Molecular and Morphological Characterizations of the Fish Parasitic Isopod Mothocya parvostis (Crustacea: Cymothoidae) Parasitizing Optional Intermediate Hosts: Juveniles of the Cobaltcap Silverside Hypoatherina tsurugae and Yellowfin Seabream Acanthopagrus latus

© 2023 Academia Sinica, Taiwan Open Access Molecular and Morphological Characterizations of the Fish Parasitic Isopod Mothocya parvostis (Crustacea: Cymothoidae) Parasitizing Optional Intermediate Hosts: Juveniles of the Cobaltcap Silverside Hypoatherina tsurugae and Yellowfin Seabream Acanthopagrus latus Hiroki Fujita1,* , Kentaro Kawai1, Diego Deville1, and Tetsuya Umino1 1Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan. *Correspondence: E-mail: [email protected] (Fujita). E-mail: [email protected] (Kawai); [email protected] (Deville); [email protected] (Umino) Received 16 June 2022 / Accepted 10 February 2023 / Published 22 May 2023 Communicated by Benny K.K. Chan Mothocya parvostis (Isopoda: Cymothoidae) is a parasitic crustacean that infests the opercular cavities of fishes. Its main final host is the Japanese halfbeak, Hyporhamphus sajori. However, M. parvostis also infests the black sea bream, Acanthopagrus schelgelii, as an optional intermediate host. Understanding the use of optional intermediate hosts is important for understanding the life history of Cymothoidae, and further information should be obtained. In this study, we aim to investigate the life cycle of M. parvostis. We collected and examined 20 mancae and 144 juveniles of M. parvostis from 129 cobaltcap silversides, Hypoatherina tsurugae, and 494 yellowfin seabreams, Acanthopagrus latus. Molecular analysis of the cytochrome c oxidase subunit I gene and 16S rRNA genes revealed that cymothoid mancae and juveniles from the two fish species were identified to be M. parvostis. All M. parvostis on H. tsurugae and A. latus might be mancae or juveniles, with no adult parasites; thus, H. tsurugae and A. latus juveniles were optional intermediate hosts of M. parvostis. In the results of morphological description, M. parvostis juveniles infesting the final host H. sajori lacked swimming setae, while juveniles parasitizing the two optional intermediate hosts had them. Mothocya parvostis mancae infested juveniles of both species just after metamorphosis, grew with the host. As the fish grows further, the parasite detached from the fish. The parasitic status of M. parvostis in the three optional intermediate hosts indicated that M. parvostis likely reproduced from June to December, and different optional intermediate hosts were used depending on the time of year in Hiroshima Bay. Therefore, a parasitic strategy involving optional intermediate hosts might increase the infestation success of M. parvostis to H. sajori. Key words: Life cycle, Manca, New host record, Parasitic cymothoid, Prevalence. Citation: Fujita H, Kawai K, Deville D, Umino T. 2023. Molecular and morphological characterizations of the fish parasitic isopod Mothocya parvostis (Crustacea: Cymothoidae) parasitizing optional intermediate hosts: juveniles of the cobaltcap silverside Hypoatherina tsurugae and yellowfin seabream Acanthopagrus latus. Zool Stud 62:21. doi:10.6620/ZS.2023.62-21. BACKGROUND Cymothoidae Leach, 1818 includes 363 species under 42 genera of cosmopolitan isopod parasites (Boyko et al. 2008 onwards). Their hosts include diverse taxa of fish inhabiting marine, brackish, and freshwater Zoological Studies 62:21 (2023) doi:10.6620/ZS.2023.62-21 1 © 2023 Academia Sinica, Taiwan environments (Yamauchi 2016). These parasites attach to their hosts at four sites: the opercular cavity, buccal cavity, abdominal cavity, and body surface (Smit et al. 2014; Aneesh et al. 2021). Cymothoids have six life stages (Brusca 1978a b 1981; Smit et al. 2014; Aneesh et al. 2015 2016 2018; Aneesh and Kappalli 2020), but are mainly identified on the basis of the morphological characteristics of adult females; thus, morphology-based species identification is difficult, and molecular analysis is the only way to identify non-female specimens (Fujita et al. 2021). Free-swimming mancae (larvae) grow into juveniles and adult males on the hosts, and then adult cymothoids change sex from male to female (Brusca 1978a b 1981; Smit et al. 2014; Aneesh et al. 2015 2016 2018; Aneesh and Kappalli 2020). Mancae of cymothoids search for hosts in a free-swimming stage. Once a host is found, they develop into juveniles. However, free-swimming juveniles of Mothocya, Nerocila, and Anilocra have been collected (Saito et al. 2014). Mancae and juveniles of some cymothoid species, such as Anilocra clupei Williams & Bunkley-Williams, 1986, A. pomacentri Bruce, 1987, Mothocya parvostis Bruce, 1986, Nerocila acuminata Schioedte and Meinert, 1881, Olencira praegustator (Latrobe, 1802), and Telotha henselii (Martens, 1869), temporarily infest fishes other than their final hosts (Adlard and Lester 1995; Lindsay and Moran 1976; Segal 1987; Taberner et al. 2003; Fogelman and Grutter 2008; Fujita et al. 2020; Fujita 2022); these hosts are called “optional intermediate hosts.” These intermediate hosts are “optional” because they are not necessary in a parasite’s normal life cycle (Fig. 1). Also, free-swimming juveniles were collected, presumably after leaving the optional intermediate hosts (Fujita et al. 2023). Mothocya parvostis is a common Cymothoidae in Japan which infests the Japanese halfbeak Hyporhamphus sajori (Temminck and Schlegel, 1846), large-scale blackfish Girella punctata Gray, 1835, and Japanese amberjack Seriola quinqueradiata Temminck and Schlegel, 1845 as final hosts (Bruce 1986). A major final host is H. sajori (Nagasawa 2020), and it shows a high prevalence of approximately 50% (Kawanishi et al. 2016; Fujita et al. 2020). In Hiroshima Bay, M. parvostis infests juveniles of the black sea bream Acanthopagrus schlegelii (Bleeker, 1854), as an optional intermediate host (Fujita et al. 2020). The presence of optional intermediate hosts has not been examined in species other than A. schlegelii. The reproduction cycles of Cymothoidae organisms vary. Anilocra pomacentri has no fixed reproduction season and reproduces throughout the year (Adlard and Lester 1995), and Mothocya epimerica Costa, 1851 has four reproduction seasons per year (Bello et al. 1997). The reproduction cycles of M. parvostis are unknown. So, there is a possibility that juvenile fishes that appear in other seasons may be used as optional intermediate hosts. Thus, we focused on juveniles of the yellowfin seabream Acanthopagrus latus (Houttuyn, 1782), a related species of A. schlegelii that appears in Hiroshima Bay in different seasons than A. schelgelii. Acanthopagrus latus is as recreationally and commercially important in the Indo-West Pacific region as A. schlegelii (Iwatsuki 2013). One of the most significant ecological differences between A. latus and A. schlegelii is their spawning season. In particular, A. latus spawns in autumn (Abol-Munafi and Umeda 1994; Nishida 2022), whereas A. schlegelii spawns in spring (Kawai et al. 2017 2020 2021). We also focused on the juveniles of cobaltcap silverside Hypoatherina tsurugae (Jordan and Starks, 1901), which grow in the period between the growth seasons of A. schlegelii and A. latus. Hypoatherina tsurugae serves as a food source for various carnivorous fishes (Mori et al. 1988) and is used for recreational fishing. Its spawning season in coastal Japan is from May to July, and the juveniles are collected from June to October (Mori et al. 1988). In this study, we performed DNA analysis to identify the cymothoids infesting juveniles of H. tsurugae and A. latus as M. parvostis. In addition, we morphologically describe M. parvostis mancae and juveniles infesting H. tsurugae and A. latus to compare them with those infesting H. sajori. Mothocya parvostis infested these two species in different seasons from its infestation in A. schlegelii; thus, we updated our Fig. 1. Diagram of cymothoid life cycles including optional intermediate and final hosts. Solid lines indicate migration by freeswimming and broken lines indicate development of cymothoids. Swimming out from brood pouch On the final host Ovigerous female Transitional Male Juvenile Jvenile Manca Manca On the optional intermediate host Manca page 2 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan knowledge about the optional intermediate hosts and parasitic strategies of M. parvostis. MATERIALS AND METHODS Sample Collection A total of 129 H. tsurugae and 494 A. latus juveniles were collected between 6 July 2021 and 14 October 2021, and between 27 October 2021 and 8 January 2022. Sampling was performed using hand, surf, and casting nets on the coast of Nomijima Island, Hiroshima Bay, Seto Inland Sea, Japan. In the case of H. tsurugae, individuals smaller than 75 mm, which corresponds to zero age (Mori et al. 1988), were considered juveniles. In the case of A. latus, individuals smaller than 160 mm were considered juveniles because they were qualified as immature (Hesp et al. 2004). Parasites infesting H. sajori were collected for morphological comparison between 26 November 2020 and 29 March 2021 in Hiroshima. Mancae swimming out from a blood pouch of ovigerous female infesting H. sajori were collected on 29 October 2020 in Hiroshima. The collected samples were preserved in 99.5% ethanol. The standard length (SL) of the fish and total length (TL) of the cymothoids were measured. The prevalence of M. parvostis to H. tsurugae and A. latus was calculated by dividing the number of the infested fishes by the total number of collected fishes. The “manca-prevalence” and “juvenile-prevalence” was calculated by dividing the number of fishes infested at each respective cymothoid stage by the total number of the collected fishes of H. tsurugae and A. latus. If a single fish was infested by mancae and juveniles, it was included in the estimation of both mancaand juvenileprevalence. Molecular identification A total of 20 cymothoids from H. tsurugae juveniles and 19 cymothoids from A. latus juveniles were randomly selected for molecular analyses. Total DNA was isolated from pereopods via an alkaline lysis method (Toyobo 2012). Eighteen microliters of NaOH (50 mM) was added to the pereopod and incubated at 95°C for 10 min. Next, 2 µL Tris-HCl (1 M, pH 8.0) was added, and the tube was vortexed thoroughly and centrifuged at 12,000 rpm for 5 min. The supernatant was removed and stored at -30°C until use in PCR. Analyses and species identification were performed following Fujita et al. (2020). Partial cytochrome c oxidase subunit I gene (COI) sequences were amplified using the primers LCO1490 (5'-GG TCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAA ATCA-3') (Folmer et al. 1994), and partial 16S rRNA sequences were amplified using the primers 16Sar (5'-CGCCTGTTTAACAAAAACAT-3') and 16Sbr (5'-CCGGTCTGAACTCAGATCATGT-3') (Simon et al. 1994). The total volume for each PCR was 8.1 μL, which was composed of 1 µL of DNA, 0.78 µL of ultrapure water, 4.06 µL of 2× PCR buffer, 1.62 µL of dNTP mix, 0.24 µL of each primer (10 μM solutions), and 0.16 µL of KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan). The thermocycler profile of COI involved an initial denaturation at 94°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 45 s, and extension at 68°C for 30 s; and a final extension at 68°C for 7 min. The thermocycler profile of 16S rRNA consisted of an initial denaturation at 94°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 50.5°C for 30 s, and extension at 68°C; and a final extension at 68°C for 7 min. The PCR products were sequenced via the dye terminator method using an ABI 3130xl genetic analyzer (Applied Biosystems, CA, USA). The sequences were aligned using MUSCLE (Edgar et al. 2004), implemented in MEGA 10 (Kumar et al. 2018), trimmed, and collapsed into haplotypes. All sequences were deposited into GenBank (accession numbers: LC757644–LC757691). Additional sequences belonging to Mothocya, which is distributed in Japan, were downloaded from GenBank (Table S1). The sequences of Anilocra Leach, 1818, Ceratothoa Dana, 1852, and Nerocila Leach, 1818, which are relative genera of Mothocya within Cymothoidae (Hata et al. 2017) that inhabit the waters of Japan (Table S1), were included to compare genetic distances within and between species. Pairwise intraand interspecific genetic distances with the Kimura two-parameter (K2P) model (Kimura 1980) were calculated using MEGA10, and a neighbor-joining tree (Saitou and Nei 1987) was generated using COI and 16S rRNA sequences. Ichthyoxenos japonensis Richardson, 1913 (NC 039713 and MF419233) were also included as outgroups. Morphological description Morphological descriptions were made with the aid of an SZX7 and BX50 (Olympus, Tokyo, Japan). Drawings were digitally inked using Illustrator (version 26.5) (Adobe, CA, USA) and DTC133 pen display (Wacom, Saitama, Japan). Measurements and terminologies essentially follow Aneesh et al. (2019a b 2020). The life stages of the cymothoids were divided into mancae, juveniles, and adults, following Aneesh et al. (2016) and Fujita (2022). page 3 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan RESULTS Cymothoids infesting H. tsurugae In this study, six mancae and 14 juveniles of Cymothoidae were collected from the opercular cavities of 129 H. tsurugae juveniles (Figs. 2 and 3). Of the 16 infested fishes, only three were simultaneously infested by more than two cymothoid individuals. The SL of H. tsurugae juveniles increased with each sampling day (Fig. 4); however, the TL of cymothoids did not significantly correlate with the SL of H. tsurugae juveniles (Fig. 5). The prevalence of cymothoids in H. tsurugae increased with each sampling day and reached a maximum of 20.8% (Fig. 6). The manca-prevalence did not change significantly during the sampling season, but the juvenile-prevalence increased (Fig. 6). The manca-prevalence in small fish (< 20 mm) was 50%, and decreased in larger fish. The juvenile-prevalence increased with larger fish (Fig. 7). Cymothoids infesting A. latus A total of 80 mancae and 64 juveniles of Cymothoidae were collected from the opercular cavity of 494 A. latus juveniles (Figs. 2 and 3). Of the 138 infested fishes, only seven were simultaneously infested by more than two cymothoid individuals. The SL of A. latus juveniles increased with each sampling day (Fig. 4). The TL of cymothoids was significantly correlated with the SL of A. latus juveniles (Fig. 5). The prevalence of cymothoids in A. latus did not significantly change during the sampling period (Fig. 6). However, after midDecember, the manca-prevalence decreased, whereas the juvenile-prevalence increased, and all cymothoids identified in early January were juveniles (Fig. 6). The manca-prevalence in small fish (< 10 mm) was 33.3%, and decreased in larger fish. The juvenile-prevalence increased in larger fish. Almost all cymothoids infesting large A. latus juveniles (> 20 mm) were juveniles (Fig. 7). Molecular identification Our alignment matrices of the COI and 16S rRNA genes consisted of 594 and 412 bp, representing seven and eight haplotypes, respectively. Our neighborjoining tree with COI and 16S rRNA genes showed that Fig. 2. Juveniles of cobaltcap silverside Hypoatherina tsurugae and yellowfin seabream Acanthopagrus latus infested with Mothocya parvostis. Arrows indicate M. parvostis. Scale bars = 5 mm. Hypoatherina tsurugae Acanthopagrus latus page 4 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan all haplotypes detected from cymothoids infesting H. tsurugae and A. latus formed a well-supported clade, along with the sequence identified as M. parvostis by Hata et al. (2017) and Fujita et al. (2020) (Fig. 8). Pairwise intraand interspecific genetic distances with the COI and 16S rRNA haplotypes revealed that the intraspecific distances were smaller than the interspecific distances (Tables 1 and 2), but they did not overlap. The minimum interspecific distances within Mothocya were 3.1% (COI) and 1.9% (16S rRNA) before and after the inclusion of haplotypes from this study. The maximum distances between our haplotypes were 1.0% (COI) and 0.8% (16S rRNA). These values were comparable with the intraand interspecific distances of Anilocra, Cerathothoa, and Nerocila (Tables 1 and 2). Morphological description Family Cymothoidae Leach, 1818 Genus Mothocya Costa, 1851 Mothocya parvostis Bruce, 1986 (Figs. 2, 3, 9–16) Material examined: Juvenile (TL 7.19 mm), from the coast of Nomijima Island, Hiroshima Bay, Seto Inland Sea, Japan, 34°13'49.6"N 132°23'18.4"E, in a opercular cavity of juvenile of Hypoatherina tsurugae (SL: 50.84 mm), 14 October 2021, coll. H. Fujita. Juvenile (TL 6.89 mm), from the coast of Nomijima Island, Hiroshima Bay, Seto Inland Sea, Japan, 34°11'43.3"N 132°26'33.4"E, collected in a opercular Fig. 3. Dorsal and ventral views of Mothocya parvostis infesting juveniles of cobaltcap silverside Hypoatherina tsurugae (A and B) and yellowfin seabream Acanthopagrus latus (C and D). A and C: mancae, B and D: M. parvostis juveniles. Scale bars = 1 mm. A C BD page 5 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan cavity of juvenile of Acanthopagrus schelgelii (SL: 23.27 mm), 8 January 2022, coll. H. Fujita. Manca (TL 3.12 mm), from the coast of Nomijima Island, Hiroshima Bay, Seto Inland Sea, Japan, 34°13'49.6"N 132°23'18.4"E, collected in a opercular cavity of juvenile of Hypoatherina tsurugae (SL: 14.5 mm), 6 July 2021, coll. H. Fujita. Manca (TL 2.96 mm), from the coast of Nomijima Island, Hiroshima Bay, Seto Inland Sea, Japan, 34°11'43.3"N 132°26'33.4"E, collected in a opercular cavity of juvenile of Acanthopagrus schelgelii (SL: 12.22 mm), 27 October 2021, coll. H. Fujita. Description of juvenile infesting H. tsurugae (Figs. 9, 10): Body elliptical, 2.9–3.0 times as long as greatest width, dorsal surfaces convex, widest at pereonite 5, most narrow at pleonite 1. Cephalon 1.4 times wider than long, semi triangle, slightly immersed in pereonite 1. Anterior margin produced moderately rostrum. Eyes oval with distinct margins, one eye 0.3–0.4 times width of cephalon; 0.3–0.8 times length of cephalon. Fig. 5. Scatter plots of the standard length of fishes and the total length of M. parvostis in juveniles of cobaltcap silverside Hypoatherina tsurugae and yellowfin seabream Acanthopagrus latus. The solid lines are regression lines. Fig. 4. Scatter plots of the standard length of non-infested and infested fish for each sampling date in juveniles of cobaltcap silverside Hypoatherina tsurugae and yellowfin seabream Acanthopagrus latus. The open circles (black) indicate non-infested fish, and the closed triangles (red) indicate infested fish. The solid lines (black) for non-infested fishes and the broken lines (red) for infested fishes are regression lines. Jul. 3, 2021 Jul. 23 Aug. 12 Sep. 1 Sep. 21 Oct. 11 20 30 40 50 60 Standard length of the fishes (mm) Oct. 31 Nov. 20 Dec. 10 Dec. 30 10 15 20 25 30 R² = 0.50 p < 0.001 R² = 0.65 p < 0.001 R² = 0.50 p < 0.001 R² = 0.41 p < 0.001 Hypoatherina tsurugae Acanthopagrus latus Sampling days 10 15 20 25 3.0 4.0 5.0 6.0 7.0 R² = 0.0086 p = 0.70 R² = 0.52 p < 0.001 20 30 40 50 3.0 4.0 5.0 6.0 7.0 8.0 Standard length of the fishes (mm) Hypoatherina tsurugae Total length of Mothocya parvostis (mm) Acanthopagrus latus page 6 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan Pereonite 1 anterior border medially straight, slightly curved laterally. Coxae 2–7 slightly visible or invisible in dorsal view; coxae strongly narrow. Pereonites 5 longest, pereonite 7 shortest; posterior margins smooth, slightly curved laterally; pereonites 7 with slightly recessed posterior margin. Pleon 0.2 times as total length, pleonites all visible in dorsal view, pleon 0.7–0.8 times as wide as greatest body width. Pleotelson 0.8–0.9 times length as wide, 1.6–1.7 times as long as pleon, posterior margin with short marginal setae. Antennula with 8 articles, reaching posterior margin of cephalon. Antenna with 8 articles, beyond anterior margin of pereonite 1. Pereopod 1, basis 1.5 times as long as greatest width; ischium 0.6 times as long as basis; merus 0.6 Fig. 7. Prevalence of the standard-length range of juveniles of cobaltcap silverside Hypoatherina tsurugae and yellowfin seabream Acanthopagrus latus. Diagonal shading bars (red), dot bars (blue), grid bars (green), and plain bars (light blue) indicate the manca-prevalence, juvenile-prevalence, the percentage of fish parasitised by both mancae and juveniles, and non-infested fishes, respectively. The asterisk indicates no data. Fig. 6. Prevalence, manca-prevalence, and juvenile-prevalence for each sampling day in juveniles of cobaltcap silverside Hypoatherina tsurugae and yellowfin seabream Acanthopagrus latus. Closed circles (green), closed triangles (blue), and closed squares (red) indicate the prevalence, the mancaprevalence, and the juvenile-prevalence, respectively. Jul. 3, 2021 Jul. 23 Aug. 12 Sep. 1 Sep. 21 Oct. 11 0 5 10 15 20 25 Prevalence (%) Sampling days 0 10 20 30 40 ● ● ●● ● ● ● ■ ■ ■ ■ ■ ■ ■ ▲ ▲ ▲ ▲ ▲ ▲ ▲ Hypoatherina tsurugae Acanthopagrus latus Oct. 31, 2021 Nov. 20 Dec. 10 Dec. 30 10 30 50 70 0 20 40 60 80 100 814 20 26 32 * Standard length of fishes (mm) Prevalence (%) 0 20 40 60 80 100 Hypoatherina tsurugae Acanthopagrus latus page 7 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan times as long as ischium; carpus 0.4 times as long as merus; propodus 5 times as long as carpus; dactylus 1.1 times as long as propodus. Pereopod 2 similar to pereopod 1. Pereopod 3, basis 1.7 times as long as greatest width; ischium 0.5 times as long as basis; merus 0.6 times as long as ischium; carpus 0.6 times as long as merus; propodus 3.3 times as long as carpus; dactylus 1.25 times as long as propodus. Pereopod 4, basis 1.8 times as long as greatest width; ischium 0.8 times as long as basis; merus 0.4 times as long as Fig. 8. Neighbor-joining trees showing seven and eight haplotypes of the cytochrome c oxidase subunit I (COI) and 16S rRNA gene infesting juveniles of cobaltcap silverside Hypoatherina tsurugae and yellowfin seabream Acanthopagrus latus along with selected sequences of other cymothoids downloaded from GenBank. Bootstrap values less than 98% are not shown. The accession numbers were deposited in GenBank (under registration). 2 4 Mothocya parvostis LC159573 Mothocya parvostis LC412904 7 Mothocya parvostis LC549123 1 Mothocya parvostis LC549148 3 6 5 Mothocya melanosticta MH395840 Mothocya collettei LC159572 Ichthyoxenos japonensis NC 039713 Ichthyoxenos japonensis MF419233 0.020 100 100 6 Mothocya parvostis LC416622 Mothocya parvostis LC549170 Mothocya parvostis LC159462 5 8 1 Mothocya parvostis LC549152 7 3 4 2 Mothocya collettei LC159461 Mothocya renardi EF422803 Ichthyoxenos japonensis MF419233 Ichthyoxenos japonensis NC 039713 100 99 100 0.020 COI 16S rRNA page 8 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan ischium; carpus 1.0 times as long as merus; propodus 2.5 times as long as carpus; dactylus 1.1 times as long as propodus. Pereopods 5 and 6 similar to pereopod 4. No robust setae on pereopods 1–6. Pereopod 7, basis 2.3 times as long as greatest width; ischium 0.7 times as long as basis, 1 robust seta on inferior margin; merus 0.6 times as long as ischium; carpus 1.0 times as long as merus, 2 robust setae on inferior margin; propodus 1.7 times as long as carpus, 4 robust setae on inferior margin; dactylus 1.0 times as long as propodus. Pleopods all lamellar, surface smooth. Pleopod 1 peduncle 1.7 times as wide as length, medial margin with 4 coupling hooks; endopod rectangular, 1.9 times as long as width; exopod trapezoidal, lateral margin almost straight, 1.9 times as long as width, 1.1 times as long as endopod, medial margin with short marginal setae. Pleopod 2 similar to pleopod 1; peduncle medial margin with 4 plumose setae; endopod with appendix masculina, slightly shorter than endopod. Uropod rami beyond posterior margin of pleotelson; uropod, peduncle triangular, 0.7 times as long as exopod, 1.7 times as long as wide. Endopod oval, 2.3 times as long as greatest width, 0.8 times as long as exopod, lateral and medial margins with short marginal setae. Exopod semitriangular, 3.2 times as long as greatest width, medial margin with short marginal setae. Description of juvenile infesting A. latus (Figs. 11, 12): Similar to juvenile infesting H. tsurugae. Body widest at pereonite 1–3, most narrow at pereonite 7. Pereonites 3 longest, pereonite 7 shortest. Each of pereopod 2 and 3 merus superior distal angle with 1 robust seta. Pereopod 6 propodus inferior margin with 1 robust seta. Pereopod 7 carpus and propodus with no robust seta. Pleopod 1 peduncle medial margin with 3 plumose setae. Description of juvenile infesting H. sajori: Similar to juvenile infesting H. tsurugae and A. latus. Absence of marginal setae of pleotelson, pleopods and uropods. Description of manca infesting H. tsurugae (Figs. 13, 14): Body elliptical, 3.4–4.1 times as long as greatest width, widest at pereonite 3, most narrow at pleonite 1. Pereonites 1 longest, pereonite 7 shortest; posterior margins straight. Pleon 0.3 times as total length, pleonites all visible in dorsal view, pleon 0.7–0.8 times as wide as greatest body width. Pleotelson 0.9–1.0 times as wide as length 0.7 times as long as pleon, posterior margin with marginal setae. Table 1. Genetic distances determined using the Kimura two-parameter model for the cytochrome c oxidase subunit I gene (COI) sequences of Cymothoidae Comparison level No. species Intraspecific Interspecific Min. (%) Max. (%) Mean (%) Min. (%) Max. (%) Mean (%) Genus Anilocra 2 0.000 0.349 0.202 10.959 11.187 11.028 Ceratothoa 4 0.169 2.593 1.221 18.398 31.589 29.224 Nerocila 2 0.000 1.280 0.444 27.307 28.836 27.721 Mothocya 4 0.000 1.020 0.261 3.126 13.902 11.752 Mothocya#4 0.000 1.020 0.310 3.126 14.122 11.781 #Including haplotypes of Mothocya parvostis collected in the present study. Table 2. Genetic distances obtained using the Kimura two-parameter model for the 16S rRNA gene sequences of Cymothoidae Comparison level No. species Intraspecific Interspecific Min. (%) Max. (%) Mean (%) Min. (%) Max. (%) Mean (%) Genus Anilocra 2 1.328 1.875 1.622 18.940 19.715 19.327 Ceratothoa 3 0.000 1.229 0.643 14.043 24.881 22.661 Nerocila 2 0.000 1.050 0.638 20.564 20.564 20.564 Mothocya 3 0.000 0.836 0.219 1.003 9.994 9.356 Mothocya#3 0.000 0.836 0.251 1.003 9.994 9.435 #Including haplotypes of Mothocya parvostis collected in the present study. page 9 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan Antennula with 8 articles, reaching beyond midpoint of cephalon. Antenna with 8 articles, reaching posterior margin of cephalon. Pereopod 1, basis 2.2 times as long as greatest width; ischium 0.5 times as long as basis; merus 0.6 times as long as ischium; carpus 0.4 times as long as merus; propodus 4.8 times as long as carpus, 3 robust setae on inferior margin; dactylus 1.2 times as long as propodus, teeth on inferior margin. Pereopod 2 similar to pereopod 1, carpus, 1 robust seta on superior distal angle. Pereopod 3 similar to pereopod 1. Pereopod 4, basis 1.8 times as long as greatest width; ischium 0.8 times as long as basis; merus 0.3 times as long as ischium; carpus 1.3 times as long as merus; propodus 2.2 times as long as carpus, 1 robust seta on inferior margin; dactylus 1.1 times as long as propodus, without teeth. Pereopod 5 similar to 4, absence of robust seta. Pereopod 6 similar to pereopod 4, carpus, 1 robust seta on inferior margin; propodus, 3 robust setae on inferior margin. No pereopod 7. Pleopods all lamellar, surface smooth. Pleopod 1 peduncle 1.7 times as wide as length, medial margin with 4 coupling hooks; endopod trapezoidal, 1.6 times as long as width; exopod trapezoidal, lateral margin almost straight, 1.8 times as long as width, subequal length of endopod, posterior margin with long marginal setae. Pleopod 2 similar to pleopod 1; endopod without appendix masculina; exopod, 1.1 times as long as endopod. Uropod rami beyond posterior margin of pleotelson; uropod, peduncle triangular, 0.7 times as long as exopod, 1.7 times as long as wide. Endopod oval,1.6 times as long as greatest width, 0.6 times as long as exopod, lateral and medial margins with long marginal setae. Exopod semitriangular, 3.2 times as long as greatest width, medial margin with long marginal setae. Description of manca infesting A. latus (Figs. 15, 16): Similar to manca infesting H. tsurugae. Pereopod 1, merus, 1 robust seta on inferior margin, 1 robust seta on superior distal angle; propodus, 2 robust setae on inferior margin; dactylus without teeth. Pereopod 2, merus, 1 robust seta on superior distal angle; carpus without robust seta; propodus, 1 robust seta on inferior margin. Pereopod 3, merus, 1 robust sera on superior distal angle; propodus, 3 robust setae on inferior margin. Pereopod 4 without robust seta and teeth. Pereopod 5, dactylus, teeth on inferior margin. Pereopod 6, carpus with 1 robust seta on inferior margin; propodus with 2 robust setae on inferior margin. Pleopod 1 peduncle, medial margin with 3 plumose setae. Pleopod 2 peduncle, medial margin with 4 plumose setae. Manca in blood pouch of ovigerous females infesting H. sajori: Similar to manca infesting H. tsurugae and A. latus. Remarks: Morphological differences between mancae and juveniles of M. parvostis infesting H. tsurugae and those infesting A. latus were mainly found in the setations on the pereopods. The morphology of robust setae may be a key to morphological species identification for cymothoid mancae and juveniles (Saito and Fujita 2022). Morphological differences of Cymothoidae among species, growth stages, and individuals should be comprehensively examined. Cymothoid juveniles infesting juveniles of A. latus and H. tsurugae have marginal setae on the posterior margin of the pleotelson, endopod of pleopods, and uropods, but absence of those of Juveniles infesting H. sajori. These marginal setae, also known as swimming setae, enhance the swimming ability of cymothoids (Tsai and Dai 1999). This indicates that juveniles infesting A. latus and H. tsurugae have better swimming ability than those infesting H. sajori. DISCUSSION Cymothoid parasites, including M. parvostis, have been described on the basis of the morphological traits of adult females; thus, morphological identification is almost impossible in other life stages and in males (Fujita et al. 2021). Fujita et al. (2020) morphologically identified and described adult M. parvostis females infesting H. sajori, a major final host, and deposited their COI and 16S rRNA sequences into GenBank. In the present study, cymothoids were collected from H. tsurugae and A. latus, a clade that encompasses an existing sequence previously identified from M. parvostis (Hata et al. 2017; Fujita et al. 2020). The intraspecific genetic distances between our collections and the sequence of M. parvostis from GenBank were lower than the interspecific genetic distances within Mothocya. Thus, the cymothoid specimens collected from H. tsurugae and A. latus were identified as M. parvostis. Mothocya parvostis has been observed in the opercular cavities of H. sajori, G. punctata, and S. quinqueradiata as final hosts (Bruce 1986) and A. schelgelii as an optional intermediate host (Fujita et al. 2020). Thus, H. tsurugae and A. latus are newly identified hosts of M. parvostis. In Hiroshima Bay, the spawning of A. schelgelii, an optional intermediate host of M. parvostis, peaks in early May (Kawai et al. 2017 2020 2021), and their juveniles settle in the surf zone from late June (Kawai et al. 2019). Mothocya parvostis mancae initially infest A. page 16 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan Fig. 15. Mothocya parvostis manca (2.96 mm) infesting a yellowfin seabream Acanthopagrus latus juvenile (12.22 mm). (A) Body, dorsal view. (B) Cephalon, ventral view. (C) Pleotelson. (D, E) Pereopods 1, 2, respectively. Scale bars: A = 1 mm; B, C = 0.2 mm; D, E = 0.1 mm. page 17 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan Fig. 16. Mothocya parvostis manca (2.96 mm) infesting a yellowfin seabream Acanthopagrus latus juvenile (12.22 mm). (A–D) Pereopods 3–6, respectively. (E, F) Pleopods 1, 2, respectively. Scale bars = 0.1 mm. page 18 of 22 Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan schelgelii juveniles after A. schelgelii metamorphoses and settles. Therefore, the prevalence of M. parvostis in A. schelgelii increases rapidly from late June to early August (Fujita et al. 2020). The spawning season of H. tsurugae in Hiroshima Bay has not been clearly determined, but it is estimated to start from May to July in other regions in Japan (Mori et al. 1988). In the present study, H. tsurugae juveniles were collected from July to October, a period consistent with the spawning season. The SL of H. tsurugae juveniles just after metamorphosis is approximately 20 mm (Tsukamoto and Kimura 1993), which is close to the minimum size of H. tsurugae juveniles collected in this study. In addition, their prevalence increased with each sampling day, similar to the pattern observed in A. schelgelii; the manca-prevalence in 10–20 mm fish was the highest. These findings suggest that H. tsurugae juveniles were collectively infested with M. parvostis mancae once metamorphosis was completed. The juvenile-prevalence was higher than the manca-prevalence in July, suggesting that M. parvostis mancae began infesting H. tsurugae juveniles before July. The spawning season of A. latus in Hiroshima Bay is poorly understood, but it likely occurs in autumn (from September to November) in another region in Japan (Abol-Munafi and Umeda 1994; Nishida 2022). In the present study, A. latus juveniles were collected from October 2021 to January 2022, a period concordant with the spawning season. The prevalence of cymothoids during the sampling period did not significantly change, but the mancaand juvenile-prevalence changed. The manca-prevalence was higher than the juvenileprevalence between late October and early December, but it decreased from late December and became zero in early January. The SL of A. latus juveniles just after they metamorphosed was approximately 10–15 mm (Tran et al. 2019), which is close to the minimum size of A. latus juveniles collected in this study. In addition, the manca-prevalence in fish with a size of 9–11 mm was the highest. These findings indicated that, similar to A. schlegelii and H. tsurugae, A. latus juveniles were collectively infested by M. parvostis mancae after metamorphosis. The prevalence of M. parvostis in A. schelgelii juveniles decreases in August, and infested A. schelgelii juveniles are rare in September (Fujita et al. 2020). Fujita et al. (2020) stated that M. parvostis infestation in A. schelgelii juveniles is temporary and M. parvostis break away from A. schelgelii juveniles; therefore, A. schelgelii juveniles might be an optional intermediate host of M. parvostis. In H. tsurugae juveniles, the juvenile-prevalence increased with each sampling day, although the manca-prevalence did not significantly change. In A. latus juveniles, the manca-prevalence was higher than the juvenile-prevalence from October to early December, but from late December, mancaprevalence decreased and juvenile prevalence increased. By early January, all M. parvostis individuals infesting A. latus were juveniles. All parasites in small fish were mancae; in larger fish, juvenile-prevalence increased. In much larger fishes (H. tsurugae: ≥ 50 mm, A. latus: ≥ 20 mm), all M. parvostis individuals were juveniles. These results suggest that M. parvostis manca infested small H. tsurugae and A. latus juveniles. As the host fish grew, infestation was not observed. In addition, these fishes are frequently observed by humans because the two species are the subject of recreational fishing, and A. latus has commercial importance (Iwatsuki 2013). However, Mothocya infesting adult H. tsurugae and A. latus were not observed. This suggests that when infested M. parvostis grew with fish juveniles, the parasites left their hosts; these fish would not be suitable hosts for M. pravostis adults. The same pattern was observed in A. schelgelii (Fujita et al. 2020). Therefore, mancae and juveniles of M. parvostis infesting juveniles of these fishes can not mature unless they move on to infest their final hosts. Based on this finding, we conclude that H. tsurugae and A. latus are optional intermediate hosts of M. parvostis. The marginal setae on the posterior margin of the pleotelson, pleopod, and uropod, also called swimming setae, enhance the swimming ability of cymothoids (Tsai and Dai 1999). Cymothoid mancae have long marginal setae for free-swimming, but generally lose them after host infestation (Smit et al. 2014). In the morphological observations in this study, M. parvostis juveniles infesting H. sajori did not have marginal setae; in contrast, M. parvostis juveniles infesting H. tsurugae and A. latus (optional intermediate hosts) did. Juveniles infesting A. schlegelii juveniles, which are optional intermediate hosts, also had marginal setae (Saito and Yoneji 2000). This indicates that M. parvostis juveniles lose marginal setae when infesting H. sajori, the final host, but may retain them when infesting the optional intermediate host. Free-swimming juveniles of Mothocya sp. were collected (Saito et al. 2014). This supports our hypothesis that M. parvostis juveniles leave optional intermediate hosts to infest H. sajori. As mentioned above, the prevalence of M. parvostis in A. schelgelii juveniles rapidly increases and then decreases (Fujita et al. 2020). Similarly, the prevalence of M. parvostis in H. tsurugae juveniles increased during the sampling period. The prevalence of M. parvostis by SL of fish in A. latus was similar to that of A. schelgelii and H. tsurugae. However, the prevalence of M. parvostis in A. latus juveniles did not change significantly during the sampling period. Although the cause is unknown, this finding page 19 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan could be attributed to multiple factors, such as the density of mancae or the period when juveniles of fish settle. Hence, the spawning ecology of A. latus and the dynamics of free-swimming mancae should be determined to explain why the prevalence of M. parvostis in A. latus juveniles did not significantly change. The reproductive cycles of cymothoid organisms vary. For example, A. pomacentri has no fixed reproduction season and reproduces throughout the year (Adlard and Lester 1995), but M. epimerica has four reproduction seasons per year (Bello et al. 1997). The reproduction cycle of M. parvostis is unknown, but mancae infest A. schelgelii juveniles from June to August (Fujita et al. 2020). Mothocya parvostis mancae can survive without a host for only 10–15 days (Hatai and Yasumoto 1980). Therefore, the reproductive season of M. parvostis must include at least June to August (Fujita et al. 2020). In this study, mancae infested H. tsurugae from July to October and A. latus from October to December. Therefore, M. parvostis could reproduce from June to December. Future collections of free-swimming mancae, as well as eggs and mancae from the brood pouch of ovigerous females throughout the year will help clarify the reproductive cycle of M. parvostis. CONCLUSIONS This study and that of Fujita et al. (2020) found that A. schelgelii, H. tsurugae, and A. latus juveniles were infested with M. parvostis from June to August, July to October, and October to January the following year, respectively. In other words, optional intermediate hosts were available to M. parvostis for at least 8 months, and M. parvostis might infest different hosts depending on the time of year. Fujita et al. (2020) hypothesized that after detaching from A. schelgelii juveniles, M. parvostis juveniles can infest H. sajori. The results of the present study support this hypothesis, but further studies will be needed to confirm the exact life cycle. If these juveniles can infest H. sajori after they detach from optional intermediate hosts, using an optional intermediate host may be an excellent strategy to increase the fitness of M. parvostis. Acknowledgments: We sincerely thank Nobuhiro Saito (Suido-sha Co. Ltd) for making helpful suggestions on drafts of this paper. We are grateful to Tatsuki Ohshita (Graduate School of Integrated Sciences for Life, Hiroshima University), Ryoya Izuta (School of Applied Biological Science, Hiroshima University), and Yuto Fujita (Faculty of Health Sciences, Hiroshima International University) for their assistance with sampling. This work was supported by JST SPRING, Grant Number JPMJSP2132. We would like to thank Editage (www.editage.com) for the English language editing. We are also grateful to two anonymous reviewers for their careful reading of our manuscript and their many insightful comments and suggestions. Authors’ contributions: HF designed and executed the experiments, collected samples, and wrote the manuscript. KK analyzed A. lutas and edited the manuscript. DD analyzed H. tsurugae and edited the manuscript. TU supervised and edited the manuscript. All authors have reviewed and approved the final manuscript. Competing interests: HF, KK, DD, and TU declare that they have no conflict of interest. Availability of data and materials: The COI and 16S rRNA sequences in this study were deposited into NCBI GenBank (accession numbers: LC757644– LC757691). Consent for publication: Not applicable. Ethics approval consent to participate: Not applicable. REFERENCES Abol-Munafi AB, Ueda S. 1994. The gonadal cycle of the yellowfin porgy, Acanthopagrus latus (Houttuyn) reared in the net cage at Tosa Bay, Japan. Aquac Sci 42(1):135–144. doi:10.11233/ aquaculturesci1953.42.135. Adlard RD, Lester RJG. 1995. The life-cycle and biology of Anilocra pomacentri (Isopoda, Cymothoidae), an ectoparasitic isopod of the coral-reef fish, Chromis nitida (Perciformes, Pomacentridae). Aust J Zoo 43:271–281. doi:10.1071/ZO9950271. Aneesh PT, Bruce NL, Kumar AB, Bincy MR, Sreenath TM. 2021. A taxonomic review of the buccal-attaching fish parasite genus Lobothorax Bleeker, 1857 (Crustacea: Isopoda: Cymothoidae) with description of a new species from southwestern India. Zool Stud 60:13. doi:10.6620/ZS.2021.60-13. Aneesh PT, Helna AK, Bijukumar A. 2019a. Redescription and neotype designation for the poorly known fish parasitic cymothoid Joryma brachysoma (Pillai, 1964) (Crustacea: Isopoda) from India. Folia Parasitol 66(014):1–6. doi:10.14411/fp.2019.014. Aneesh PT, Helna AK, Trilles JP, Chandra K. 2019b. A taxonomic review of the genus Joryma Bowman and Tareen, 1983 (Crustacea: Isopoda: Cymothoidae) parasitizing the marine fishes from Indian waters, with a description of a new species. Mar Biodivers 49:1449–1478. doi:10.1007/s12526-018-0920-7. Aneesh PT, Kappalli S. 2020. Protandrous hermaphroditic reproductive system in the adult phases of Mothocya renardi (Bleeker, 1857) (Cymothoidae: Isopoda: Crustacea) – light and electron microscopy study. Zool Stud 59:61. doi:10.6620/ZS.2020.59-61. page 20 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan Aneesh PT, Kappalli S, Kottarathil HA, Gopinathan A, Paul TJ. 2015. Cymothoa frontalis, a cymothoid isopod parasitizing the belonid fish Strongylura strongylura from the Malabar Coast (Kerala, India): redescription, description, prevalence and life cycle. Zool Stud 54:1–28. doi:10.1186/s40555-015-0118-7. Aneesh PT, Helna AK, Kumar AB, Trilles JP. 2020. A taxonomic review of the branchial fish parasitic genus Elthusa Schioedte & Meinert, 1884 (Crustacea: Isopoda: Cymothoidae) from Indian waters, with the description of three new species. Mar Biodivers 50:65. doi:10.1007/s12526-020-01084-6. Aneesh PT, Sudha K, Helna AK, Anilkumar G. 2016. Mothocya renardi (Bleeker, 1857) (Crustacea: Isopoda: Cymothoidae) parasitizing Strongylura leiura (Bleeker) (Belonidae) off the Malabar coast of India: re-description, occurrence and life cycle. Syst Parasitol 93:583–599. doi:10.1007/s11230-016-9646-8. Aneesh PT, Sudha K, Helna AK, Anilkumar G. 2018. Agarna malayi Tiwari 1952 (Crustacea: Isopoda: Cymothoidae) parasitising the marine fish, Tenualosa toli (Clupeidae) from India: redescription/description of parasite life cycle and patterns of occurrence. Zool Stud 57:25. doi:10.6620/ZS.2018.57-25. Baillie C, Welicky RL, Hadfield KA, Smit NJ, Mariani SD, Beck RM. 2019. Hooked on you: shape of attachment structures in cymothoid isopods reflects parasitic strategy. BMC Evol Biol 19(1):207. doi:10.1186/s12862-019-1533-x. Bello G, Vaglio A, Piscitelli G. 1997. The reproductive cycle of Mothocya epimerica (Isopoda: Cymothoidae) a parasite of the sand smelt, Atherina boyeri (Osteichthyes: Atherinidae), in the Lesina Lagoon, Italy. J Nat Hist 31(7):1055–1066. doi:10.1080/00222939700770551. Bleeker P. 1854. Faunae Ichthyologicae japonicae. Species Novae. Natuurwet Tijdschr Ned Indie 6:395–426. Boyko CB, Bruce NL, Hadfield KA, Merrin KL, Ota Y, Poore GCB, Taiti S, Schotte M, Wilson GDF. (eds) 2008. onwards. World Marine, Freshwater and Terrestrial Isopod Crustaceans database. Cymothoidae Leach, 1818. Accessed through: World Register of Marine Species. Available at: http://www.marinespecies.org/ aphia.php?p=taxdetails&id=118274. Accessed 8 May 2022. Bruce NL. 1986. Revision of the isopod crustacean genus Mothocya Costa, in Hope, 1851 (Cymothoidae: Flabellifera), parasitic on marine fishes. J Nat Hist 20:1089–1192. doi:10.1080/00222938600770781. Bruce NL. 1987. Australian Pleopodias Richardson, 1910, and Anilocra Leach, 1818 (Isopoda: Cymothoidae), crustacean parasites of marine fishes. Rec Aust Mus 39:85–130. doi:10.3853/j.0067-1975.39.1987.166. Brusca RC. 1978a. Studies on the cymothoid fish symbionts of the Eastern Pacific (Crustacea: Isopoda: Cymothoidae). I. Biology of Nerocila californica. Crustaceana 34:141–154. doi:10.1163/156854078X00718. Brusca RC. 1978b. Studies on the cymothoid fish symbionts of the Eastern Pacific (Crustacea: Isopoda: Cymothoidae). II. Systematics and biology of Lironeca vulgaris Stimpson 1857. Allan Hancock Occasional Papers New Series 2:1–19. Brusca RC. 1981. A monograph on the Isopoda Cymothoidae (Crustacea) of the eastern Pacific. Zool J Linn Soc 73:117–199. doi:10.1111/j.1096-3642.1981.tb01592.x. Costa A. 1851. Catalogo dei crotacei Italiani e di moltri altri del Mediterraneo per Fr. Gugl. Hope. Napoli, pp. 1–48. Dana JD. 1852. On the classification of the Crustacea Choristopoda or Tetradecapoda. Am J Sci 2:297–316. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. doi:10.1093/nar/gkh340. Fogelman RM, Grutter AS. 2008. Mancae of the parasitic cymothoid isopod, Anilocra apogonae: early life history, host-specificity, and effect on growth and survival of preferred young cardinal fishes. Coral Reefs 27:685–693. doi:10.1007/s00338-008-0379-2. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotech 3:294–299. Fujita H. 2022. Infestation of the aegathoid stage of Anilocra clupei (Isopoda: Cymothoidae) on a Japanese halfbeak Hyporhamphus sajori (Beloniformes: Hemiramphidae) collected from Japan. Cancer 31:29–36. (in Japanese with English abstract) doi:10.18988/cancer.31.0_29. Fujita H, Kawai K, Deville D, Umino T. 2023. Quatrefoil light traps for free-swimming stages of cymothoid parasitic isopods and seasonal variation in their species compositions in the Seto Inland Sea, Japan. Int J Parasitol Parasites Wildl 20:12–19. doi:10.1016/j.ijppaw.2022.12.002. Fujita H, Kawai K, Taniguchi R, Tomano S, Sanchez G, Kuramochi T, Umino T. 2020. Infestation of the parasitic isopod Mothocya parvostis on juveniles of the black sea bream Acanthopagrus schlegelii as an optional intermediate host in Hiroshima Bay. Zool Sci 37:544–553. doi:10.2108/zs190147. Fujita H, Umino T, Saito N. 2021. Molecular identification of the aegathoid stage of Anilocra clupei (Isopoda: Cymothoidae) parasitizing sweeper Pempheris sp. (Perciformes: Pempheridae). Crustacean Res 50:29–31. doi:10.18353/crustacea.50.0_29. Hata H, Sogabe A, Tada S, Nishimoto R, Nakano R, Kohya N, Takeshima H, Kawanishi R. 2017. Molecular phylogeny of obligate fish parasites of the family Cymothoidae (Isopoda, Crustacea): evolution of the attachment mode to host fish and the habitat shift from saline water to freshwater. Mar Biol 164:1–15. doi:10.1007/s00227-017-3138-5. Hatai K, Yasumoto S. 1980. A parasitic isopod, Irona melanosticta isolated from the gill chamber of fingerlings of cultured yellowtail, Seriola quinqueradiata. Bull Nagasaki Pref Institute Fish 6:87–96. (in Japanese with English title) Hesp SA, Potter IC, Hall NG. 2004. Reproductive biology and protandrous hermaphroditism in Acanthopagrus latus. Environ Biol Fish 70(3):257–272. doi:10.1023/B:EBFI.0000033344. 21383.00. Houttuyn M. 1782. Beschrijving van eenige Japanse visschen en andere zee-schepzelen (Vol. 1). Iwatsuki Y. 2013. Review of the Acanthopagrus latus complex (Perciformes: Sparidae) with descriptions of three new species from the Indo-West Pacific Ocean. J Fish Biol 83(1):64–95. doi:10.1111/jfb.12151. Jones CM, Miller TL, Grutter AS, Cribb TH. 2008. Natatory-stage cymothoid isopods: description, molecular identification and evolution of attachment. Int J Parasitol 38(3–4):477–491. doi:10.1016/j.ijpara.2007.07.013. Jordan DS, Starks EC. 1901. A review of the atherine fishes of Japan. Proc US Natl Mus 24:199–206. doi:10.5479/SI.00963801.241250.199. Kawai K, Fujita H, Sanchez G, Furusawa S, Umino T. 2020. Estimating the spawning season of black sea bream Acanthopagrus schlegelii in Hiroshima Bay, Japan, from temporal variation in egg density. Fisheries Sci 86(4):645–653. doi:10.1007/s12562-020-01433-1. Kawai K, Fujita H, Sanchez G, Umino T. 2021. Oyster farms are the main spawning grounds of the black sea bream Acanthopagrus schlegelii in Hiroshima Bay, Japan. PeerJ 9:e11475. doi:10.7717/ peerj.11475. Kawai K, Fujtia H, Umino T. 2019. Horizontal distribution and annual fluctuations in abundance of settled juveniles of the black sea bream Acanthopagrus schlegelii in Hiroshima Bay, Japan. Bull Hiroshima Univ Mus 11:1–5. Kawai K, Okazaki R, Tomano S, Umino T. 2017. DNA identification page 21 of 22Zoological Studies 62:21 (2023) © 2023 Academia Sinica, Taiwan and seasonal changes of pelagic fish eggs in Hiroshima Bay. Nippon Suisan Gakk 83:215–217. (in Japanese with English abstract). doi:10.2331/suisan.16-00069. Kawanishi R, Sogabe A, Nishimoto R, Hata H. 2016. Spatial variation in the parasitic isopod load of the Japanese halfbeak in western Japan. Dis Aquat Organ 122:13–19. doi:10.3354/dao03064. Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16(2):111–120. doi:10.1007/BF01731581. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35(6):1547–1549. doi:10.1093/molbev/ msy096. Latrobe BH. 1802. A drawing and description of the Clupea tyrannus and Oniscus praegustator. T Am Philos Soc 5:77–81. doi:10.2307/1004979. Leach WE. 1818. Cymothoadées. In: Cuvier, F. (Ed), Dictionnaire des Sciences Naturelles, Vol. 12. Paris, pp. 338–354. Lindsay JA, Moran LR. 1976. Relationships of parasitic isopods Lironeca ovalis and Olencira praegustator to marine fish host in Delaware Bay. T Am Fish Soc 105:327–332. doi:10.1577/15488659(1976)105%3C327:ROPILO%3E2.0.CO;2. Martens E. 1869. Südbrasilische Süssund Brackwasser-Crustaceen nach den Sammlungen des Dr. Reinh. Hensel. Archiv für Naturgeschichte 35:1–37. Mori K, Kimura S, Tsukamoto Y, Kohno Y, Yoshida M. 1988. Growth of the atherinid fish Hypoatherina tsurugae in Ago Bay, Central Japan. Aquac Sci 36(2):87–90. (in Japanese). doi:10.11233/ aquaculturesci1953.36.87. Nagasawa K. 2020. Mothocya parvostis (Isopoda: Cymothoidae) parasitic on Japanese halfbeak, Hyporhamphus sajori, in the central Seto Inland Sea, Japan, with a brief summary of the hosts, geographical distribution, and pathogenic effects of the isopod. Nature of Kagoshima 47:51–57. Nishida Y. 2022. Acanthopagrus latus. In: Kondo Y, Ohtsuka S, Sato M. (eds) Life in the tidal flats of Hachi-no-higata: the well-preserved natural ecosystem at Takehara in the Seto Inland Sea, Japan, 1st edn. NextPublishing Authors Press, Tokyo. p. 46. (in Japanese) Saito N, Fujita H. 2022. Morphological description and molecular barcoding of the aegathoid stage of Nerocila japonica Schioedte & Meinert, 1881 (Crustacea: Isopoda: Cymothoidae) infesting red seabream Pagrus major (Temminck & Schlegel, 1843). Crust Res 51:47–54. doi:10.18353/crustacea.51.0_47. Saito N, Fujita H, Aiba S. 2022. First host record of Ceratothoa oxyrrhynchaena (Isopoda: Cymothoidae) infesting the serranid fish, Sacura margaritacea (Perciformes: Serranidae). Taxa 53:59–63. (in Japanese with English abstract). doi:10.19004/ taxa.53.0_59. Saito N, Yamauchi T, Ariyama H, Hoshino O. 2014. Descriptions and ecological notes of free-swimming forms of cymothoid isopods (Crustacea: Peracarida) collected in two waters of Japan. Crus Res 43:1–16. doi:10.18353/crustacea.43.0_1. Saito N, Yoneji T. 2000. The genus of Mothocya (Isopoda: Cymothoidae) parasitic on black sea bream juveniles Acanthopagrus schlegelii. Umiushi-tsushin 29:4–6. (in Japanese). Saitou N, Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. doi:10.1093/oxfordjournals.molbev.a040454. Schioedte JC, Meinert FW. 1881. Symbolae ad monographiam Cymothoarum Crustaceorum Isopodum Familiae 2. Anilocridae. Naturhistorisk Tidsskrift 13:1–166. Segal E. 1987. Behaviour of juvenile Nerocila acuminata (Isopoda, Cymothoidae) during attack, attachment and feeding on fish prey. Bull Mar Sci 41:351–360. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Floors P. 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am 87:651–701. doi:10.1093/aesa/87.6.651. Smit NJ, Bruce NL, Hadfield KA. 2014. Global diversity of fish parasitic isopod crustaceans of the family Cymothoidae. Int J Parasitol Parasites Wildl 3:188–197. doi:10.1016/j.ijppaw.2014. 03.004. Taberner R, León PDR, Volonterio O. 2003. Description of the pulli stages of Telotha henselii (Von Martens, 1869) (Isopoda, Cymothoidae), with new hosts and locality records from Uruguay and Argentina. Crustaceana 76:27–37. doi:10.1163/156854003321672809. Temminck CJ, Schlegel H. 1846. Pisces. Fauna Japonica, sive descriptio animalium quae in itinere per Japoniam suscepto annis 1823-30 collegit, notis observationibus et adumbrationibus illustravit P. F. de Siebold, Parts 10–14, 173–269. Temminck CJ, Schlegel H. 1845. In: Temminck and Schlegel 1843. Fauna Japonica, sive description animalium quae in itinere per Japoniam. Parts 7–9:113–172. Toyobo. 2012. Special feature on PCR protocols by material and application. Available at: https://lifescience.toyobo.co.jp/upload/ upld99/feature/pcr99fe01.pdf. Accessed 26 May 2022. (in Japanese) Tsai ML, Dai CF. 1999. Ichthyoxenus Fushanensis, New species (Isopoda: Cymothoidae), parasite of the fresh-water fish Varicorhinus Barbatulus from northern Taiwan. J Crust Biol 19(4):917–923. doi:10.1163/193724099X00600. Tran TT, Tran HD, Kinoshita I. 2019. Simultaneous and sympatric occurrence of early juveniles of Acanthopagruslatus and A. schlegelii (Sparidae) in the estuary of northern Vietnam. Limnology 20(3):321–326. doi:10.1007/s10201-019-00581-3. Tsukamoto Y, Kimura S. 1993. Development of laboratory-reared eggs, larvae and juveniles of the atherinid fish, Hypoatherina tsurugae, and comparison with related species. Jpn J Ichthyol 40(2):261–267. doi:10.11369/jji1950.40.261. Williams EH, Bunkley-Williams L. 1986. The 1st Anilocra and Pleopodias isopods (Crustacea: Cymothoidae) parasitic on Japanese fishes, with 3 new species. P Biol Soc Wash 99(4):647– 657. Yamauchi T. 2016. Cymothoid isopods (Isopoda: Cymothoidae) from fishes in Japanese waters. Cancer 25:113–119. (in Japanese with English title). doi:10.18988/cancer.25.0_113. Supplementary materials Table S1. List of sequences from cymothoid species distributed in Japan downloaded from GenBank, with the corresponding accession numbers (Jones et al. 2008; Hata et al. 2017; Baillie et al. 2019; Fujita et al. 2020, 2021; Fujita 2022; Saito and Fujita 2022; Saito et al. 2022). (download) page 22 of 22Zoological Studies 62:21 (2023)