Introduction
Trematodes are parasites which use vertebrates, including marine fishes, as definitive hosts, and many species require more than two host individuals to complete their life cycles (Olsen, Reference Olsen1967). However, information on the intermediate and paratenic hosts remains insufficient for many trematode species, largely because their morphological identification of non-adult stages is challenging. Because metacercariae are transmitted to their definitive hosts via predation, identification of the second intermediate, paratenic and definitive hosts may provide insights of predator–prey relationships in marine ecosystems, which often remain poorly understood (e.g., Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026).
Recent advances in molecular methods have enabled species-level identification of sporocyst, redia, and metacercaria stages of trematodes in marine ecosystems (Ansai et al., Reference Ansai, Sekine, Munakata, Sase, Sasaki, Nitta, Suzuki, Abe, Takano, Fukumori, Nakatsubata, Suzuki and Waki2025; Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026). Particularly, identification of metacercariae enables prediction of predator–prey relationships between the intermediate and definitive hosts (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026), thereby improving understanding of trematode life cycles and marine food webs. These previous studies have shown that sequences of the nuclear 28S ribosomal RNA (rDNA) and mitochondrial cytochrome c oxidase subunit 1 (COI) regions, especially when used in combination, are effective for identifying trematode species. The COI region is particularly suitable for species identification across a broad range of trematode taxa (Ansai et al., Reference Ansai, Sekine, Munakata, Sase, Sasaki, Nitta, Suzuki, Abe, Takano, Fukumori, Nakatsubata, Suzuki and Waki2025; Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Martin et al., Reference Martin, Downie and Cribb2020; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026).
Pelagic gastropods (Mollusca) exhibit holoplanktonic or neustonic lifestyles throughout their life cycles (Lalli and Gilmer, Reference Lalli and Gilmer1989). The pelagic gastropods include gelatinous zooplankton such as members of the order Pteropoda Cuvier, 1804 (e.g., Cavoliniidae J. E. Gray, 1850 (1815)) and certain Littorinimorpha A. N. Golikov & Starobogatov, 1975 (e.g., Pterotracheidae Rafinesque, 1814), as well as neustons such as Janthina spp. (Littorinimorpha: Epitoniidae S. S. Berry, 1910 (1812)) and Glaucus spp. (Nudibranchia Cuvier, 1817: Glaucidae J. E. Gray, 1827) (Lalli and Gilmer, Reference Lalli and Gilmer1989). Pelagic gastropods have been reported as the intermediate hosts of trematodes, including metacercariae (Leuckart, Reference Leuckart1854; Lester and Newman, Reference Lester and Newman1986; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Morales-Ávila et al., Reference Morales-Ávila, Saldierna-Martínez, Moreno-Alcántara and Violante-González2018; Reimer, Reference Reimer1976), although most of these reported metacercariae have not been identified to the species, genus, or even family level (Morales-Ávila et al., Reference Morales-Ávila, Saldierna-Martínez, Moreno-Alcántara and Violante-González2018). Therefore, the definitive hosts of trematodes infecting pelagic gastropods remain largely unknown. In the present study, metacercariae parasitising pelagic gastropods were surveyed in Japanese waters. The taxonomic positions of the detected metacercariae were determined using molecular analyses, and their definitive hosts were clarified for several trematode species. These results provide insight into predator–prey relationships between some pelagic gastropods and marine fishes. Additionally, rediae obtained from benthic gastropods were analysed to improve understanding of the trematode life cycles.
Materials and methods
Sample collections and observations
Pelagic gastropods were collected from 2016 to 2025 in coastal regions of Japan (Table 1). Most specimens were transported to the laboratory alive (Figure 1), whereas others were frozen or fixed in 70% ethanol and subsequently identified based on morphological characteristics following Okutani (Reference Okutani2000, Reference Okutani2017). The lifestyle of each gastropod species (holoplankton or neuston) was determined according to previous records (Lalli and Gilmer, Reference Lalli and Gilmer1989; Okutani, Reference Okutani2017) (Table 1). The gastropods were subsequently examined and dissected under a stereomicroscope. When metacercariae were detected, they were observed alive, fixed, and kept in 70% or 99% ethanol and were used for molecular identification. Selected metacercariae were mounted between a glass slide and a coverslip and fixed in 70% or 99% ethanol or 5% formalin. Whole-mount specimens were observed under a light microscope after staining with acetocarmine or Heidenhein’s iron-hematoxylin, dehydrating in an ethanol series, clearing in xylene or creosote, and mounting on slides with Canada balsam. Line drawings were prepared using a camera lucida (U-DA, OLYMPUS CORPORATION) attached to a light microscope. Some permanent mounts were prepared after a portion of tissue was cut from each specimen for molecular analyses. Some host pelagic gastropods that were transported frozen or fixed in 70% ethanol were processed as described above.
Identification, sampling locality, and date of metacercariae detected from pelagic gastropods
* Number of metacercariae identified based on molecular analyses.
** Number of paragenophore specimens in parentheses.
*** Unclear.
Dt, Dinurus tornatus; Cs, Cephalolepidapedon saba; O2, Opechona sp. 2; Pro1, Prodistomum orientale (Prodistomum Type 1 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024)); Pro2, Prodistomum orientale (Prodistomum Type 2 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024)); Pro5, Prodistomum Type 5 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024); Pre1, Preptetos sp. 1; L1, Lepocreadiidae gen. sp. 1 sensu Waki et al. (Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026).
Host pelagic gastropods and metacercariae. (a) The holoplanktonic gastropod Pterotrachea coronata. (b) Lepocreadiid metacercariae in Pterotrachea coronata. (c) Metacercaria of Prodistomum orientale (Prodistomum Type 2 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024)) isolated from the pelagic gastropod Cavolinia uncinata. (d) The neustonic gastropod Glaucus atlanticus. (e) Metacercaria of Dinurus tornatus isolated from Glaucus atlanticus.
Adult trematodes from fish hosts were collected opportunistically and used for molecular comparison with the metacercariae from pelagic gastropods. From 2017 to 2025, fishes were obtained by angling and commercial fishing in waters surrounding Japan and transported to the laboratory on ice. Some specimens were frozen in plastic bags pending further parasitological examination. The fish were identified according to Nakabo (Reference Nakabo2013), and their scientific names follow Froese and Pauly (Reference Froese and Pauly2025). Trematodes were extracted from the fish using forceps under a dissecting microscope or collected from the digestive tract using the double-netting method (Madinabeitia and Nagasawa, Reference Madinabeitia and Nagasawa2013). The adult trematodes were fixed and preserved on glass slides in 70% or 99% ethanol and subsequently utilised for morphological and molecular analyses as described above. Permanent mounts of the adults and metacercariae were deposited in the Ibaraki Nature Museum (INM-1-131993–132020).
To detect lepocreadiid rediae and sporocysts, we collected benthic gastropods of the family Cypraeidae Rafinesque, 1815 (cowries) at Otsuki, Kochi Prefecture, Japan, on 29 April 2025 (Table 3), as cowries are known to serve as hosts for lepocreadiid rediae in Japanese waters (Ansai et al., Reference Ansai, Sekine, Munakata, Sase, Sasaki, Nitta, Suzuki, Abe, Takano, Fukumori, Nakatsubata, Suzuki and Waki2025). A pelagic gastropod with a heavy metacercaria infection was also collected at this locality (Table 1: Pterotrachea coronata). Shells of the cowries were crushed individually with a hammer, and their internal organs were dissected under a stereomicroscope in 1% NaCl solution. Detected rediae or sporocysts were preserved in 70% or 99% ethanol for subsequent molecular and morphological analyses. The ethanol-preserved specimens were mounted in Hoyer’s medium and observed under a light microscope (BX53, OLYMPUS CORPORATION). Line drawings and measurements of rediae were prepared as described above. Measurements and illustrations of sporocysts and cercariae were not provided because these specimens were immature.
DNA barcoding and phylogenetic analyses
Alkaline lysates of whole bodies or small tissue fragments of individual metacercariae and adults were individually prepared as templates for polymerase chain reaction (PCR) in accordance with Nakao et al. (Reference Nakao, Waki, Sasaki, Anders, Koga and Asakawa2017). Partial fragments of the 28S rRNA and mitochondrial COI regions were amplified using the following primer sets: for 28S rRNA, dig12 and 1500R (Tkach et al., Reference Tkach, Kudlai and Kostadinova2016) or LSU5 (Littlewood et al., Reference Littlewood, Curini-Galletti and Herniou2000) and 1600R (Williams et al., Reference Williams, Reid and Littlewood2003); for COI, JB3 (Bowles et al., Reference Bowles, Hope, Tiu, Liu and McManus1993) and CO1-R trema (Miura et al., Reference Miura, Kuris, Torchin, Hechinger, Dunham and Chiba2005), JB3 and JB4.5 (Bowles et al., Reference Bowles, Blair and McManus1992), or JB3 and trem.cox1.rrnL (Králová-Hromadová et al., Reference Králová-Hromadová, Špakulová, Horáčková, Turčeková, Novobilský, Beck, Koudela, Marinculić, Rajský and Pybus2008). Additional COI primer sets, targeting non-overlapping regions with those of the COI primers above, were used exclusively for comparison with previously published data (Bray et al., Reference Bray, Cutmore and Cribb2022; Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024): Dig_cox1Fa and Dig_cox1R (Wee et al., Reference Wee, Cribb, Bray and Cutmore2017). PCR amplification was performed in a 25 μL reaction volume using Tks Gflex DNA polymerase (TaKaRa) and the manufacturer-supplied reaction buffer, as described by Nakao et al. (Reference Nakao, Waki, Sasaki, Anders, Koga and Asakawa2017). The same primers used for amplification were employed for sequencing. When necessary, internal sequencing primers for 28S rDNA were used as follows: ECD2S (Williams and Ozawa, Reference Williams and Ozawa2006), D3m (Takano and Kano, Reference Takano and Kano2014), and 900F (Lockyer et al., Reference Lockyer, Olson, Østergaard, Rollinson, Johnston, Attwood, Southgate, Horak, Snyder, Le, Agatsuma, McManus, Carmichael, Naem and Littlewood2003). Sanger sequencing of PCR amplicons was performed by Eurofins Genomics Inc. (Tokyo, Japan). The sequences obtained in this study were deposited in the International Nucleotide Sequence Database Collaboration (INSDC) through the DNA Data Bank of Japan (DDBJ) under accession numbers LC914968–LC915045. Sequence alignment matrices were prepared using BioEdit version 7.0.5.3 (Hall, Reference Hall1999).
In the present study, hemiurid and lepocreadiid metacercariae were detected as described later. The hemiurid metacercaria exhibited morphological characteristics consistent with species of Dinurus Looss, 1907 (Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026). Since Dinurus species can be reliably distinguished based on partial sequences of 28S rDNA (Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026), the 28S rDNA sequence obtained in this study was compared with those of Dinurus species deposited in the INSDC. P-distance was calculated in MEGA X (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018), and intraspecific divergence values of related species were used as thresholds for species assignment. Amplification of the COI fragment was unsuccessful.
For lepocreadiid metacercariae, COI sequences generated using the primer set JB3 and corresponding reverse primers were analysed based on an overlapping region of 305 sites. An alignment dataset comprising COI sequences of metacercariae, adult specimens, and rediae obtained in this study, together with reference sequences from online databases, was constructed using BioEdit. A maximum likelihood phylogenetic tree was constructed in MEGA X under the selected best-fit substitution model (HKY + G + I; Hasegawa et al., Reference Hasegawa, Kishino and Yano1985). Nodal support was determined with 1000 bootstrap replicates. Operational taxonomic units (OTUs) were recognized in the tree as described below. Each OTU was considered to represent a single species based on intraspecific divergence values calculated in MEGA X as thresholds for species assignments. When necessary, divergence values of COI sequences obtained using an alternative primer set (Dig_cox1Fa and Dig_cox1R after Bray et al., Reference Bray, Cutmore and Cribb2022; Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) were also used for species identifications of each OTU. If species was not determined, the genus was examined by phylogenetic analyses using 28S rDNA sequences (1030-site overlapping region) from specimens in this study and reference sequences from online databases. These analyses were conducted in MEGA X under the General Time Reversible (GTR) + G + I model (Nei and Kumar, Reference Nei and Kumar2000) selected as the best-fit model.
Results
Trematode infections
A total of 10 species of pelagic gastropods (415 individuals) were examined (Table 1). Metacercariae infections were detected in four host species: one neustonic and three holoplanktonic gastropod species (Figure 1; Table 1). Based on morphological observations and molecular analyses of 28S rDNA and COI sequences, the metacercariae were identified as one hemiurid species and six lepocreadiid species. Thirty-one adult trematodes (including presumably immature adults; see below) were collected from 15 fish hosts and were used for molecular comparison (Table 2). These adults were morphologically identified as Prodistomum orientale (Layman, 1930) and Opechona sp. 2 (see below for species name). Rediae and one sporocyst were detected in three of 80 host individuals of cowries examined (Table 3). Rediae from two individuals of Monetaria caputserpentis (Linnaeus, 1758) were identified molecularly as members of the genus Prodistomum Linton, 1910. The taxonomic position of a sporocyst from Naria erosa (Linnaeus, 1758) was uncertain because only immature cercariae were found from the sporocyst and amplification of sporocyst DNA was unsuccessful.
Species, sampling locality, and date of adults and immature adults detected from marine fishes
* Amplified by the primer set JB3 and its reverse primers.
** Amplified by the primer set Dig_cox1Fa and Dig_cox1R.
*** Sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024)
Rediae and sporocysts in molluscs of the family Cypraeidae sampled from Otsuki, Kochi Prefecture, Japan on 29 April 2025
* Amplified by the primer set JB3 and its reverse primers.
** Amplified by the primer set Dig_cox1Fa and Dig_cox1R.
*** Not amplified.
Identification of trematodes with morphological descriptions
We provide ecological and morphological data for metacercariae from the pelagic gastropods, adult trematodes from fish hosts, and rediae from cowrie hosts. Unless otherwise stated, results of COI sequences are based on fragments amplified using the primer set of JB3 and the corresponding reverse primers. All measurements in the descriptions are expressed as mean values, with ranges in parentheses, and are in micrometres unless otherwise stated.
Family Hemiuridae Looss, 1899
A hemiurid metacercaria detected in the neustonic gastropod Glaucus atlanticus Forster, 1777, was identified as Dinurus tornatus (Rudolphi, 1819).
Genus Dinurus Looss, 1907
Species Dinurus tornatus (Rudolphi, 1819)
Description of metacercaria (Figure 4a): Body proper (soma) subcylindrical, 1060 in length. Oral sucker rounded, subterminal, 114 × 119. Pharynx muscular, 61 × 52. Esophagus not visible. Caeca filled with blue material. Ventral sucker rounded, larger than oral sucker, 203 × 209. Testes and ovary not visible, possibly because of use for template of molecular analyses. Ecsoma shrunken.
Remarks: Intra- and interspecific divergence values of the 28S rDNA sequences (616 sites) were zero and ranged from 0.007 to 0.0169, respectively (Supplementary Table S1). The divergence values between our metacercaria from Glaucus atlanticus and Dinurus tornatus in the online databases were zero (Supplementary Table S1), indicating our metacercaria was Dinurus tornatus. Its COI sequence was not determined. The morphological characteristics of our metacercaria, such as sizes of its organs and body proper, were consistent with those recorded in previous studies (Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026), supporting our molecular identifications.
Host pelagic gastropod of metacercaria in Japan: Glaucus atlanticus Forster, 1777 (present study)
Additional host of metacercaria in Japan: Physalia utriculus (Gmelin, 1788) (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026)
Host fish of adult in Japan: Coryphaena hippurus Linnaeus, 1758 (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026)
Deposition of metacercaria: INM-1-132020
DNA markers: 28S rDNA, LC914968 (metacercaria)
Family Lepocreadiidae Odhner, 1905
Metacercariae of this family were detected in three species of holoplanktonic gastropods (Table 1). The phylogenetic analysis of COI sequences (305-site overlapping region) resolved nine OTUs (Figure 2). Intraspecific divergence values for this overlapping region of this family ranged from 0 to 0.0752 based on the COI sequences of adult specimens generated in this study and reference sequences from the online databases. The divergence values of this overlapping region within each OTU fell within this range (Supplementary Table S2), whereas the minimum inter-OTU divergence (0.0832) exceeded the maximum of the intraspecific divergence value (0.0752). These results indicate that each OTU represents a single species. Metacercariae from the pelagic gastropods were included in OTUs 1, 2, 5, 6, 7, and 9, whereas OTU 4 consisted solely of a sequence derived from a redia recovered from a cowrie. OTUs 3 and 8 comprised only sequences obtained from the online databases. Among the seven OTUs containing specimens from this study (OTUs 1, 2, 4–7, and 9), species-level identification was achieved for OTUs 1 and 7. Genus-level identification was determined for OTUs 2, 4, 6, and 9 through subsequent phylogenetic analyses of 28S rDNA sequences (Figure 3), although their species names could not be determined. The genus corresponding to OTU 5 remained unresolved. OTU 1 included sequences of Prodistomum Type 1 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) and Prodistomum Type 2 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024), both of which were identified as Prodistomum orientale in the previous study (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) based on additional molecular analyses. Here, we reported each type (Prodistomum Type 1 and Type 2) separately because slight morphological differences have been noted between them (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024).
Unrooted maximum likelihood tree of Lepocreadiidae inferred from partial sequences of COI (305 sites). Bootstrap values above 50% are shown. Numbers of each OTU are indicated within parentheses, and database accession numbers are shown before species names. A single asterisk (*) in red represents newly obtained sequences. Double-asterisks (**) in red represent OTUs or Types that include sequences of metacercariae in pelagic gastropods. Scale bar indicates substitutions per site.
Maximum likelihood tree of Lepocreadiidae inferred from partial sequences of 28S rDNA (1030 sites), with Stegodexamene anguillae (Aephnidiogenidae) (KF484005) as outgroup taxon. Bootstrap values above 50% are shown. Database accession numbers are shown after species names. A single asterisk (*) in red represents our newly generated sequences. Scale bar indicates substitutions per site.
A paragenophore and a hologenophore were successfully made for lepocreadiid metacercariae of Cephalolepidapedon saba Yamaguti, 1970 (OTU 7) and Opechona sp. 2 (OTU 9), respectively. We could not prepare hologenophores for the remaining lepocreadiid OTUs because of their small body sizes (approximately 200–350 μm in length after fixation) and high morphological similarity. Paragenophores could not be assigned to these OTUs because multiple metacercarial species were detected within a single host individual. Accordingly, we provided a brief description of an unidentified lepocreadiid metacercaria under ‘Genus uncertain’, representing specimens with morphological characters similar to those of the other lepocreadiid OTUs.
Genus Cephalolepidapedon Yamaguti, 1970
Cephalolepidapedon saba Yamaguti, 1970 (OTU 7)
Two metacercariae detected in Pterotrachea coronata Forsskål, 1775, were identified as this species based on molecular and morphological evidence.
Description of metacercaria (Figure 4b): Body subcylindrical, 678 × 224. Oral sucker oval, 91 × 108. Prepharynx long, 78 in length. Pharynx oval, 57 × 49. Oesophagus 39 in length. Ventral sucker at middle of body, 91 × 87. Eye-spot pigment, situated at level of prepharynx. Testes tandemly situated. Anterior testis 34 × 22. Posterior testis 36 × 21. Ovary just anterior to anterior testis, 19 × 11. Excretory pore opening posterior terminal of body. Excretory vesicle close to oral sucker.
Trematodes examined in the present study. (a) Metacercaria of Dinurus tornatus (Hemiuridae). (b–i) Trematodes of family Lepocreadiidae. (b) Metacercaria of Cephalolepidapedon saba. (c) Adult of Opechona sp. 2. (d) Metacercaria of Opechona sp. 2. (e, f) Adults of Prodistomum orientale: (e) and (f) represent Prodistomum Type 1 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) and Prodistomum Type 2 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024), respectively. (g) Redia of Prodistomum Type 5 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024). (h) Redia of Prodistomum Type 7. (i) Unidentified lepocreadiid metacercaria. (a, d, e, f) Partial parts of worms were drawn because some tissues were used for a template of PCR. Abbreviations: c, caecum; ec, ecsoma; ep, excretory pore; es, oesophagus; gp, genital pore; o, ovary; os, oral sucker; ph, pharynx; sr, seminal receptacle; sv, seminal vesicle; t, testis; u, uterus; v, vitellarium; vs, ventral sucker.
Remarks: In the COI tree, these metacercariae formed OTU 7 with Cephalolepidapedon saba retrieved from the online database (Figure 2), and they are thought to be conspecific by the comparison of COI sequences (305 sites, Supplementary Table S2). Our paragenophore specimen was morphologically similar to those in previous studies (e.g., large oral sucker with small papillae, Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Kondo et al., Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016), supporting our molecular identifications.
Host pelagic gastropod of metacercaria in Japan: Pterotrachea coronata Forsskål, 1775 (present study)
Other hosts of metacercaria in Japan: Aurelia coerulea von Lendenfeld, 1884; Chrysaora pacifica (Goette, 1886); Cyanea nozakii Kishinouye, 1891 (Kondo et al., Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016); Physalia utriculus (Gmelin, 1788) (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026)
Host fish of adults in Japan: Scomber japonicus Houttuyn, 1782 (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Shimazu, Reference Shimazu1989; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026)
Deposition of metacercaria: INM-1-132019
DNA markers: COI, LC915038 (metacercaria)
Genus Opechona Looss, 1907
Opechona sp. 2 (OTU 9)
Fourteen adult trematodes recovered from the host fishes Labracoglossa argenteiventris Peters, 1866, and Pterocaesio trilineata Carpenter, 1987, were morphologically identified as a member of the genus Opechona. Molecular analyses indicated that these adults and a metacercaria detected in Pterotrachea coronata were conspecific. OTU 9 formed a sister clade to OTU 8 (Figure 2), which included a single sequence of Opechona sp. 1 sensu Waki et al. (Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026), originating from a metacercaria infecting the cnidarian Urashimea globosa Kishinouye, 1910 (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026). Therefore, we labelled the present specimens as Opechona sp. 2.
Description of adult (Figure 4c): Body elongate, subcylindrical. Oral sucker large, infundibuliform. Prepharynx distinct. Pharynx oval and large. Pseudoesophagus present. Intestinal bifurcation in forebody, and caeca reaching close to posterior margin of body, connecting excretory vesicle and forming uroproct. Ventral sucker small and rounded, without elaborate musculature. Testes oval or rounded, smooth. Testes tandem, separated from each other in mid hindbody. Seminal receptacle and anterior testis separated from each other. Cirrus-sac claviform. Ovary rounded and small. Uterus without mature eggs. Vitelline follicles from posterior margin of body to level of ventral sucker. Genital pore opening anterolateral to ventral sucker. Excretory pore opening at posterior end of body. Measurements in Table 4.
Comparison of morphological measurements of Opechona species. All measurements are in μm unless indicated otherwise
*Sizes of two testes.
Description of metacercaria (Figure 4d): Body subcylindrical. Oral sucker infundibuliform and triangular, 158 × 139. Prepharynx 34 in length. Pharynx oval, 83 × 57. Oesophagus 58 in length. Ventral sucker 83 × 78. Eye-spot pigment, at level of pharynx.
Remarks: None of the trematodes found from fish host individuals had eggs in their uteri. This suggests that the host fish species are unsuitable hosts for this trematode species, not due to insufficient periods for the trematodes to mature in the fishes. These immature adults were thought to be the genus Opechona based on the following morphological characteristics: pseudoesophagus present, ventral sucker without elaborate musculature, cirrus-sac claviform, ovary rounded, genital pore opening anterolateral to ventral sucker, uroproct present, excretory pore opening terminal of body. The genus Opechona includes 14 species (WoRMS, 2026a). Among them, our worms are morphologically similar to Opechona bacillaris (Molin, 1859), Opechona kahawai Bray and Cribb, Reference Bray and Cribb2003, and Opechona olssoni (Yamaguti, Reference Yamaguti1934) in having the following features: body elongate, oral sucker large and infundibuliform, testes oval or rounded. However, the present species can be distinguished from Opechona bacillaris and Opechona kahawai based on positions of seminal receptacle and anterior testis (very close to each other in the two species vs separated from each other in our adults). Moreover, Opechona bacillaris has testes situated close to each other (vs separated in our worms) and Opechona kahawai has a small pharynx (length 1/4 of oral sucker length vs 1/2 in our worms). Opechona olssoni differed from our worms because it has a heart-shaped ovary (vs oval in our worms). The morphological features of our worms suggested that they are an undescribed species. However, as our worms were immature, their morphological features may be different from those of fully matured adult individuals. In the COI tree, the immature adults and the metacercaria were included in OTU 9 (Figure 2), and comparisons of divergence values indicated that they were conspecific (305 sites, Supplementary Table S2). The morphological characteristics of our metacercaria, such as large oral sucker and pharynx, were similar to metacercaria of the genus Opechona in previous studies (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026), supporting the result of our molecular analyses.
Host pelagic gastropod of metacercaria in Japan: Pterotrachea coronata Forsskål, 1775 (present study)
Host fishes of immature adult in Japan: Labracoglossa argenteiventris Peters, 1866; Pterocaesio trilineata Carpenter, 1987 (present study)
Depositions: Immature adults INM-1-132009–132016; metacercaria INM-1-132017
DNA markers: 28S rDNA, LC914972–LC914974 (immature adults); COI, LC915040–LC915043 (immature adults), LC915044 (metacercaria)
Genus Preptetos Pritchard, 1960
Preptetos sp. 1 (OTU 6)
Metacercariae detected in Firoloida desmarestia Lesueur, 1817, and Pterotrachea coronata formed OTU 6 and were considered conspecific, although its species name could not be determined (Figure 2; Supplementary Tables S2 and S3). The 28S rDNA tree showed that the metacercariae were members of the genus Preptetos (Figure 3). Accordingly, they are labelled as Preptetos sp. 1.
Host pelagic gastropods of metacercaria in Japan: Pterotrachea coronata Forsskål, 1775; Firoloida desmarestia Lesueur, 1817 (present study)
DNA markers: 28S rDNA, LC914976 and LC914977 (metacercariae); COI, LC914987, LC915034–LC915037 (metacercariae)
Genus Prodistomum Linton, 1910
A total of 16 adult specimens were collected from two fish hosts—Scomber australasicus Cuvier, 1832, and Scomber japonicus—and identified morphologically as a single species of the genus Prodistomum. These adults were divided into two Types: namely, Prodistomum Type 1 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) and Prodistomum Type 2 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024), by molecular analyses (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024), both of which have been identified as Prodistomum orientale (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024), although slight morphological differences between them have been reported (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024). Thirty-three metacercariae from Cavolinia uncinata (A. d’Orbigny, 1835) and Pterotrachea coronata, and rediae detected from the cowrie Monetaria caputserpentis were identified as this genus based on the molecular analyses.
Prodistomum orientale (Layman, 1930) (Prodistomum Type 1 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024)) (OTU 1)
Description of adult (Figure 4e): Body elongate oval. Eye-spot pigment, from level of oral sucker to posterior to pharynx or at level of intestinal bifurcation. Oral sucker small. Prepharynx present. Pharynx muscular, relatively short. Pseudoesophagus present. Ventral sucker rounded. Uterus anterior to testes. Testes oval, nearly tandem but slightly oblique. Ovary trilobed or oval, just anterior to anterior testis. Internal seminal vesicle oval. Genital pore anterior to ventral sucker. Anterior margin of vitellaria posterior to ventral sucker, at level of anterior margin of seminal vesicle. Egg length equal to or less than 61. Excretory vesicle running right of uterus and reaching intestinal bifurcation. Excretory pore at posterior terminal of body. Measurements in Table 5.
Comparison of morphological measurements of Prodistomum species. All measurements are in μm unless indicated otherwise
* Sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024).
Remarks: Fifteen adult specimens from Scomber spp. were identified as Prodistomum orientale based on the following morphological characteristics: oral sucker small, prepharynx present, anterior margin of vitellaria posterior to ventral sucker, testes and oval, ovary trilobed or oval, egg length ≤61. Currently, 19 species are recognized in the genus Prodistomum (WoRMS, 2026b). Among them, Prodistomum mohsini (Ahmad, 1984) resembles Prodistomum orientale in having a small oral sucker, vitellaria extending posterior to the ventral sucker, and oval testes and ovary. However, Prodistomum mohsini differs from Prodistomum orientale based on the size of oral sucker (large in Prodistomum mohsini vs small in Prodistomum orientale) and sizes of eggs (equal to or more than 100 in length in Prodistomum mohsini vs ≤61 in Prodistomum orientale). Molecular analyses based on COI sequences amplified with the primer set Dig_cox1Fa and Dig_cox1R (428-site overlapping region; Supplementary Table S4) assigned these adults to Prodistomum Type 1 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) as follows. Intraspecific divergence values for this COI region ranged from 0 to 0.0304 within each type, whereas divergence values between the two types ranged from 0.0888 to 0.1098. The divergence values of this COI region between the present specimens and database sequences of Prodistomum Type 1 ranged from 0.0234 to 0.0280, whereas comparisons with Type 2 sequences ranged from 0.0888 to 0.0958 (Supplementary Table S4). Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) reported that the two types can be distinguished morphologically by pharynx length (<44 μm long in Prodistomum Type 1 vs >46 μm in Prodistomum Type 2). Although the pharynx lengths of the present specimens exceeded those reported by Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024), they remained shorter in Prodistomum Type 1 (50–68 μm) than in Prodistomum Type 2 (75 μm; Table 5), consistent with the morphological distinction between the two types. However, these results demonstrated that the measurements of the pharynx itself fail to distinguish these two types morphologically.
In the COI tree based on the 305-site overlapping region (Supplementary Table S2), two metacercariae obtained from the pelagic gastropod Pterotrachea coronata formed a clade with adult specimens of Prodistomum Type 1 within OTU 1 (Figure 2). Based on COI sequences from the present study and reference sequences in the online databases, the divergence values within each type ranged from 0 to 0.0237, whereas divergence between the two types ranged from 0.0561 to 0.0752. The COI sequences of the two metacercariae were identical to those of adults of Prodistomum Type 1, indicating our metacercariae can be labelled as this type (Supplementary Table S2).
Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) reported that Prodistomum Type 1 distributes only in Australia; however, the adults and metacercariae were reported from host fishes and pelagic gastropods in the present study.
Host pelagic gastropod of metacercaria in Japan: Pterotrachea coronata Forsskål, 1775 (present study)
Host fishes of adult in Japan: Scomber australasicus Cuvier, 1832; Scomber japonicus Houttuyn, 1782 (present study)
Deposition of adult: INM-1-131993–132007
DNA markers: 28S rDNA, LC914975 (adult); COI, LC914982, LC914984–LC914986, LC914988–LC914999 (adults), LC914979, LC915000, LC915001 (metacercariae)
Prodistomum orientale (Layman, 1930) (Prodistomum Type 2 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024)) (OTU 1)
Description of adult (Figure 4f): Morphological characteristics similar to those of Prodistomum orientale (Prodistomum Type 1), except length of pharynx (75 in this type) as described in section of Prodistomum Type 1. Measurements are in Table 5.
Remarks: One adult specimen recovered from Scomber japonicus in Saga Prefecture, Japan, was identified morphologically as Prodistomum orientale (Table 2). The COI sequence analysis of the 305-site overlapping region (Supplementary Table S2) revealed that this specimen was Prodistomum Type 2 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024). Metacercariae recovered from Cavolinia uncinata and Pterotrachea coronata were also determined as Prodistomum Type 2 based on COI sequence comparisons (Supplementary Table S2).
Host pelagic gastropods of metacercaria in Japan: Pterotrachea coronata Forsskål, 1775; Cavolinia uncinata (A. d’Orbigny, 1835) (present study)
Additional hosts of metacercaria in Japan: Aurelia coerulea von Lendenfeld, 1884; Clytia sp.; Chrysaora pacifica (Goette, 1886); Physalia utriculus (Gmelin, 1788); Urashimea globosa Kishinouye, 1910 (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026)
Host fishes of adult in Japan: Scomber japonicus Houttuyn, 1782 (present study; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026), Coryphaena hippurus Linnaeus, 1758 (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026)
Deposition of adult: INM-1-132008
DNA markers: 28S rDNA, LC914971 (adult); COI, LC915004 (adult), LC914978, LC915002, LC915003, LC915005–LC915008 (metacercariae)
Prodistomum Type 5 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) (OTU 2)
In the COI tree, OTU 2 comprised 24 metacercariae recovered from the pelagic gastropod Pterotrachea coronata (Figure 2; Supplementary Table S2). The divergence values of COI sequences generated using the primer sets Dig_cox1Fa and Dig_cox1R revealed that these metacercariae were conspecific with Prodistomum Type 5, whose adults were reported from Scomber japonicus by Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) (Supplementary Table S4).
The redia detected in one cowrie individual of Monetaria caputserpentis (Figure 4g) was also thought to be Prodistomum Type 5 based on COI sequence comparisons (Supplementary Table S2). Mature cercariae were not observed in this redia.
Host pelagic gastropod of metacercaria in Japan: Pterotrachea coronata Forsskål, 1775 (present study)
Host cowrie of redia in Japan: Monetaria caputserpentis (Linnaeus, 1758) (present study)
Host fish of adult in Japan: Scomber japonicus Houttuyn, 1782 (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024)
DNA markers: 28S rDNA, LC914969 (metacercaria); COI, LC914980, LC914981, LC915009, LC915011–LC915033 (metacercariae), LC915010 (redia).
Prodistomum Type 7 (OTU 4)
COI sequences of the redia recovered from the cowrie Monetaria caputserpentis did not match any newly generated sequences from either primer set or any sequences available in online databases (Figure 4h; Supplementary Tables S2 and S4). Phylogenetic analysis of 28S rDNA sequences placed this redia within a clade of the genus Prodistomum, closely related to Prodistomum Type 4 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) and Prodistomum Type 5 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) (Figure 3). We labelled this redia as Prodistomum Type 7 as a newly determined OTU of the genus, positioned adjacent to Prodistomum Type 6 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) (= Prodistomum keyam). Mature cercariae were not observed in the redia.
Host cowrie of redia in Japan: Monetaria caputserpentis (Linnaeus, 1758) (present study)
DNA markers: 28S rDNA, LC914970 (redia); COI, LC914983 and LC915039 (redia)
Lepocreadiidae gen. sp. 1 sensu Waki et al. (Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026) (OTU 5)
A single metacercaria was obtained from Firoloida desmarestia. Based on the comparisons of COI sequences, the metacercaria was thought to be conspecific with metacercariae previously reported from the cnidarian Physalia utriculus (LC889270 and LC889271), Lepocreadiidae gen. sp. 1 sensu Waki et al. (Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026).
Host pelagic gastropod in Japan: Firoloida desmarestia Lesueur, 1817 (present study)
Additional host of metacercaria in Japan: Physalia utriculus (Gmelin, 1788) (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026)
DNA marker: COI, LC915045 (metacercaria)
Genus uncertain
As noted above, we provided a brief description of an unidentified lepocreadiid metacercaria under ‘Genus uncertain’.
Description of metacercaria (Figure 4i): Body subcylindrical, 313 × 168. Oral sucker oval, 43 × 51. Prepharynx 15 in length. Pharynx oval, 21 × 22. Ventral sucker at half of body, 41 × 49. Eye-spot pigment, from level of prepharynx to oesophagus. Testes tandemly situated. Anterior testis 13 × 14. Posterior testis 14 × 19. Ovary 12 × 11, anterior to anterior testis. Excretory pore opening posterior terminal of body. Excretory vesicle close to intestinal bifurcation.
Host pelagic gastropod of metacercaria in Japan: Pterotrachea coronata Forsskål, 1775 (present study)
Deposition of metacercaria: INM-1-132018
Discussion
In the present study, six species of lepocreadiid metacercariae were detected in pelagic gastropods, all of which represent new host-parasite records. Four of these were considered conspecific with adults or immature adults recovered from fish hosts in the present study and with trematodes identified to the species level in previous studies (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026). Accordingly, their definitive fish hosts have been determined. The definitive hosts of the remaining two trematode species, Lepocreadiidae gen. sp. 1 sensu Waki et al. (Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026) and Preptetos sp. 1 (Lepocreadiidae), remain unknown. However, they are likely to infect marine fishes, as members of Lepocreadiidae typically use fishes as their definitive hosts (Jones et al., Reference Jones, Bray and Gibson2005). These results suggest that the lepocreadiid metacercariae use planktonic gastropods as the intermediate or paratenic hosts and reach definitive host fishes through trophic transmission. The host fishes and pelagic gastropods in the present study were thought to co-occur through Japanese coastal regions and therefore the gastropod hosts can be preyed upon by the fish hosts. Notably, a single individual of Pterotrachea coronata (Pterotracheidae) harboured five of the six detected lepocreadiid species and contained 115 metacercariae. Although we examined only one individual, the heavy infection intensity suggested that this host species is frequently exploited by these metacercariae. This gastropod species may therefore represent a major transmission pathway for definitive hosts in their lifecycles (Figure 5). Holoplanktonic gastropods of the family Pterotracheidae are widely distributed in the pelagic zone (Blackburn, Reference Blackburn1956; Lemus-Santana et al., 2015), indicating that marine fishes have frequent opportunities to prey on these gastropods. The marine fish Coryphaena hippurus, reported as the definitive host of Prodistomum orientale (Prodistomum Type 2) (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026), is also reported to prey on holoplanktonic gastropods (Lalli and Gilmer, Reference Lalli and Gilmer1989). This fish species is therefore likely infected with this trematode through predation (Figure 5). Metacercariae of Cephalolepidapedon saba, Prodistomum orientale (including Prodistomum Type 1 and Type 2 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024)), and Lepocreadiidae gen. sp. 1 sensu Waki et al. (Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026) have also been reported from cnidarian jellyfish (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Kondo et al., Reference Kondo, Ohtsuka, Hirabayashi, Okada, Ogawa, Ohkouchi, Shimazu and Nishikawa2016; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026). Therefore, marine fishes may acquire infection not only through holoplanktonic gastropods but also through cnidarians (Figure 5). Adults of the genus Preptetos have been reported from herbivorous reef fishes, particularly those of the family Acanthuridae (Bray et al., Reference Bray, Cutmore and Cribb2022). This suggests that infections in pelagic gastropods are transmission dead-ends for metacercariae of Preptetos sp. 1, although its definitive host fish is currently unknown. To determine whether holoplanktonic gastropods represent a major source of metacercaria infection, future studies should evaluate fish predation on pelagic gastropods through behavioural observations and/or stomach content analyses.
Hypothetical transmission routes of trematodes associated with pelagic gastropods and marine fish hosts in coastal regions of Japan. Pink, white, and green lines represent transmission routes of Prodistomum Type 5 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) (Lepocreadiidae), the other lepocreadiid species, and Dinurus tornatus (Hemiuridae), respectively. Solid and dotted lines indicate the infection route of the metacercariae and cercariae, respectively. A white-outlined dark arrow represents a possible transmission route of metacercaria of Dinurus tornatus to the definitive host. (a) Monetaria caputserpentis, the first intermediate host of Prodistomum Type 5. (b) Unknown mollusc, the first intermediate host of the other trematodes. (c–h) Various second intermediate hosts or paratenic hosts. (c–e) Pelagic gastropods. (c) Cavolinia uncinata. (d) Species of Pterotracheidae. (e) Glaucus atlanticus. (f) Unknown copepod, the second intermediate host of Dinurus tornatus. (g) Planktonic cnidarian jellyfishes. (h) Neustonic cnidarian jellyfish, Physalia utriculus. (i–k) Marine fishes, the definitive hosts. (i) Scomber spp. (j) Labracoglossa argenteiventris and Pterocaesio trilineata. (k) Coryphaena hippurus.
Lepocreadiid cercariae have been reported to invade gelatinous plankton directly and develop into metacercariae (Køie, Reference Køie1975; Stunkard, Reference Stunkard1969, Reference Stunkard1980a, Reference Stunkard1980b). Pelagic gastropods harbouring lepocreadiid metacercariae may therefore act as the second intermediate hosts (Figure 5). However, pelagic gastropods of the family Pterotracheidae have been reported to feed on gelatinous plankton (Hamner et al., Reference Hamner, Madin, Alldredge, Gilmer and Hamner1975), suggesting that Firoloida desmarestia and Pterotrachea coronata belonging to this family may acquire infection through predation on infected planktonic hosts and may also act as paratenic hosts.
The metacercariae and rediae of Prodistomum Type 5 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) have not previously been reported, although adults of this lineage were reported from Scomber japonicus in Japan (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024). The present findings suggest that metacercariae of Prodistomum Type 5 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) specifically infect holoplanktonic gastropods, whereas metacercariae of other Prodistomum species have been recorded from cnidarian and ctenophoran plankton (Cribb et al., Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026). In addition, Prodistomum Type 5 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) and Prodistomum Type 7 were shown here to use the cowrie Monetaria caputserpentis as the first intermediate host. Prodistomum Type 4 sensu Cribb et al. (Reference Cribb, Cutmore, Wee, Browne, Morales and Pitt2024) has been reported to use the cowrie Purpuradusta gracilis as the first intermediate host (Ansai et al., Reference Ansai, Sekine, Munakata, Sase, Sasaki, Nitta, Suzuki, Abe, Takano, Fukumori, Nakatsubata, Suzuki and Waki2025). These findings suggest that rediae of Prodistomum species may exhibit specificity to cowries. Confirmation of host specificity will require comprehensive surveys of gelatinous plankton and marine molluscs, including cowries.
We detected one metacercaria of Dinurus tornatus in the gut of the neustonic gastropod Glaucus atlanticus. This trematode species has been reported in the marine fish Coryphaena hippurus (Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024; Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026). Although we cannot deny that these fish are infected with Dinurus tornatus through predation on the gastropod host as discussed in a previous study (Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024), Glaucus atlanticus is unlikely to be a major source of infection for Coryphaena hippurus, as no published reports indicated that neustonic gastropods, including Glaucus atlanticus, are preyed upon by other organisms including fishes (Figure 5). In contrast, Dinurus tornatus has been reported from the neustonic cnidarian Physalia utriculus in Japanese waters (Waki et al., Reference Waki, Sekine, Aou, Nitta, Yamamoto, Toshino, Nakano, Okawa, Takano, Yamazaki and Hagihara2026), and Coryphaena hippurus has been reported to consume neustonic cnidarians (Kojima, Reference Kojima1961). These observations suggest that cnidarians are the major infection source of Dinurus tornatus for Coryphaena hippurus (Figure 5). Glaucus atlanticus has been reported to feed on Physalia cnidarians (Lalli and Gilmer, Reference Lalli and Gilmer1989), suggesting that metacercariae of Dinurus tornatus are transmitted to Glaucus atlanticus through predation on infected cnidarians. Planktonic copepods commonly serve as the second intermediate hosts for hemiurid trematodes (Køie, Reference Køie1979, Reference Køie1989, Reference Køie1990, Reference Køie1991; Marcogliese, Reference Marcogliese1995). Planktonic copepods are known prey of planktonic jellyfish (Nagasawa, Reference Nagasawa1987; Zenitani et al., Reference Zenitani, Kono, Watari and Tsukamoto2013), although records of neustonic jellyfish feeding on copepods are lacking. Neustonic jellyfish may therefore acquire infection by consuming unidentified organisms that have previously ingested infected copepods. As the infection routes of Dinurus tornatus to the host fish Coryphaena hippurus may involve multiple unidentified organisms harbouring metacercariae, the lifecycle of this trematode species could be more complicated than discussed above.
Metacercariae have been reported from pelagic gastropods in previous studies, but only a limited number of trematode species has been identified (Morales-Ávil et al., 2018; Louvard et al., Reference Louvard, Yong, Cutmore and Cribb2024), and the predator–prey relationships between pelagic gastropods and fishes have rarely been discussed. This study revealed that pelagic gastropods harboured metacercariae of several fish trematode species, suggesting that the gastropods are frequently preyed upon by marine fishes. Conversely, metacercariae were not detected in 6 of the 10 pelagic gastropod species examined in this study. Given the limited number of host specimens examined, further study with a larger number of host individuals may reveal additional metacercaria infection from these gastropods.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26101564.
Acknowledgements
The present study was kindly helped by Takumi Miyashita and Risa Niida. Besides, we appreciate the officers and crew of the research vessel Shinyo-maru of the Tokyo University of Marine Science and Technology. We are also indebted to Eiji Yamasaki (Blue Line Tajiri) and RCMB technicians Hirokazu Abe and Masahiko Washio (Tohoku University) for collecting the pelagic gastropods.
Financial support
This research received no specific grant from any funding agency, commercial entity, or not-for-profit sector.
Competing interests
The authors declare none.
Ethical standard
Formal ethical approval was not required because the study involved unregulated fish and invertebrate species.
