論文
Pseudanthocotyloides mamaevi Williams, 1988 (Polyopisthocotyla: Mazocraeidae) from Wild and Cultivated Japanese Anchovy Engraulis japonicus (Clupeiformes: Engraulidae) in Japan
2026 年 61 巻 1 号 p. 13-22
詳細
2026 年 61 巻 1 号 p. 13-22
The polyopisthocotylan responsible for anemia in the Japanese anchovy Engraulis japonicus has not previously been identified. In this study, specimens collected from both healthy wild Japanese anchovies and anemic cultured fish raised from eggs were identified as Pseudanthocotyloides mamaevi (Polyopisthocotyla: Mazocraeidae). This represents the first record of this species in Japan and provides a redescription accompanied by molecular data. Although P. mamaevi exhibits relatively high pathogenicity to Japanese anchovy, disease outbreaks have not been reported in aquaculture operations that use wild-caught larvae. This study therefore constitutes the first report of disease caused by this polyopisthocotylan in Japanese anchovy aquaculture.
The Japanese anchovy Engraulis japonicus (Clupeiformes: Engraulidae) is a small pelagic fish that is widely distributed in the Western Pacific, primarily along the southern Sakhalin Islands, Sea of Japan, and Pacific coast of Japan, extending southward near Taiwan, with occasional occurrences in the Philippines and Indonesia (Aonuma and Yagishita, 2013). Being a major commercial fish species in Japan, it has annual catches exceeding 120,000 tons (Ministry of Agriculture, Forestry and Fisheries of Japan, 2024). This species is utilized not only as food, but also as aquaculture feed and as bait in fisheries for carnivorous fish (Miyahara, 1997). In the 2010s, aquaculture techniques were developed to ensure a stable supply of Japanese anchovy (Oda and Inaba, 2023).
Despite their economic importance, little is known about the diseases affecting Japanese anchovies (Fujii et al., 2015). However, in autumn 1983, a significant anemia-associated mortality event among wild Japanese anchovies was reported in the Iyo-Nada area of the Seto Inland Sea (Yamamoto et al., 1984). This mortality was attributed to gill parasitism by the monogenean Pseudanthocotyloides sp. (Mazocraeidae) (Yamamoto et al., 1984). Later, based on photographs provided by Yamamoto et al. (1984), Williams (1988) hypothesized that this Japanese monogenean was either P. mamaevi Williams, 1988 or a closely related species. Zhang et al. (1999, 2001) reported P. mamaevi from Japanese anchovy collected in Fujian Province, China, but there was no discussion as to whether it is the same species as the Japanese species. In 2006, another case of anemia in wild Japanese anchovies was reported off the coast of Kyoto Prefecture in the Sea of Japan, associated with the polyopisthocotylan parasites (Nakatsugawa, 2007). Despite these reports, detailed identification and descriptions of the polyopisthocotylans parasitizing Japanese E. japonicus have not yet been conducted.
To date, four species of Pseudanthocotyloides have been described: P. heterocotyle (Van Beneden, 1871), P. banghami Price, 1959, P. dossae Caballero y C. and Bravo-Hollis, 1965, and P. mamaevi Williams, 1988 (Price, 1959; Caballero y C. and Bravo-Hollis, 1965; Euzet and Prost, 1969; Williams, 1988). Among these, P. heterocotyle has been reported to cause mass mortality and anemia in the Atlantic herring Clupea harengus at infestation levels exceeding 300 parasites per fish in a public aquarium in Scotland (Longshaw, 1996).
In 2023, a mass mortality event involving Japanese anchovies associated with anemia occurred at a trial fish farm in Kyoto Prefecture, Japan. The anemic symptoms began to appear in late September, and the mortality rate reached 85% by October. Upon dissection of diseased fish, numerous monogenean parasites were collected from the gills. This study aimed to identify gill parasites infesting cultivated Japanese anchovies from Kyoto Prefecture and wild Japanese anchovies from the Seto Inland Sea and North Pacific coast of Japan, using morphological characteristics and DNA data.
The cultivated Japanese anchovy consisted of 60 individuals hatched from the eggs of wild-caught fish in July 2023. The fish were reared in a semi-closed recirculating system with formulated feed at a water temperature of around 20°C until November 2023. Gills from 40 dead captive anchovies were examined for pale gills (a symptom of anemia) and for polyopisthocotylan infection. Polyopisthocotylan specimens collected from two moribund Japanese anchovies were fixed in Davidson’s solution or 99% ethanol in vials. Wild Japanese anchovies were collected from the Seto Inland Sea and the North Pacific by angling and commercial fishing (Table 1). The polyopisthocotylans were removed from the gills using needles or forceps under a dissecting microscope. The bodies of one and two parasite specimens from cultivated and wild fish, respectively, were separated from their haptors using needles and were preserved in 99% ethanol for molecular analysis. The remaining haptors and other specimens were flattened under coverslips and fixed in acetic acid-formalin-alcohol (AFA) or modified picrate glycerin (Nitta and Nagasawa, 2018).
| Prefecture | Site | n* | Body length (mm) | Date | Prevalence and intensity |
|---|---|---|---|---|---|
| Kyoto | Miyazu City (land-based fish farm) | 2 | ca. 70 (in total length)** | 4 October, 20 November 2023 | 100% (2/2), >20 per fish |
| Hiroshima | Off Kegoya, Kure City*** | 11 | 46.7–70.9 (in standard length [SL]) | 15 October 2014 | 36.4% (4/11), 1.75 (1–2) |
| Off Utsumi-chō, Fukuyama City**** | 34 | No data | 21 July 2019 | No data | |
| Mie | Off Minami-Ise**** | 1 | 94.0 (in SL) | 15 July 2022 | 100% (1/1), 1 |
For morphological analysis, polyopisthocotylan specimens fixed in Davidson’s solution or AFA were stained with Heidenhain’s iron hematoxylin or alum carmine. The specimens were dehydrated using a graded ethanol series, cleared in xylene, and mounted in Canada balsam. Drawings were prepared using a drawing tube attached to a Nikon Optiphot-2 compound microscope equipped with a phase-contrast microscope (Nikon, Japan). Images were captured with an Olympus DP28 digital camera (Olympus, Japan) mounted on an Olympus BX51 light microscope (Olympus, Japan). Measurements were taken from the images using ImageJ software (version 1.54g, https://imagej.net/ij/) and expressed in micrometers, with the range followed by the mean and number (n) of specimens examined in parentheses. The specimens were deposited at the Meguro Parasitological Museum (MPM) in Tokyo, Japan.
Fish identification was based on Aonuma and Yagishita (2013), and the scientific names of the fish mentioned in this study followed those of Froese and Pauly (2024). The prevalence and intensity of infection were determined as described by Bush et al. (1997). In terms of higher-level taxonomy, this study adopts the framework supported by recent phylogenetic analyses, which demonstrated the polyphyly of “Monogenea.” It follows the revision proposed by Brabec et al. (2023), in which the former “Monogenea” was divided and the two subclasses, Monopisthocotyla and Polyopisthocotyla, were elevated to class rank.
DNA analysisDNA extraction, and amplification of the 18S rDNA (18S), 28S rDNA (28S), internal transcribed spacer 1, 5.8S rDNA, internal transcribed spacer 2, and the 5′ end of the 28S rDNA (ITS1-5.8S-ITS2), as well as the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), were performed using the method described by Nitta and Ota (2025). Sequences were deposited in the International Nucleotide Sequence Database Collaboration (INSDC) via the DNA Data Bank of Japan and were compared with other sequences in the INSDC using BLASTn searches conducted on 26 October 2024.
Separate phylogenetic analyses were conducted using the 18S and 28S datasets. These datasets included newly obtained sequences and previously available Mazocraeidae sequences from INSDC (Table 2). The sequence alignments were performed using MAFFT version 7 (Katoh et al., 2019) with the “unalignlevel: 0.8” and “Leave gappy regions” options under the G-INS-i strategy. Ambiguous regions within the alignments were removed using Gblocks version 0.91b (Castresana, 2000) with the “Allow gap positions within the final blocks” option. Two hexabothriid species, Narcinecotyle longifilamentus Torres-Carrera, Ruiz-Escobar, García-Prieto, and Oceguera-Figueroa, 2020 and Pseudohexabothrium taeniurae Agrawal, Chisholm, and Whittington, 1996, were used as outgroups.
| Species | Host | Locality | 18S rDNA | 28S rDNA | Reference |
|---|---|---|---|---|---|
| Mazocraeidae (Mazocraeidea) | |||||
| Mazocraeinae | |||||
| Mazocraes alosae Hermann, 1782 | – | Mediterranean Sea | – | MT508780 | unpublished |
| Mazocraes bengalensis Sailaja, Shameem and Madhavi, 2019 | Opisthopterus tardoore (Pristigasteridae) | India: Bay of Bengal | – | KF306255 | unpublished |
| Heteromazocraes engrauliae Sailaja, Shameem and Madhavi, 2017 | Thryssa setirostris (Engraulidae) | India: Bay of Bengal | – | KF208425 | Sailaja et al. (2017) |
| Heteromazocraes lingmueni Zhang, Ding, Pang and Liu, 1998 | Coilia nasus (Engraulidae) | China: Yangtze River | – | KC412069 | unpublished |
| Heteromazocraes thryssaensis Sailaja, Shameem and Madhavi, 2017 | Thryssa setirostris | India: Bay of Bengal | – | KF306256 | Sailaja et al. (2017) |
| Kuhnia scombri (Kuhn, 1829) | Scomber scombrus (Scombridae) | United Kingdom | AJ228783 | AF382044 | Littlewood et al. (1998), Olson and Littlewood (2002) |
| Kuhnia sprostonae Price, 1961 | Scomber japonicus | Chile | – | MT890124 | unpublished |
| Leptomazocraes orientalis (Chauhan, 1952) | Dussumieria hasseltii (Dussumieriidae) | India: Bay of Bengal | – | KF147881 | unpublished |
| Mazocraeoides dussumieri Mamaev, 1975 | Dussumieria hasselti | India | – | KF306257 | unpublished |
| Mazocraeoides prashadi Chauhan, 1950 | – | – | KT267182 | – | unpublished |
| Paramazocraes setipinna Ding & Zhang in Zhang, Yang and Liu, 2001 | Setipinna taty (Engraulidae) | India: Bay of Bengal | – | JX984605 | Sailaja et al. (2016) |
| Paramazocraes thrissocles Tripathi, 1959 | Thryssa mystax (Engraulidae) | India: Bay of Bengal | – | KF147880 | Sailaja et al. (2016) |
| Pseudanthocotyloides mamaevi Williams, 1988 | Engraulis japonicus (Engraulidae) | Japan: Hiroshima | LC860876 | LC860879 | This study |
| Engraulis japonicus | Japan: Hiroshima | LC860877 | LC860880 | This study | |
| Engraulis japonicus | Japan: Kyoto | LC860878 | LC860881 | This study | |
| Grubeinae | |||||
| Neogrubea seriolellae Dillon and Hargis, 1968 | Seriolella porosa (Centrolophidae) | Argentina: off Patagonian coast | KM190942 | KM190943 | Hernandez-Orts et al. (2014) |
| Hexabothriidae (Diclybothriidea) | |||||
| Narcinecotyle longifilamentus Torres-Carrera, Ruiz-Escobar, García-Prieto and Oceguera-Figueroa, 2020 | Narcine entemedor (Narcinidae) | Mexico: Oaxaca | MN447330 | MN367803 | Torres-Carrera et al. (2020) |
| Pseudohexabothrium taeniurae Agrawal, Chisholm and Whittington, 1996 | Taeniura lymma (Dasyatidae) | Australia | AJ228791 | AF382035 | Littlewood et al. (1998), Olson and Littlewood (2002) |
The final alignments consisted of 1,655 positions (18S) and 768 positions (28S). The best-fit substitution models were selected using the Bayesian information criterion in IQ-TREE version 2.3.6 (Kalyaanamoorthy et al., 2017; Minh et al., 2020). Bayesian inference (BI) and posterior probability (PP) estimations were performed using MrBayes version 3.2.6 (Ronquist et al., 2012) with the following models: SYM + I + G4 (18S) and GTR + F + G4 (28S). Two independent runs of four Markov chains were executed for 1,000,000 generations with the trees sampled every 100 generations. The convergence and parameter estimates were assessed using Tracer version 1.6.0 (Rambaut et al., 2018). The first 25% of the trees from each run were discarded as burn-in. Maximum likelihood (ML) trees were generated using IQ-TREE version 2.3.6 with 1,000 bootstrap replicates, using TIM3e + I (18S) and TPM2u + F + G4 (28S) as models.
The land-based Japanese anchovy population (60 individuals) was maintained from July to November 2023. Parasitism by the polyopisthocotylan, accompanied by symptoms of anemia (pale gills), was observed in late September with a mortality rate of 85% by October, when the fish had grown to approximately 3 g in weight and 70 mm in total length. All 40 deceased fish examined were infested with the polyopisthocotylan. More than 20 parasites from two of these individuals were collected and preserved for identification (Table 1). In contrast, no signs of anemia, such as pale gills, were observed in the wild fish examined.
Redescription (based on eight stained and eight unstained specimens)Mazocraeidea Bychowsky, 1937
[Japanese name: Tsukami-mushi-moku]
Mazocraeidae Price, 1936
[New Japanese name: Tsukami-mushi-ka]
Mazocraeinae Price, 1961
[New Japanese name: Tsukami-mushi-aka]
Pseudanthocotyloides mamaevi Williams, 1988
[New Japanese name: Katakuchi-iwashi-tsukami-mushi]
Body (Fig. 1A) elongate, total length 1,488–3,147 (2,404, n = 9), maximum width 478–845 (617, n = 9) at level of germarium, length: width ratio 1: 0.21–0.36 (0.26, n = 9). Haptor (Fig. 1A) symmetrical, 373–860 (622, n = 15) long, 426–901 (727, n = 15) wide, consisting of 4 pairs of clamps with short peduncle, one pair of hamuli, one pair of marginal hooks I, and one pair of marginal hooks II. Clamp consisting of six elements (Fig. 1F): scleritum arcuatum posterius horseshoe shape; scleritum antero-supplementarium fusiform with notches in the center of the upper and lower edges and holes at both ends; scleritum medio-supplementarium bulging five-radiating shape with a rows of slightly larger holes in the center, smaller holes scattered to each side of the rows, and larger holes near the lower edge; scleritum medio-supplementarium heart-shaped with collapsed lower edge; scleritum postero-supplementarium semicircular with slightly protruding ends at lower edge and large hole in the center; scleritum arcuatum posterius U-shaped, with small holes at both ends. Clamp IV (Fig. 1A) anterior pair, largest, 63–167 (114, n = 16) long, 71–182 (128, n = 16) wide; clamp III (Fig. 1A), 44–109 (70, n = 16) long, 58–110 (83, n = 16) wide; clamp II (Fig. 1A) 44–89 (63, n = 16) long, 58–97 (72, n = 16) wide; clamp I (Fig1A) posterior pair, smallest, 34–77 (56, n = 16) long, 53–89 (68, n = 16) wide. Hamulus (Fig. 1G) with wide handle, 26–32 (28, n = 14) long. Marginal hook I (Fig. 1H) consisting of hook integrated with slightly thick handle, 16–19 (18, n = 7) long; marginal hook II (Fig. 1I) with thin handle, 12–14 (13, n = 3) long.
Mouth (Fig. 1A, B) terminal. Eye absent. A pair of buccal suckers (Fig. 1A, B) oval, muscular, lying in each lateral wall of buccal cavity, 27–36 (31, n = 7) long, 24–35 (29, n = 7) wide. Pharynx (Fig. 1B) oval to elliptical, 28–50 (42, n = 7) long, 22–42 (32, n = 7) wide. Esophagus (Fig. 1A) 135–289 (229, n = 5) long. Intestinal bifurcation dorsal to copulatory organ. Intestinal ceca with lateral and median diverticula, extending into haptor, not confluent posteriorly.
Testis (Fig. 1A) elongate, follicular, 348–725 (530, n = 4) long, 88–133 (115, n = 4) wide; testicular zone intercecal, posterolateral to germarium, leaching anterior side of haptor. Vas deferens (Fig. 1A, C) leaving from anterior margin of testis, running anteriorly in dorsal side, connecting copulatory organ. Copulatory organ (Fig. 1D, E) consisting of central muscular part and two lateral muscular parts on both sides, 15–26 (21, n = 14) long, 14–35 (26, n = 14) wide; central muscular part associated 4–7 (5, n = 13) hooks; two muscular parts with each one lateral hook.
Germarium (Fig. 1A, C) elongate, U-shaped, intercecal, middle of the body; germarium area 251–417 (343, n = 4) long, 88–117 (102, n = 4) wide. Oviduct (Fig. 1C) arising to the left anterior terminal of germarium, running anteriorly, connecting posterior side of seminal receptacle. Genitointestinal canal (Fig. 1A, C) leaving from right intestinal ceca, connecting left side of seminal receptacle. Right side of seminal receptacle (Fig. 1C) receiving the vitelline duct to form the ovovitelline duct. Ovovitelline duct (Fig. 1C) running posteriorly, turning upwards and forming oötype. Oötype (Fig. 1C) surrounded Mehlis’ grand at the base and continuing to uterus. Uterus (Fig. 1A) running midline anteriorly to copulatory organ. Vagin absent. Vitelline reservoir (Fig. 1A) Y-shaped, slightly anterior from median; vitellarium coextensive with intestinal ceca, extending into haptor. Egg (Fig. 1J) elliptical with short filament at both ends in uterus, 122 (n = 1) long, 73 (n = 1) wide without filament; length of two filaments along curve 69 and 97 (n = 1).
SynonymsPseudanthocotyloides mamaevi Williams, 1988: 95–97, Fig. 1; Zhang et al., 1999: 215; Zhang et al., 2001: 226–227, Fig. 15-2.
Pseudanthocotyloides mamaeve (sic) (without attribution): Liu and Chen, 2012: 212, unnumbered fig. (incorrect subsequent spelling).
Pseudanthocotyloides sp.: Yamamoto et al., 1984: 122–123, Figs 2, 3.
Monogenea gen., sp.: Nakatsugawa, 2007: 21, unnumbered fig.
Taxonomic summaryType host: Australian anchovy Engraulis australis (Clupeiformes: Engraulidae) (Williams, 1988)
Other host: Japanese anchovy Engraulis japonicus (Yamamoto et al., 1984; Zhang et al., 2001; Nakatsugawa, 2007; this study)
Type locality: Australia: Swan River Estuary, Perth (Williams, 1988).
Other localities: Japan: Iyo-nada, Ehime Prefecture (Yamamoto et al., 1984), off Kegoya, Kure City and off Numakuma, Fukuyama City, Hiroshima Prefecture, in Seto Inland Sea (this study); Kurita Bay, Sea of Japan (Nakatsugawa, 2007) and a land-based fish farm in Miyazu City (this study), Kyoto Prefecture; off Minami-Ise Town in North Pacific (this study). China: Chongwu Town, Fujian Province (Zhang et al., 1999, 2001).
Materials examined: MPM Coll. Nos. 25387–25391.
Representative DNA sequences: The 18S (LC860876–LC860878, 1,791 bp), 28S (LC860879–LC860881, 1,249 bp), ITS1-5.8S-ITS2 (LC860882–LC860884, 1,501 bp), and cox1 (LC860885–LC860887, 420 bp). DNA sequences were determined from one individual from cultivated Japanese anchovy (MPM Coll. No. 25390) and two individuals from the Seto Inland Sea (MPM Coll. No. 25388), and all sequences were identical.
Japanese name: Nitta et al. (2019) named the order Mazocraeidea “Tsukami-mushi-moku”, referring to their use of clamps to attach to hosts (tsukami-mushi = grasping worms; moku = order). Following this convention, new Japanese names are proposed for the family (-ka) and subfamily (-aka). The name “Katakuchi-iwashi-tsukami-mushi” combines the Japanese host name with tsukami-mushi.
RemarksPseudanthocotyloides mamaevi was originally described from the gills of the Australian anchovy Engraulis australis collected in the Swan River Estuary, Perth, Western Australia (Williams, 1988). The measurements and morphological features of the reproductive system and sclerotized structures of the specimens in this study were nearly identical to those described by Williams (1988) and Zhang et al. (2001) (Table 3). This polyopisthocotylan species is distinguished from the other three Pseudanthocotyloides species by the position of the testis, which lies lateral and posterior to the germarium (as opposed to being posterior to the germarium in other species; Table 3). Based on morphological features, measurements, and comparisons with newly collected specimens, the unidentified species parasitizing Japanese anchovies in the Seto Inland Sea and the Sea of Japan off Kyoto, as reported in the study by Yamamoto et al. (1984) and Nakatsugawa (2007), were confirmed as P. mamaevi.
| Species | P. mamaevi | P. heterocotyle | P. banghami | P. dossae | |||||||||
| Reference | This study | Yamamoto et al. (1984) | Williams (1988) | Zhang et al. (2001) | Van Beneden (1871); Linstow (1885) | Euzet and Prost (1969) | Mamaev (1982) | Kohn et al. (1992) | Timi et al. (1999) | Rahimian et al. (1999) | Price (1959, 1961) | Caballero y C. and Bravo-Hollis, 1965 | |
| Host | Engraulis japonicus | E. japonicus | E. australis | E. japonicus | Sprattus sprattus as Clupea sprattus | S. sprattus as C. sprattus | S. sprattus; E. enchrasicholus | Ctengraulis edentulous; Decapterus punctatus | E. anchoita; Anchoa marini, | Clupea harengus; S. sprattus | Dorosoma cepedianum | A. hepsetus | |
| Locality | Seto Inland Sea, North Pacific, Japan | Seto Inland Sea, Japan | Western Australia, Australia | Fujian, China | Ostende, Belgium | Mediterranean Sea, Sète, France | Mediterranean Sea (including Sète, France) | Governador, Brazil | Argentine Sea, Argentina | Swedish coast, Sweden; North Sea, UK | Tennessee and Alabama, USA | Gulf of Mexico, Mexico | |
| Body | length (L) | 1,488–3,147 | 2,400–2,900 | 1,287–1,773 | 1,613–2,864 | 2,500 | 2,500–2,800 | — | 899–1,613 | 1,320–2,200 | 1,481–3,488 | 1,400–3,500 | 954 |
| width (W) | 478–845 | 580–680 | 343–601 | 259–724 | 120 | 500–600 | — | 148–480 | 320–680 | 111–1610 | 300–500 | 223 | |
| Haptor | L × W | 373–860 × 426–901 | — | — | 310–672 × 372–724 | — | — | — | — | — | — | 500 (L) | 298 × 432 |
| Clamp VI | L × W | 63–167 × 71–182 | 100–150 × 75–100 | 91–115 (W) | 108–175 × 152–188 | 130 (D) | 100–110 × 105–120 | — | 79–135 × 102–159 | 130–183 × 149–206 | 115–165 × 100–165 | 120–140 (D) | 122 × 129 |
| Clamp III | L × W | 44–109 × 58–110 | 50–60 × 45–50 | 63–78 (W) | — | 66 (D) | 50–55 × 70–75 | — | 44–67 × 48–67 | 40–57 × 57–69 | 68–102 × 48–80 | 80–90 (D) | 74 × 78 |
| Clamp II | L × W | 44–89 × 58–97 | — | 51–70 (W) | — | 66 (D) | 50–55 × 70–75 | — | 41–66 × 48–67 | 42–55 × 48–65 | 65–90 × 44–70 | 70 (D) | 63 × 74 |
| Clamp I | L × W | 34–77 × 53–89 | — | 48–34 (W) | 41–82 × 54–95 | 66 (D) | 50 × 60 | — | 36–60 × 48–60 | 38–48 × 42–57 | 60–83 × 48–63 | 60 (D) | 63 × 74 |
| Marginal hook I | L | 16–19 | — | 12–13 | 13–15 | 16 | 15–17 | — | — | 17–21 | 8–20 | 20–24 | 15 |
| Marginal hook II | L | 12–14 | — | 12 | — | — | 10–12 | — | — | 6–8 | 6–12 | 10 | — |
| Hamulus | L | 26–32 | — | 23–34 | 22–26 | 29 | 25–33 | — | — | 23–31 | 22–34 | 60–68 | 26 |
| Buccal sucker | L × W | 27–36 × 24–35 | — | — | 38–50 × 31–43 | 33 (D) | 25 (D) | — | 14–41 × 19–40 | 25–40 × 23–38 | 23–45 (L) | 40–60 (D) | 26 × 31 |
| Pharynx | L × W | 28–50 × 22–42 | — | 31–55 (diameter [D]) | — | — | 40 (D) | — | 32–47 × 26–44 | 38–48 × 29–38 | 40–71 × 28–57 | 60 × 36–40 | 33 × 30 |
| Esophagus | L | 135–289 | — | — | — | — | — | — | — | — | 365 | — | — |
| No. of copulatory organ hooks | 2 lateral, 4–7 medial | — | 2 lateral, 5–6 medial | 2 lateral, 4 medial | — | 2 lateral, 4 medial | — | — | 2 lateral, 4 medial, 1–4 marginal | 2 lateral, 4–5 medial | 2 lateral, 4 anteromedial, 5 posteromedial | 2 lateral, 6 medial | |
| Copulatory organ (CO) | L × W | 15–26 × 14–35 | — | 16–24 (D) | 23–28 × 26–34 | — | 20–25 (D) | — | — | 29–38 × 25–38 | 20–40 × 18–31 | — | — |
| Distance from anterior end to CO | 211–285 | — | — | — | — | — | — | 178–248 | — | About 120 | — | ||
| Testis area | Lateral and posterior to germarium | — | Lateral and posterior to germarium | — | — | Posterior to germarium | — | — | Posterior to germarium | Posterior to germarium | Posterior to germarium | Posterior to germarium | |
| Germarium area | L × W | 251–417 × 88–117 | — | — | — | — | — | — | — | 224–400 × 80–128 | — | — | 377 × 59 |
| Vagina | Absent | — | Absent | — | — | Absent | — | — | Present, mediodorsal | Absent | Absent | Not observed | |
| Genito-intestinal canal connection | Right intestinal cecum | — | Right intestinal cecum | — | — | Right intestinal cecum | — | — | Right intestinal cecum | Left intestinal cecum | Right intestinal cecum | — | |
| Egg | L × W | 122 × 73 | — | — | 129 × 49 | — | — | — | 210 × 68 | — | 228–234 (L) | 200 × 70 | — |
A BLAST query of the 18S rDNA fragment of P. mamaevi revealed its closest match to Mazocraeoides prashadi Chauhan, 1950 (Mazocraeidae) (KT267182), with 97.60% identity and 100% coverage (1,751/1,794 bp). The closest match for the 28S rDNA sequence was Leptomazocraes orientalis (Chauhan, 1952) (Mazocraeidae) (KF147881), with 96.57% identity and 72% coverage (873/904 bp). The ITS1-5.8S-ITS2 and cox1 sequences showed similarity to Mazocraes alosae Hermann, 1782 (Mazocraeidae) (KT267184: 92.25% identity, 28% coverage, 393/426 bp) and Winkenthughesia bramae (Parona and Perugia, 1896) (Pseudodiclidophoridae) (ON787609: 80.29% identity, 100% coverage, 338/421 bp), respectively.
Phylogenetic analysisPhylogenetic trees constructed using BI and ML methods displayed nearly identical topologies. The BI trees based on the 18S and 28S datasets are presented in Fig. 2. Both of the analyses indicated that Neogrubea seriolellae Dillon and Hargis, 1968 diverged earlier than other mazocraeids. In the 18S tree, Kuhnia scombri (Kuhn, 1829) branched off, followed by a sister group of Mazocraeoides prashadi and P. mamaevi.
The 28S BI tree showed a similar topology, with the clade containing P. mamaevi forming a sister group to Mazocraeoides species. In contrast, the ML tree for the 28S dataset placed Kuhnia and Mazocraeoides species as sister groups, although the bootstrap support (BS) value for this relationship was low at 34.
Pseudanthocotyloides heterocotyle has been reported in Europe, Brazil, and Argentina (Table 4), and their records may include multiple species. Notably, specimens collected by Timi et al. (1999) from Engraulis anchoita and Anchoa marini in Argentina possessed a vagina not observed in other Pseudanthocotyloides species, and their copulatory organs featured additional marginal spines (1–4) along with two lateral and four medial spines. Molecular data are invaluable for detecting cryptic species, and this study demonstrates the utility of major DNA markers (28S rDNA, ITS1-5.8S-ITS2, and cox1) for Pseudanthocotyloides species. Further taxonomic research using molecular tools and the re-evaluation of P. heterocotyle are warranted. Additionally, the subfamily classification of Mazocraeidae by Price (1961) and its revision by Mamaev (1982) merit consideration. Our phylogenetic analysis revealed that the subfamily Kuhninae sensu Price (1961) is polyphyletic, supporting the revised system of Mamaev (1982).
Infestations by P. mamaevi can induce anemia in cultivated Japanese anchovies. Members of the genus Pseudanthocotyloides are polyopisthocotylans, which are blood-sucking parasites that are likely to induce severe anemia in heavily infected hosts (Whittington and Chisholm, 2008). For example, Longshaw (1996) reported that infections of P. heterocotyle exceeding 300 individuals per fish caused mass mortality associated with anemia in Atlantic herring weighing approximately 50 g. Given that Japanese anchovies are much smaller (ca. 10 g~ in mature fish; Matsuda et al., 2014), even relatively low parasite burdens may produce a similar effect. Supporting this, Yamamoto et al. (1984) documented anemic wild populations of Japanese anchovy in Iyo-nada with a mean body weight of 1.1–2.0 g (fork length: 58–62 mm) and a mean parasite intensity of 11.2 (range: 2–62). In the present study, all 40 fish examined from the tank (body weight: ~3 g; total length: ~70 mm) were parasitized, with parasite counts exceeding 20 individuals per fish indicating that the mass mortality observed at the fish farm was most likely caused by this parasite.
Infection with the polyopisthocotylan may also causes mortality in larval Japanese anchovies, which may explain why parasitism at this early stage appears to be rare. In the wild, adult Japanese anchovies, which live for 2–3 years (Yasumi et al., 2007), are known to harbor P. mamaevi throughout the year (Yamamoto et al., 1984; Nakatsugawa, 2007; this study). Juvenile to young fish (1–3 g body weight) are susceptible to anemia when infested with approximately 10 parasites (Yamamoto et al., 1984; this study), indicating that larval fish are unlikely to survive infestation by this polyopisthocotylan. Consistent with this, no disease outbreaks attributed to P. mamaevi have been reported in Japanese anchovies reared from wild-caught larvae in marine net cages or flow-through tanks (Matsuda et al., 2014; Fujii et al., 2015; Oda and Inaba, 2023). This suggests that wild larval Japanese anchovies remain free of the polyopisthocotylan, as any infested larvae would not survive. An alternative possibility is that the seed larvae used in previous aquaculture operations simply had limited opportunities to encounter the parasite. However, given that Japanese anchovy is widely distributed across the Sea of Japan, the Seto Inland Sea, and the Pacific coast of Japan with P. mamaevi (Yamamoto et al., 1984; Nakatsugawa, 2007; this study), this explanation seems unlikely. In the present study as well, Japanese anchovies reared from July were not found to be infested until they had grown to juveniles in September. Because the trial farm employed a semi-circulating system, parasite eggs likely entered the tank from the sea and continuously exposed the fish to infective stages, yet only juveniles, survived long enough for infestations to become apparent. To test the hypothesis that P. mamaevi is lethal pathogenic to Japanese anchovy larvae, experimental infestation at different fish stages, and target parasite surveys of wild larval and juvenile Japanese anchovies from regions where this polyopisthocotylan occurs would provide critical information.
I thank Fumitaka Kudoh (Fisheries Technology Department; Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Center) for providing information about cultured fish and assisting with the shipment of specimens. I would like to thank Takuya Sato (Kyoto University), Kazuya Nagasawa, and Susumu Ohtsuka (Hiroshima University) for allowing me to use their laboratory facilities. I am grateful to Editage (www.editage.com) for English language editing. I also thank anonymous reviewers for valuable comments to improve the manuscript. This study was supported by JSPS KAKENHI grants (no. JP18J00466); the Food Safety and Consumer Affairs Bureau, the Ministry of Agriculture, Forestry, and Fisheries of Japan; and the Japan Fisheries Research and Education Agency.
