Introduction
The world’s deepest recorded depth for trematodes is approximately 6200 m, as reported by Takano et al. (Reference Takano, Fukumori, Kuramochi and Kano2023). They identified 28S ribosomal DNA (rDNA) of a trematode from the genus Lepidapedon in a DNA extract obtained from a deep-sea gastropod of the family Velutinidae, sampled via a trawl in depth from 6185 to 6221 m in the Kuril–Kamchatka Trench in the Northwest Pacific. As Lepidapedon species use fish as definitive hosts (eg, McCauley, Reference McCauley1968), the trematode found in the gastropod was presumed to be in the larval stage, although its development stage remains uncertain. Furthermore, the specific species of the trematode remained unknown because adult-stage sequences had not been reported. In the present study, we obtained an adult Lepidapedon oregonense from the macrourid fish Coelorinchus gilberti in Japan. Based on a comparison of 28S rDNA sequences, we determined that L. oregonense is the trematode species reported by Takano et al. (Reference Takano, Fukumori, Kuramochi and Kano2023). Overall, the objective of the present study was to identify the species of the trematode recorded at the deepest depth.
Materials and methods
On February 18 and April 1, 2023, one individual Coelorinchus gilberti was sampled through angling at 1000 and 1000 – 1100 m depths, respectively, in Sagami Bay, North Pacific, Japan. The collected fishes were dissected immediately, and their internal organs were transported to the laboratory. These organs were examined under a stereomicroscope, and 4 trematodes were detected. Three of the 4 trematode individuals were damaged. The trematodes were mounted on slides, gently pressed between the slides and coverslips, and fixed in 70% ethanol for several days. Subsequently, a piece of body tissue was cut from each trematode for polymerase chain reaction (PCR), as described later. For morphological observation, the remaining tissues of the trematodes were stained with iron-hematoxylin, dehydrated with ethanol series, cleared with xylene, and mounted on slides with Canada balsam. The intact trematode specimen was identified as L. oregonense, as described later, although the remaining 3 species could not be identified morphologically because of the extent of damage. The trematodes were measured using a light microscope (BX53, Olympus) equipped with a digital camera unit (AdvanCam-U3II, Advan vision). All measurements are in micrometers.
Alkaline lysates of the trematode tissues were individually made, as previously reported by Waki et al. (Reference Waki, Nakao, Sasaki, Ikezawa, Inoue, Ohari and Miyazaki2022), and used as templates for PCR. Genes for nuclear 28S rDNA, mitochondrial cytochrome c oxidase subunit 1 (COI), and mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) were selected. Primers, PCR amplification, subsequent DNA sequencing, and alignment data preparation were performed as described by Pérez-del-Olmo et al. (Reference Pérez-del-Olmo, Dallarés, Georgieva, Constenla, Kostadinova and Carrassón2019) and Waki et al. (Reference Waki, Nakao, Sasaki, Ikezawa, Inoue, Ohari and Miyazaki2022). For the 28S rDNA (794 bp), Bayesian inference analyses employing sequences of related species from GenBank were conducted using MrBayes v.3.2.7 (Ronquist et al., Reference Ronquist, Teslenko, Van Der Mark, Ayres, Darling, Höhna and Huelsenbeck2012), including Markov chain Monte Carlo searches on simultaneous runs for 800,000 generations, with trees sampled every 1000 generations under the Hasegawa–Kishono–Yano + G model (Hasegawa et al. Reference Hasegawa, Kishino and Yano1985). In this analysis, Gorgocephalus spp. (MW353882 and MW353889), closely related to the family Lepidapedidae, was selected for the outgroup. Pairwise comparison of divergence values was performed using MEGA X (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018) for both 28S rDNA and nad1 datasets to identify the species of our trematode, setting the p-distance. The determined DNA sequences were deposited in the DDBJ/ENA/GenBank databases under the accession numbers.
Results and discussion
Molecular analysis
We amplified 4 DNA fragments (1205 – 1257 bp) of 28S rDNA from 4 trematode individuals. Two sequences, including that from the trematode identified as L. oregonense, were identical. By comparing these sequences (796 bp) with those from previous studies, we found that the divergence values for intraspecific and interspecific variations within the family Lepidapedidae were 0 − 0.0013 and 0.0026 − 0.0771, respectively. Our 2 identical sequences matched with Lepidapedon sp. TT-2023 reported in Takano et al. (Reference Takano, Fukumori, Kuramochi and Kano2023) (LC767449) with a divergence value of zero, indicating that they belonged to the same species: L. oregonense. Regarding nad1 analysis, intraspecific divergence values within the genus Lepidapedon were 0 − 0.0467. Both sequences from our L. oregonense adults were identical (divergence values of zero), confirming their identity as the same species. The remaining 2 broken adults were phylogenetically classified as members of the genus Lepidapedon, as detailed below; however, they could not be identified at the species level through molecular analysis because of the absence of closely matching sequences in the online database (divergence values between the unidentified adults and Lepidapedon spp. from the database: 0.0151 − 0.0465 [28S rDNA] and 0.1729 − 0.2664 [nad1]). The 28S rDNA tree, based on 794 bp, showed that our 4 trematode individuals belonged to the genus Lepidapedon because they formed a clade with other species of this genus supported by a high posterior probability value (0.99) (Fig. 1A). Therefore, the 2 unidentified trematodes were labeled “Lepidapedon sp. TK1” and “Lepidapedon sp. TK2.” Determining the DNA sequences of 28S rDNA and nad1 for these 2 species will aid in identifying their species once adults that can be identified by morphology are obtained and their DNA sequences established.
Bayesian inference phylogram based on trematodes inferred from partial nucleotide sequences of nuclear 28S ribosomal DNA (794 bp) (A) and Lepidapedon oregonense sampled in the present study (B). In A, posterior probability values lower than 0.95 are omitted. Database accession numbers are shown after the names of the sequences. Asterisks represent the sequences of the present study (LC797520-797523). The outgroups are Gorgocephalus spp. The scale bar indicates the number of substitutions per site. In B, the dotted line shows the outline of tissue excised for use in polymerase chain reaction (arrowhead).
Morphologic descriptions of L. oregonense
This study used a gravid adult with no damage for morphologic description. The specimen was observed ventrally. All measurements, unless indicated otherwise, are in micrometers.
Family Lepidapedidae Yamaguti, 1958
Genus Lepidapedon Stafford, 1904
Species Lepidapedon oregonense McCauley, Reference McCauley1968
Descriptions (Figure 1B, Table 1): Body narrow and long, subcylindrical. Body 7.1 mm in length and 340 in greatest width. Oral sucker situated anterior terminal of body, 223 in length and 159 in width. Prepharynx long, 280 in length. Pharynx large and clearly observed, 116 in length and 92 in width. Esophagus long, 299 in length. Caeca bifurcation middle between pharynx and ventral sucker. Ventral sucker at about an anterior quarter of body length, 153 in length and 130 in width. Distance between caeca bifurcation and ventral sucker 569. Testis oval and slightly elongated, width close to body width. Distance between two testes 436. Anterior testis 330 in length and 272 in width. Posterior testis 334 in length. External seminal vesicle 512 in length and 164 in width. Cirrus sac 280 in length and 138 in width. Genital opening situated posterior to caeca bifurcation. Ovary anterior two-thirds of body, slightly elongated oval with irregular outline, 250 in length and 114 in width. Distance between ovary and anterior testis 294. Anterior margin of vitelline not reaching ventral sucker. Eggs oval, 64 — 73 in length and 45 — 50 in width. Excretory vesicle reaching close to the posterior margin of the posterior testis.
Morphological comparisons between the adult trematode in the present study and related Lepidapedon species
All measurements, unless indicated otherwise, are in micrometers. Dashes indicate no description in the cited reference.
a The width could not be measured because part of the testis was lost when the tissue was cut out for polymerase chain reaction.
Remarks: The genus Lepidapedon currently includes 47 species (WoRMS 2024). In our specimen, the vitellarium does not extend to the ventral sucker, and the excretory vesicle is close to the posterior margin of the posterior testis. These morphologic features characterize our specimen as a member of the “Desclersae subgroup,” which includes 10 species, such as L. blairi (Bray et al., Reference Bray, Faliex, Allienne and Mouahid2013). In this subgroup, our specimen resembles L. filiforme and L. oregonense because of the narrow body. Among them, we identified our specimen as L. oregonense based on specific morphologic features, such as ovaries with a slightly irregular outline (vs spherical in L. filiforme), slightly elongated testes (vs oval almost circular), and small eggs (egg length 72 — 85 μm in L. oregonense and 95 — 108 μm in L. filiforme).
Hosts and trematode stages: Coelorinchus gilberti (adult, this study). Coryphaenoides filifer (adult, McCauley Reference McCauley1968), Coryphaenoides leptolepis (adult, McCauley, Reference McCauley1968). Velutinidae gen. sp. (larval stage, Takano et al., Reference Takano, Fukumori, Kuramochi and Kano2023)
Sampling locations: North Pacific Ocean. Sagami Bay (1000 – 1100 m depth) (this study). 166 – 165 km off Oregon, USA (2800 – 2865 m depth) (McCauley, Reference McCauley1968). The Kuril–Kamchatka Trench (6185–6221 m depth) (Takano et al., Reference Takano, Fukumori, Kuramochi and Kano2023).
Date of collection: April 1, 2023
DNA markers: 28S rDNA, LC797520 – 797521. COI, LC797524. Nad1, LC810999–LC811000.
Japanese name: Sokodara shinkai kyuchu (trematode of macrourid fish in deep sea)
Lepidapedon oregonense distributed across a wide range of depths and a broad area of the North Pacific Ocean (McCauley, Reference McCauley1968; Takano et al., Reference Takano, Fukumori, Kuramochi and Kano2023). Given the presence of L. oregonense in 2 genera of fish belonging to the family Macrouridae, this trematode species possibly infects various species of macrourid fish. The intermediate host gastropod Velutinidae gen. sp. has only been reported at depths of approximately 6200 m (Fukumori et al., Reference Fukumori, Takano, Hasegawa and Kano2019; Takano et al., Reference Takano, Fukumori, Kuramochi and Kano2023). Considering the extensive depth range of L. oregonense, it is plausible that other mollusc species inhabiting shallower waters may also serve as intermediate hosts of this trematode.
Morphologic descriptions of the remaining 2 species
Gravid adult of each species, referred to as Lepidapedon sp. TK1 and TK2 were used for morphologic descriptions.
Lepidapedon sp. TK1
Descriptions (Supplementary Fig. 1A) Body long, anterior and posterior sides of body damaged. Body 1.2 mm in maximum width. Pharynx slightly damaged, 218 in length and 202 in width. Esophagus long, 567 in length. Ventral sucker small, 216 in length and 193 in width. Testis oblong and its width one-third of body width. Anterior testis 642 in length and 415 in width. Posterior testis 687 in length and 411 in length. External seminal vesicle large. Cirrus sac 440 in length and 274 in width. Ovary damaged. Anterior margin of vitelline not reaching ventral sucker. Eggs oval, 57 – 62 in length and 24 – 28 in width. Excretory vesicle reaching close to the posterior margin of the posterior testis.
Remarks: This species differs from L. oregonense in body size (maximum width 1.2 mm vs 340μm in L. oregonense) and testes size (ca. half to one-third of body width vs close to body width in L. oregonense). This specimen’s vitellarium does not reach the ventral sucker, and the excretory vesicle reaches the posterior margin of anterior testis; therefore, it is classified to “elongatum subgroup” in Bray (Reference Bray, Faliex, Allienne and Mouahid2013). Lepidapedon sp. TK1 shares similarities with L. arlenae, L. mariannae, and L. sereti given its elongated body and small testes. However, Lepidapedon sp. TK1 can be distinguished from L. arlenae by divergence values of 0.0289 for 28S rDNA and 0.1752 for nad1, which are all outside of the ranges of intraspecific values in this genus.
DNA markers: 28S rDNA, LC797522. Nad1, LC810997.
Lepidapedon sp. TK2
Descriptions (Supplementary Fig. 1B) Anterior and posterior sides of body damaged. Body 1.9 mm in maximum width. Testis round, width ca. half of body width. Anterior testis 850 in length and 773 in width. Posterior testis 785 in length and 855 in length. Ovary oval, 470 in length and 405 in width. Eggs oval, 61 – 69 in length and 32–38 in width. Excretory vesicle reaching close to the posterior margin of the posterior testis.
Remarks: This species differs from L. oregonense in body size (maximum width 1.9 mm vs 340 μm in L. oregonense) and testes shape and size (round and approximately half of the body width vs oval and slightly elongated with a width close to that of the body in L. oregonense). Due to substantial damage or loss of many organs in Lepidapedon sp. TK2, finding species with similar morphologic characters is exceedingly challenging.
DNA markers: 28S rDNA, LC797523. Nad1, LC810998.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000269.
Acknowledgements
We greatly appreciate Mr Hirokazu Taniguchi, Mr Shuzo Yumoto and Mr Takashi Nishino for their help with sample collection and fish identification for this study. We also greatly appreciate Dr Tsuyoshi Takano for helping our research.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interest
The author(s) declare none.
Ethical standard
No approval of research ethics committees was required to accomplish the goals of this study because experimental work was conducted with an unregulated fish and invertebrate species.
