected exclusively with G. japonicus, whereas post-metamorphic individuals harboured C. kasugaiensis n. sp., suggesting a substantial turnover in parasite fauna during host development. Most surveys of amphibian helminths focus primarily on adult hosts, potentially overlooking an important component of parasite biodiversity. Incorporating tadpoles into parasite surveys may therefore improve our understanding of helminth diversity, host specificity, transmission strategies, and the evolutionary processes shaping amphibian–parasite associations.
From an evolutionary perspective, host developmental stages may represent distinct ecological opportunities for parasite colonization. Recent studies on the evolution of animal parasitism in nematodes have suggested that major evolutionary transitions are often associated with shifts in host use and transmission pathways (Nagae and Hasegawa Reference Nagae and Hasegawa2026). The marked difference between the nematode communities observed in tadpoles and post-metamorphic frogs in the present study suggests that amphibian metamorphosis may act as an ecological filter shaping parasite assemblages. Examining parasite communities across host developmental stages may therefore provide valuable insights into host switching, parasite specialization, and the evolution of host–parasite associations.
This study expands current knowledge of cosmocercid and gyrinicolid diversity in East Asia and provides a robust taxonomic foundation for future studies on amphibian–nematode associations. Further research is needed to clarify the life cycles of both nematode species and to elucidate the ecological and physiological factors underlying their occurrence in different host developmental stages.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26102478.
Data availability
The sequence data for Gyrinicola japonicus and Cosmocerca kasugaiensis obtained herein are deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) (G. japonicus: PP627555, PP627556, OQ878561–OQ878565 for 28S; PP590615 for ITS; PP549858–PP549860 for cox1. C. kasugaiensis: PP627202–PP627204, OQ878512–OQ878514, OQ878493–OQ878499 for 28S; PP627552–PP627554, OQ877238, OQ877239 for ITS; PP627207, OQ466536–OQ466539 for cox1).
Acknowledgements
We thank Professor Akihiko Moriyama (Chubu University), Yuto Koike, Hiroki Nagaya, and Koshiro Kaga (Hasegawa Lab, Chubu University) for their help with sample collection.
Author contribution
Koichi Hasegawa and Liang Li contributed to the study design and wrote the manuscript. Masato Mizukoshi and Seiya Nagae executed the field investigation and collected parasite specimens. Seiya Nagae, Hui-Xia Chen, Xue-Feng Ni, and Liang Li observed morphology, sequenced and analysed genetic data, and identified nematode specimens; Hui-Xia Chen, Seiya Nagae, and Liang Li conducted the ASAP and BI analyses. David Gibson revised the manuscript. All authors have read and approved the final manuscript.
Financial support
This study was supported by the National Natural Science Foundation of China (Grant No. 32300361) for Dr. Hui-Xia Chen, the Key Development Foundation of Hebei Normal University (L2024ZD17), and the research funding from the Research Institute for Biological Function and the College of Bioscience and Biotechnology, Chubu University, for Dr. Koichi Hasegawa.
Competing interests
The authors declare that they have no competing interests.
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