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Article

Description of Life Cycle Stages of Fish Parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA Barcode Linked to Morphological Details

by
Hiroki Fujita
1,*,
Haruki Shinoda
1,2 and
Yuzumi Okumura
1,2,3
1
Seto Marine Biological Laboratory, Field Science Education and Research Center, Kyoto University, 459 Shirahama, Wakayama 649-2211, Wakayama, Japan
2
Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Kyoto, Japan
3
Faculty of Science, Ehime University, 2-5 Bunkyo, Matsuyama 790-8577, Ehime, Japan
*
Author to whom correspondence should be addressed.
Fishes 2025, 10(4), 155; https://doi.org/10.3390/fishes10040155
Submission received: 28 February 2025 / Revised: 23 March 2025 / Accepted: 30 March 2025 / Published: 1 April 2025
(This article belongs to the Section Fish Pathology and Parasitology)
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Abstract

Cymothoidae (Crustacea: Isopoda) infest fish in marine, brackish, and freshwater environments. Cymothoa pulchrum Lanchester, 1902 is a cymothoid commonly found in the buccal cavity of mainly Tetraodontiformes fishes, distributed in the central and western Indo-Pacific region. This study describes the morphology of each life cycle stage of C. pulchrum: adult female, transitional, adult male, juvenile, and manca. In addition, we obtained DNA sequences linked to the morphological information of this species. We compared it with the sequences in the database using the neighbor-joining tree based on cytochrome c oxidase subunit I (COI) and 16S rRNA. Prior to this study, morphological data on the immature stages of Japanese Cymothoa was limited to juveniles of Cymothoa indica Schioedte and Meinert, 1884. The research identified 12 distinct morphological features that differentiate juvenile C. pulchrum from juvenile C. indica. Molecular analysis revealed that the COI sequences obtained in this study matched some of the C. pulchrum sequences in the database, whereas other sequences in the database formed a clade with Cymothoa eremita (Brünnich, 1783). In the phylogenetic tree based on 16S rRNA, C. pulchrum was also divided into two groups. In the COI phylogenetic tree, C. pulchrum and C. eremita form a total of five groups, and these two species might need to be re-examined taxonomically and molecularly.
Keywords:
Diodon holocanthus; fish disease; juvenile; manca; pathology
Key Contribution: Life cycle stages of Cymothoa pulchrum were described.

1. Introduction

To manage fishery resources, it is necessary to understand various ecological positions, including relationships with parasites. The cymothoids are parasites that damage fish [1,2,3,4,5]. Cymothoidae Leach, 1818 (Crustacea: Isopoda) family comprises over 360 species across 49 genera of globally distributed fish parasites [6]. It is one of the largest families among isopods, and found in all seas [7,8]. Their host range includes various fish species inhabiting marine, brackish, and freshwater ecosystems [8,9]. Cymothoid parasites infest their hosts at four locations: the branchial cavity, buccal cavity, burrowed flesh, and external body surface [8].
Cymothoidae feed on fish tissues or blood for nourishment, depending on their hosts [10,11,12]. Previous research has demonstrated that Nerocila phaiopleura Bleeker, 1857, an externally attaching species, can damage the host’s body surface and decrease its body fat content [13]. Furthermore, injuries caused by cymothoids may result in viral infections [14]. These parasites also pose a threat to aquaculture environments [15,16,17,18], with some studies reporting higher fish mortality rates owing to cymothoid infestations [2].
The life cycle of cymothoids involves free-swimming mancae (=mancae II) developing into juveniles and maturing into adult males on their hosts. Subsequently, the adult male changes their sex from male to female [19,20,21]. The identification of cymothoid species primarily relies on the morphological features of the adult females. Consequently, traditional morphometric methods for species identification are challenging, making molecular analysis the sole reliable approach for identifying non-female specimens [22,23]. To address this limitation in species identification, it is crucial to gather more morphological data on cymothoid juveniles and mancae. Furthermore, for accurate species identification, it is essential to accumulate the DNA sequences associated with the morphological characteristics of adult females.
Cymothoa pulchrum Lanchester, 1902 is a cymothoid commonly found in the buccal cavity of fishes, and is distributed in the central and western Indo-Pacific region [24]. Cymothoa pulchrum is found from six species of Tetraodontiformes, one species of Carangiformes, one species of Eupercaria incertae sedis, and one species of Acanthuriformes (see Yamauchi and Hoshino [25]). However, there is no information on the morphology of life cycle stages other than that of adult females and adult males. In addition, the nucleotide sequence information for C. pulchrum was obtained by Hata et al. [26], but this identification has been called into question [27] as this sequence is not linked to a basis for morphological species identification.
In this study, adult female and adult male C. pulchrum were collected from the wild longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. In addition, C. pulchrum mancae and juveniles were obtained by culturing parasitized D. holocanthus. Here, we describe the morphological characteristics of each life cycle stage. We also sequenced the cytochrome c oxidase subunit I (COI) and 16S rRNA sequences linked to the morphological information of adult females and compared them with sequences in the database.

2. Materials and Methods

2.1. Sampling and Rearing

Six D. holocanthus individuals infested with C. pulchrum were collected from Kashiwajima port, Kashiwajima Island, Otsuki, Kochi Prefecture, Japan, using a hand net (Table 1). Cymothoa pulchrum were removed from the buccal cavity (basihyal side) of three D. holocanthus (Fish ID: a–c) and fixed in 99.5% ethanol on the site. The remaining three D. holocanthus (Fish ID: 1–3) were transported alive in aerated seawater to the Seto Marine Biological Laboratory (SMBL), Field Science Education and Research Center, Kyoto University. To obtain the manca, three D. holocanthus were reared in a tank (620 mm × 370 mm × 300 mm) with a continuous flow of seawater. On 10 July 2024, D. holocanthus (Fish ID 1) spat out C. pulchrum, and the spat-out C. pulchrum was collected (the adult female was damaged by being bitten by the host, but the male was alive). Diodon holocanthus spitting out C. pulchrum was isolated from the other individuals (died on 18 July). On 15 August, there were no cymothoids in the buccal cavity of D. holocanthus (Fish ID 2, SMBL-V0852), and the remaining cymothoids were at the bottom of the tank. On 28 August, D. holocanthus (Fish ID 3) died, and the cymothoids were lying at the bottom of the tank and decomposed. On 30 September, cymothoids were again found to infest the buccal cavity of D. holocanthus (Fish ID 2), and several cymothoids were also found to infest the body surface (Figure 1A,B). Three cymothoids infesting the body surface were removed and fixed with 99.5% ethanol. In addition, three juveniles that had fallen off the body surface were found in the aquarium; therefore, they were also fixed. On 17 October, D. holocanthus (Fish ID 2) died, and as there were more cymothoids attached to its body than confirmed on 30 September, the cymothoids and fish were fixed with 99.5% ethanol (Figure 1C,D). The life stage of each individual was determined according to Aneesh et al. [28].

2.2. Morphological Observation

We morphologically observed the manca, male, transitional, and female. Photographs of cymothoids were captured using a SMZ18 stereomicroscope (Nikon, Tokyo, Japan) with the real-time EDF function in NIS-Elements Documentation (version 5.30.00) (Nikon). The images were subsequently processed and combined using Photoshop 2025 (version 26.2.0) (Adobe, San Jose, CA, USA). Morphological descriptions were made with the aid of a SMZ800 stereomicroscope with a P-IDT drawing tube and an ECLIPSE E200 upright microscope with a Y-IDT drawing tube (Nikon). The illustrations were digitally inked utilizing Illustrator 2024 (version 28.4.1) (Adobe) in conjunction with a DTC133 pen display (Wacom, Saitama, Japan). The measurements and terminologies are primarily based on Aneesh et al. [28] and Fujita et al. [29]. Species descriptions were formulated using the DEscription Language for TAxonomy (DELTA) as outlined by Coleman et al. [30]. Taxonomic data are also provided in DELTA format (Supplementary File S1). These individuals were deposited at SMBL (SMBL-V0841–V0852).

2.3. DNA Analysis

The COI and 16S rRNA sequences of three individuals were determined to accumulate DNA information linked to morphological details. The COI and 16S rRNA regions of a female (SMBL-V0841), a juvenile (SMBL-V0845), and a manca (SMBL-V0849) were sequenced. The host DNA attached to the sample surface was removed according to Fujita and Nakano [31]. DNA was extracted from the pereopods using an alkaline lysis method according to the recommended protocol for the KOD One PCR Master Mix and KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan).
The COI region was amplified using primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [32]. The total volume for COI 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. The PCR conditions for COI were as follows: 35 cycles of 98.0 °C for 10 s, 45.0 °C for 30 s, and 68.0 °C for 45 s. The 16S rRNA region was amplified using primers 16Sar (5′-CGCCTGTTTAACAAAAACAT-3′) and 16Sbr (5′-CCGGTCTGAACTCAGATCATGT-3′) [33]. The total volume for 16S rRNA PCR was 12.5 μL, which was composed of 1 μL of DNA, 4.5 μL of distilled water, 0.375 μL of each primer (10 μM solutions), and 6.25 μL of KOD One PCR Master Mix. The PCR conditions for 16S rRNA were 35 cycles of 98.0 °C for 10 s, 50.0 °C for 5 s, and 68.0 °C for 1 s. The PCR products were sent to Eurofins Genomics (Tokyo, Japan) sequencing services and sequenced using the dye terminator method. The sequences were deposited in GenBank (accession numbers: PV235260–PV235262, PV241149–PV241151).
The nucleotide sequences of six Cymothoa species were downloaded from GenBank for both the COI and 16S rRNA. The sequences were aligned using MUSCLE [34], implemented in MEGA 11 [35]. Neighbor-joining trees [36] were generated based on COI (total of 658 bp) and 16S rRNA (total of 524 bp including gaps) sequences using the Kimura two-parameter (K2P) model [37] and the “pairwise deletion” option. Nerocila japonica Schioedte and Meinert, 1881, and N. phaiopleura were also included as outgroups.
Pairwise intra- and inter specific genetic distances were calculated based on COI using K2P in genus Cymothoa. The values were calculated for each species and lineage. The K2P value was calculated using the same 27 sequences of Cymothoa as the phylogenetic tree.

3. Results

3.1. Taxonomy

Order: Isopoda Latreille, 1816
Superfamily: Cymothooidea Leach, 1814
Family: Cymothoidae Leach, 1814
Genus: Cymothoa Fabricius, 1793
Cymothoa pulchrum Lanchester, 1902
[Japanese name: Fugu-noe]
(Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8, Supplementary File S1)
Cymothoa pulchrum Lanchester, 1902: 377, pl. 35, Figures 8 and 8a [38].—Monod, 1924: 100 [39].—Trilles, 1975: 991, pl. II (16) [40]; 1994: 148 [41].—Galzin and Trilles, 1979: 257, Figures 1–52 [42].—Avdeev, 1982b: 69 [43].—Williams, Bunkley-Williams and Dyer, 1996: 1, Figure 5 [44].—Kensley, 2001: 233 [45].—Trilles and Bariche, 2006: 228 [46].—Martin, Bruce and Nowak, 2016 [24].
Cymothoa pulchra—Nierstrasz, 1915: 92, Figures 11–13 [47]; 1931: 133, Figures 1–4 [48].—Monod, 1934: 12, pl. 26 (a–b), 30 (a) [49].—Shiino, 1951: 81, 85, Figure 4a–h [50].—Avdeev, 1978b: 281 [51].—Saito, Itani and Nunomura, 2000: 65 [52].—Bruce, Lew Ton and Poore, 2002: 176 [53].—Kuramochi, Ikeda and Watanabe, 2003: 1, 2, Figure 1 [54].—Nagasawa and Uyeno, 2012: 139, Figure 1 [55].—Nagasawa and Doi, 2012 [56].—Yamauchi and Hoshino, 2021 [25].
Material examined: Two non-ovigerous females (TL 29.59, 28.66 mm), SMBL-V0841, V0843, one transitional (TL 31.30 mm), SMBL-V0842, hand net, 5 July 2024, buccal cavity (basihyal side) of Diodon holocanthus Linnaeus, 1758, Kashiwajima port, Kashiwajima Island, Otsuki, Kochi Prefecture (32°46′08.7″ N 132°37′36.0″ E). One male (TL 19.02 mm), SMBL-V0844, one juvenile (TL 5.97 mm), SMBL-V0845, reared from 5 July 2024 to 10 July 2024. One transitional (TL 23.23 mm), SMBL-V0846, two juveniles (TL 3.99, 4.54 mm), SMBL-V0847, V0848, three mancae (TL 2.63, 2.80, 2.89 mm), SMBL-V0849–V0851, reared from 5 July 2024 to 17 October 2024.
Additional material: One female (damaged), 142 juveniles, and eight mancae. These individuals were only used for simple external observations.
Description of non-ovigerous females: Body subparallel, 1.8 times longer than maximum width, widest at pereonite 3 or pereonite 4, pereonite 7 narrowest, with convex dorsal surfaces. Cephalon 2.0 times as wide as long, trapezoidal, moderately immersed in pereonite 1. Eyes degenerate, with indistinct margins. Anterior margin of pereonite 1 extends forward. Coxae nearly visible in dorsal view, coxae of pereonite 2–4 posteroventral margins subtruncate, coxae of pereonite 5–7 with moderately acute carinae; pereon longest at pereonite 4, shortest at pereonite 7; pereonites 1–5 posterior margins smooth and slightly curved laterally, that of pereonite 6 and 7 slightly recessed, pereonite 7 without penis. Pleonites 1–5 (pleon) 0.2 times longer than total length (TL), 0.8 times wider than maximum body width, with all pleonites visible in dorsal view. Pleotelson 0.3 times longer than wide, 0.8 times longer than pleon, without seta.
Antennula 8 articles, not extending beyond mid-length of cephalon. Antenna 7–8 articles, extending beyond mid-length of cephalon.
Pereopod 1 basis 1.5 times longer than maximum width; ischium 0.6 times longer than basis; merus 0.3 times longer than ischium, without robust seta; carpus 1.2 times longer than merus; propodus 3.1 times longer than carpus, without robust seta; dactylus 1.1 times longer than propodus, without teeth. Pereopod 7 basis 1.5 times longer than maximum width; ischium 0.6 times longer than basis, without robust seta; merus 0.4 times longer than ischium, without robust seta on superior distal angle, without robust seta; carpus 0.8 times longer than merus, without robust seta; propodus 2.3 times longer than carpus, without robust seta; dactylus same length as propodus, without teeth.
Pleopod 1 peduncle 3.6 times wider than long, without coupling hook, without plumose seta; endopod rectangular, 1.4 times longer than wide, no swimming seta; exopod circular, same length as wide, same length as endopod, no swimming seta. Pleopod 2 peduncle 0.3 times wider than long, without coupling hook, without plumose seta; endopod oval, 1.4 times longer than wide, without appendix masculine, no swimming seta; exopod semicircular, 1.2 times longer than endopod, same length as wide, no swimming seta.
Uropodal peduncle triangular, 1.7 times longer than wide, same length as exopod, without seta on distal corner; endopod rami 0.8 times longer than that of exopod, extending beyond mid-length of pleotelson; endopod oval, 2.3 times longer than maximum width, no swimming seta; exopod oval, 3.1 times longer than maximum width, no swimming seta.
Description of transitional: Body subparallel, 1.8 times longer than maximum width, pereonite 4 widest, pereonite 7 narrowest, with convex dorsal surfaces. Cephalon 1.7 times as wide as long, trapezoidal, moderately immersed in pereonite 1. Eyes degenerate, with indistinct margins. Anterior margin of pereonite 1 extends forward. Coxae nearly visible in dorsal view, coxae of pereonite 2–4 posteroventral margins subtruncate, coxae of pereonite 5–7 with moderately acute carinae; pereon longest at pereonite 1, shortest at pereonite 7; pereonites 1–5 posterior margins smooth and slightly curved laterally, that of pereonite 6 and 7 slightly recessed, pereonite 7 without penis. Pleon 0.2 times longer than TL, 0.8 times wider than maximum body width, with all pleonites visible in dorsal view. Pleotelson 0.4 times longer than wide, 0.9 times longer than pleon, without seta.
Antennula 8 articles, not extending beyond mid-length of cephalon. Antenna 8–9 articles, extending beyond mid-length of cephalon.
Pereopod 1 basis 1.7 times longer than maximum width; ischium 0.4 times longer than basis; merus same length as ischium, without robust seta; carpus 0.9 times longer than merus; propodus 2.5 times longer than carpus, without robust seta; dactylus 1.2 times longer than propodus, without teeth. Pereopod 7 basis 1.4 times longer than maximum width; ischium 0.5 times longer than basis, without robust seta; merus 0.4 times longer than ischium, without robust seta; carpus same length as merus, without robust seta; propodus 2.2 times longer than carpus, without robust seta; dactylus 1.1 times longer than propodus, without teeth.
Pleopod 1 peduncle 2.6 times wider than long, without coupling hook, without plumose seta; endopod rectangular, same length as wide, no swimming seta; exopod circular, same length as wide, same length as endopod, no swimming seta. Pleopod 2 peduncle 0.3 times wider than long, without coupling hook, without plumose seta; endopod oval, 1.3 times longer than wide, with appendix masculine longer than or shorter than endopod rami, no swimming seta; exopod circular, 1.1 times longer than endopod, 1.1 times longer than wide, no swimming seta.
Uropodal peduncle triangular, 1.2 times longer than wide, 0.9 times longer than exopod, distal corner without seta; endopod rami 0.8 times longer than that of exopod, extending beyond mid-length of pleotelson; endopod oval, 2.1 times longer than maximum width, no swimming seta; exopod oval, 3.2 times longer than maximum width, no swimming seta.
Description of male: Body elliptical, 1.9 times longer than maximum width, pereonite 4 widest, pleonite 2 narrowest, with smooth dorsal surfaces. Cephalon 1.6 times as wide as long, semi-oval, slightly immersed in pereonite 1. Eyes are bean-shaped, with indistinct margins. Anterior margin of pereonite 1 extends forward. Coxae nearly visible in dorsal view, coxae of pereonite 2–4 posteroventral margins subtruncate, coxae of pereonite 5–7 with moderately acute carinae; pereon longest at pereonite 1, shortest at pereonite 7; pereonites 1–5 posterior margins smooth and slightly curved laterally, that of pereonite 6 and 7 slightly recessed, pereonite 7 with penis. Pleon 0.2 times longer than TL, 0.7 times wider than maximum body width, with all pleonites visible in dorsal view. Pleotelson 0.4 times longer than wide, 0.9 times longer than pleon, without seta.
Antennula 8 articles, extending beyond mid-length of cephalon. Antenna 8 articles, extending beyond mid-length of cephalon.
Pereopod 1 basis 1.6 times longer than maximum width; ischium 0.5 times longer than basis; merus 0.4 times longer than ischium, without robust seta; carpus 1.4 times longer than merus; propodus 2.7 times longer than carpus, without robust seta; dactylus 0.9 times longer than propodus, without teeth. Pereopod 7 basis 1.2 times longer than maximum width; ischium 0.5 times longer than basis, without robust seta; merus 0.5 times longer than ischium, without robust seta; carpus 0.8 times longer than merus, without robust seta; propodus 2.3 times longer than carpus, without robust seta; dactylus same length as propodus, without teeth.
Pleopod 1 peduncle 2.8 times wider than long, without coupling hook, without plumose seta; endopod elliptical, 1.6 times longer than wide, no swimming seta; exopod semicircular, 1.4 times longer than wide, 1.4 times longer than endopod, no swimming seta. Pleopod 2 peduncle 0.5 times wider than long, without coupling hook, without plumose seta; endopod rounded triangular, 1.4 times longer than wide, with appendix masculine extending beyond endopod rami, no swimming seta; exopod quadrilateral, 1.2 times longer than endopod, 1.1 times longer than width, no swimming seta.
Uropodal peduncle triangular, 1.4 times longer than wide, same length as exopod, distal corner without seta; endopod rami 0.8 times longer than that of exopod, extending beyond mid-length of pleotelson; endopod oval, 2.4 times longer than maximum width, no swimming seta; exopod oval, 3.3 times longer than maximum width, no swimming seta.
Description of juvenile: Body elliptical, 2.0–2.2 times longer than maximum width, pereonite 6 widest, pleonite 2 narrowest, with smooth dorsal surfaces. Cephalon 1.8–2.5 times as wide as long, semi-oval, not immersed in pereonite 1. Eyes oval, with distinct margins, with long axis of each eye 0.8–1.1 times as long as cephalon, short axis 0.2 times maximum width of cephalon. Anterior margin of pereonite 1 straight. Coxae nearly visible in dorsal view, narrow; pereon longest at pereonite 1, shortest at pereonite 7; pereonites 1–5 posterior margins smooth and slightly curved laterally, that of pereonite 6 and 7 slightly recessed, pereonite 7 with penis. Pleon 0.2 times longer than TL, 0.7 times wider than maximum body width, with all pleonites visible in dorsal view. Pleotelson 0.4–0.6 times longer than wide, 0.7–1.0 times longer than pleon, with several short swimming setae on posterior margins.
Antennula 8 articles, extending beyond anterior border of pereonite 1. Antenna 9 articles, extending beyond anterior border of pereonite 1.
Pereopod 1 basis 2.1 times longer than maximum width; ischium 0.4 times longer than basis; merus 0.9 times longer than ischium, with 1 robust seta on superior distal angle; carpus 0.4 times longer than merus; propodus 6.1 times longer than carpus, without robust seta; dactylus same length as propodus, without teeth. Pereopod 7 basis 1.5 times longer than maximum width; ischium 0.7 times longer than basis, without robust seta; merus 0.5 times longer than ischium, superior distal angle without robust seta, inferior margin with 1 robust seta; carpus 0.8 times longer than merus, inferior margin with 0–2 robust setae; propodus 2.7 times longer than carpus, inferior margin with 0–3 robust setae; dactylus 0.8 times longer than propodus, without teeth.
Pleopod 1 peduncle 1.5 times wider than long, with 4 coupling hooks on medial margin, without plumose seta; endopod rectangular, 2.3 times longer than wide, with several short swimming setae on posterior margins; exopod elliptical, 1.4 times longer than wide, same length as endopod, with several short swimming setae on posterior margins. Pleopod 2 peduncle 0.5 times wider than long, with 4 coupling hooks on medial margin, without plumose seta; endopod oval, 1.7 times longer than wide, with appendix masculine longer than or shorter than endopod rami, with several short swimming setae on posterior margins; exopod elliptical, 1.4 times longer than endopod, 1.1 times longer than wide, with several short swimming seta on posterior margins.
Uropodal peduncle triangular, 1.4 times longer than wide, 0.8 times longer than exopod, distal corner with 0–1 seta; endopod rami 0.9 times longer than that of exopod, extending beyond posterior margin of pleotelson; endopod elliptical, 2.9 times longer than maximum width, with several swimming setae on posterior margins; exopod elliptical, 3.6 times longer than maximum width, with several swimming setae on posterior margins.
Description of manca: Body oval, 2.4 times longer than maximum width, pereonite 5 widest, pleonite 1 narrowest, with smooth dorsal surfaces. Cephalon 1.6 times as wide as length, triangle, not immersed in pereonite 1. Eyes oval, with distinct margins, with long axis of each eye 0.6 times the as long as cephalon, short axis 0.2 times the maximum width of cephalon. Anterior margin of pereonite 1 straight. Coxae nearly visible in dorsal view, narrow; pereon longest at pereonite 1, shortest at pereonite 7; pereonites 1–5 posterior margins smooth and slightly curved laterally, that of pereonite 6 and 7 slightly recessed, pereonite 7 without penis. Pleon 0.2 times longer than TL, 0.6 times wider than maximum body width, with all pleonites visible in dorsal view. Pleotelson 0.7 times longer than wide, 0.8 times longer than pleon, with several short swimming setae on posterior margins.
Antennula 8 articles, extending beyond anterior border of pereonite 1. Antenna 9 articles, extending beyond anterior border of pereonite 1.
Pereopod 1 basis 2.1 times longer than maximum width; ischium 0.5 times longer than basis; merus 0.7 times longer than ischium, with 1 robust seta on superior distal angle; carpus 0.5 times longer than merus; propodus 5.1 times longer than carpus, inferior margin with 0–1 robust seta; dactylus same length as propodus, without teeth. Pereopod 6 basis 2.0 times longer than maximum width; ischium 0.6 times longer than basis, inferior margin with 1 robust seta; merus 0.4 times longer than ischium, without robust seta on superior distal angle, inferior margin with 0–1 robust seta; carpus 0.9 times longer than merus, inferior margin with 1 robust seta; propodus 2.8 times longer than carpus, inferior margin with 2–5 robust setae; dactylus 0.8 times longer than propodus, without teeth. Pereopod 7 underdeveloped, narrower and shorter; dactylus not recurved.
Pleopod 1 penduncle 1.8 times wider than long, with 4 coupling hooks on medial margin, without plumose seta; endopod narrow, 2.8 times longer than wide, with several short swimming setae on posterior margins; exopod elliptical, 1.6 times longer than wide, same length as endopod, with several short swimming setae on posterior margins. Pleopod 2 peduncle 0.5 times wider than long, 4 coupling hooks on medial margin, without plumose seta; endopod narrow, 2.5 times longer than wide, without appendix masculine, with several short swimming setae on posterior margins; exopod elliptical, 1.7 times longer than endopod, 1.1 times longer than wide, with several short swimming setae on posterior margins.
Uropodal peduncle triangular, 1.7 times longer than wide, 0.7 times longer than exopod, distal corner without seta; endopod rami same length as that of exopod, extending well beyond posterior margin of pleotelson; endopod narrow, 3.9 times longer than maximum width, with several swimming setae on posterior margins; exopod narrow, 5.4 times longer than maximum width, with several swimming setae on posterior margins.
Coloration: Pearl yellow in preserved ethanol.
Hosts: All individuals used in this study were obtained from D. holocanthus. Cymothoa pulchrum is found from six species of Tetraodontiformes–stellate puffer, Arothron stellatus (Bloch and Schneider, 1801), guineafowl puffer, Arothron meleagris (Lacepéde, 1798), spotfin burrfish, Chilomycterus reticulatus (Linnaeus, 1758), D. holacanthus, spot-fin porcupinefish, Diodon hystrix Linnaeus, 1758, black-blotched porcupinefish, Diodon liturosus Shaw, 1804; one species of Carangiformes–Caranx sp.; one species of Eupercaria incertae sedis–Japanese parrotfish, Calotomus japonicus (Valenciennes, 1840); one species of Acanthuriformes–little spinefoot, Siganus spinus (Linnaeus, 1758) [25].
Distribution: Central and western Pacific and Indian Ocean (see Martin et al. [24]). The distribution of this species in the seas around Japan is thought to be dependent on the Kuroshio Current [55].
Remarks: The individuals in this study were generally consistent with the characteristics of C. pulchrum (subparallel body, widest at pereonite 3–5, cephalon deeply immersed in pereonite 1, anterior border of pereonite 1 subtruncate, coxae almost visible in dorsal view, posterior margin of pleotelson round, basis of pereopod 1 with large carina, uropodal endopod rami reaching half of pleotelson), which were redescribed in Martin et al. [24]. However, a prominent lobe of the pereopod 7 ischium, which was observed by Martin et al., 2016 [24], was not observed in this study. As the same observation was made by Shiino [50], this may be an intraspecific variation in C. pulchrum in Japan.
The manca was distinguished from the juvenile by (1) triangular cephalon (semi-oval in juvenile), (2) pereonite 7 without penis (with penis in juvenile), (3) pereopod 7 underdeveloped (complete pereopod 7 in juvenile), (4) pleopod 1 endopod narrow (rectangular in juvenile), (5) pleopod 2 endopod narrow (oval in juvenile), (6) pleopod 2 endopod without appendix masculine (with appendix masculine in juvenile), (7) uropodal peduncle with no seta (0–1 seta on distal corner in juvenile), (8) uropod extending well beyond posterior margin of pleotelson (extending slightly in juvenile), and (9) uropodal endopod and exopod narrow (elliptical in juvenile).
The juvenile was distinguished from the male by (1) body widest at pereonite 6 (pereonite 4 in male), (2) cephalon not immersed in pereonite 1 (slightly immersed in pereonite 1 in male), (3) distinct oval eyes (indistinct bean-shaped eyes in male), (4) anterior border of pereonite 1 straight (protrudes forward in male), (5) pleotelson, pleopod, uropod, with several short swimming setae (without seta in male), (6) antennula extending beyond anterior border of pereonite 1 (reaching half of cephalon in male), (7) antenna 9 articles (8 article in male), (8) antenna extending beyond anterior border of pereonite 1 (reaching half of cephalon in male), (9) pereopods with robust setae (without robust seta in male), (10) pereopod 7 basis with no carina on superior proximal margin (with large carina in male), (11) pleopod 1 and 2 peduncle with 4 coupling hooks (without coupling hook in male), (12) uropod extending beyond posterior margin of pleotelson (reaching half of pleotelson in male).
The male was distinguished from the transitional by (1) elliptical body (subparallel in transitional), (2) narrowest at pleonite 2 (pereonite 7 in transitional), (3) smooth dorsal surfaces (convex dorsal surfaces in transitional), (4) semi-oval cephalon (trapezoidal in transitional), (5) cephalon slightly immersed in pereonite 1 (deeply immersed in transitional), (6) bean-shaped eyes (degenerate eyes in transitional), pereonite 7 with penis (without penis in transitional), (7) antennula extending beyond the mid-length of cephalon (not reaching mid-length of cephalon in transitional), and (8) pleopod 2 endopod appendix masculine extending beyond endopod rami (shorter than endopod rami in transitional).
The transitional was distinguished from the female by (1) pereon longest at pereonite 1 (pereonite 4 in female), (2) antenna 8–9 articles (7–8 articles in female), (3) pleopod 2 endopod with appendix masculine (without appendix masculine in female).
For many species in Cymothoa, no morphological information is available, except for adults (Table 2). Three species of Cymothoa have been reported from Japanese waters [9,57], but only Cymothoa indica has morphological information of the juveniles [57,58]. The juvenile of C. pulchrum was distinguished from that of C. indica by (1) body rounder (narrower in C. indica), (2) widest at pereonite 6 (pereonite 4 in C. indica), (3) narrowest at pleonite 2 (pleonite 5 in C. indica), (4) antennula extending beyond anterior border of pereonite 1 (mid-length of pereonite 1 in C. indica), (5) antenna extending beyond anterior border of pereonite 1 (mid-length of pereonite 1 in C. indica), (6) pereopod 7 ischium without robust seta (with 1–3 robust setae on inferior margin), (7) pereopod 7 propodus with 0–3 robust setae on inferior margin (4–7 robust setae in C. indica), (8) pleopod 2 endopod with appendix masculine longer than or shorter than endopod rami (shorter than endopod rami in C. indica), (9) uropodal peduncle with 0–1 seta on distal corner (3 setae in C. indica), (10) uropodal endopod elliptical (oval in C. indica), (11) uropodal exopod elliptical (triangular in C. indica). Roy et al. [59] described manca and juvenile of C. indica, but there is some doubt about their life cycle stage identification (e.g., manca having well developed pereopod 7, juvenile not having any swimming setae). Therefore, we did not use the information of mancae and juveniles described by Roy et al. [59].

3.2. DNA Analysis

In the neighbor-joining phylogenetic tree, the three individuals sequenced in this study matched C. pulchrum infesting D. holocanthus (Amami Islands) (LC159560, LC159449) and C. japonicus (Chiba) (LC159561, LC159450), which were registered by Hata et al. [26] (Figure 9). In contrast, C. pulchrum parasitic on S. spinus (Yoron Island) (COI only, LC160321), D. hystrix (Okinawa Island) (COI only, LC160322), and C. reticulatus (Kochi) (16S rRNA only, LC160307), formed a different clade (Figure 9). In COI, Cymothoa eremita (Brünnich, 1783), registered by Martin et al. [27], formed the same clade as C. pulchrum parasitizing S. spinus (Yoron Island) and D. hystrix (Okinawa Island).
K2P values were calculated based on the combined group 1–5 of C. pulchrum and C. eremita shown in the phylogenetic tree (Figure 9, Table 3). Only when each group was treated as a separate species did the maximum value of intraspecific distance become smaller than the minimum value of interspecific distance (Table 3).

4. Discussion

Previously, morphological information on C. pulchrum was available only for adult females and males. In this study, we reported the morphology of the manca, juveniles, and transitional of C. pulchrum for the first time. The manca and juveniles of cymothoids do not have morphological characteristics that are used to identify species, and morphological species identification is currently difficult for most species [22,23]. To enable the morphological species identification of mancae and juveniles, it is necessary to construct a new system for species identification. There are few species for which morphological information on mancae and juveniles is available for Cymothoa (Table 2), and more information needs to be accumulated. In this process, molecular species identification would be useful.
In molecular species identification (DNA barcoding), the problem is that sequences have been registered in databases based on incorrect species identification [64]. To solve this problem, it is necessary to accumulate nucleotide sequence data linked to morphological information. The COI and 16S rRNA sequences of the adult female C. pulchrum registered in this study will be useful for future molecular species identification.
In this study, we observed the morphology of C. pulchrum obtained from wild D. holocanthus as well as from that reared in the laboratory. The mancae and juveniles observed in this study were obtained from D. holocanthus (Fish ID 2) in September and October 2024. Diodon holocanthus (Fish ID 2) expelled C. pulchrum from its buccal cavity in August 2024. However, when D. holocanthus (Fish ID 2) died in October, the buccal cavity of D. holocanthus (Fish ID 2) was parasitized by the transitional of C. pulchrum, and its body surface was covered with numerous C. pulchrum mancae and juveniles. It is not known exactly where these C. pulchrum came from.
Diodon holocanthus (Fish ID 1 and 2) expelled C. pulchrum. In particular, when D. holocanthus (Fish ID 1) expelled C. pulchrum, the female was damaged by D. holocanthus, but the male remained alive. It is unclear whether the detachment was caused by C. pulchrum or D. holocanthus. Further research is needed to determine the significance of the relationship between C. pulchrum and D. holocanthus in the wild and the duration of this relationship with its host.
Currently, the life cycle of cymothoids is generally recognized as the same for the entire family [8]. Williams and Bunkley-Williams [65] proposed a hypothesis about the life cycle of the Anilocra, in which juveniles function as males, and juveniles (males) migrate between multiple host individuals, mating when they parasitize a female-parasitized fish. Cymothoa pulchrum is considered to be a cymothoid that parasitizes the buccal cavity of its host. In contrast, the mancae and juveniles obtained in this study were parasitic on the body surface of D. holocanthus. This indicates that the mancae of C. pulchrum can also obtain nutrients from its host and grow into juveniles on the body surface of D. holocanthus. Furthermore, there are some points that differ from the previously thought life cycle of cymothoids, such as juveniles with long appendix masculine of pleopod 2 endopod. It is necessary to clarify the parasitic sites of each life cycle stage and reproductive methods of C. pulchrum.
In this study, DNA sequences of C. pulchrum linked to morphological information were obtained. Martin et al. [27] reported that the C. eremita they identified and sequenced matched C. pulchrum (Yoron Island) infesting S. spinus sequenced by Hata et al. [26]. Martin et al. [27] state that the C. pulchrum identified by Hata et al. [26] is a misidentification, because they thought C. pulchrum is specific to the Tetraodontiformes (although it is not actually correct [25]); C. pulchrum and C. eremita are morphologically similar, and Hata et al. [26] did not provide any evidence for identifying cymothoid species. However, Martin et al. [27] only added the sequences of C. eremita identified by them or by Hata et al. [26] of one individual each to the phylogenetic tree. In this study, we analyzed all available COI sequences of C. eremita and C. pulchrum sequences, so the situation is more complex. Cymothoa eremita clade of Martin et al. [27] included C. pulchrum infesting S. spinus (Yoron Island) and D. hystrix (Okinawa Island) by Hata et al. [26]. On the other hand, the C. pulchrum identified in this study formed a different clade from the C. eremita by Martin et al. [27], and this clade also included the sequences of C. pulchrum infesting D. holocanthus (Amami Islands) and C. japonicus (Chiba) reported by Hata et al. [26]. Because both D. holocanthus and D. hystrix belong to the genus Diodon (Diodontidae), there was no correspondence between the host taxa and the phylogenetic relationship of C. pulchrum. Therefore, it is necessary to verify the possibility that C. eremita also infests D. hystrix and S. spinus. It is also possible that the C. pulchrum lineage separates into two groups. In the 16S rRNA phylogenetic tree, C. pulchrum was also divided into two clades. It is necessary to sample at more locations, conduct molecular analyses, and observe their morphology. In addition, the fact that no C. pulchrum sequences have been obtained from outside Japan is also a problem.
In Japan, C. eremita has only been obtained from the Japanese thread-sail fish, Hime japonica (Günther, 1877) [as Aulopus japonicus Günther, 1877] [9,50], and there are no other cymothoids reported from H. japonica. However, C. eremita (LC159559, LC159447) that parasitizes H. japonica, sequenced by Hata et al. [26], is very distant from that registered by Martin et al. [27] on the phylogenetic tree. A taxonomic re-examination combining morphological and molecular analyses may be necessary for C. eremita and C. pulchrum. Incidentally, Hadfield et al. [66] stated that the host of C. eremita in Shiino [50] is the Taiwan thread-sail fish, Hime formosana (Lee and Chao, 1994) rather than H. japonica. However, we simply followed the synonym list and assumed that the host is H. japonica because Shiino [50] does not describe the characteristics of the fish and there is no evidence that it is H. formosana.

5. Conclusions

In this study, we described the morphology of C. pulchrum: manca, juvenile, adult male, transitional, and adult female. It becomes possible to distinguish between juveniles of C. pulchrum and those of C. indica based on morphology. Additionally, COI and 16S rRNA sequences linked to the morphology of C. pulchrum were obtained. In the molecular phylogenetic tree, C. pulchrum and C. eremita were mixed together and divided into several groups; therefore, a taxonomic re-examination of these two species using a combination of morphological and molecular methods is necessary.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fishes10040155/s1, Supplementary File S1: DELTA format file describing Cymothoa pulchrum Lanchester, 1902 collected from the buccal cavity of the longspined porcupinefish, Diodon holocanthus Linnaeus, 1758.

Author Contributions

H.F., data curation, funding acquisition, investigation, methodology, resources, writing—original draft; H.S., investigation, methodology, writing—review and editing; Y.O., resources, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was partially supported by grants-in-aid from the Japan Society of Promotion of Science (KAKENHI No. 23KJ1170).

Institutional Review Board Statement

Ethical review and approval were waived following Notice (No. 71) of the Japanese Ministry of Education, Culture, Sports, Science and Technology, as well as Regulations on Animal Experimentation at Kyoto University, because the research target species were invertebrates, and the fish were collected in accordance with the Fishery Act of Japan.

Data Availability Statement

The DNA sequences in this study were deposited in NCBI GenBank (accession numbers: PV235260–PV235262, PV241149–PV241151). The specimens used in this study are deposited in the Seto Marine Biological Laboratory (SMBL), Field Science Education and Research Center, Kyoto University (SMBL-V0841–V0852). Taxonomic data is contained in Supplementary File S1.

Acknowledgments

We would like to thank Tomoyuki Nakano (Kyoto University) for providing us with the molecular experimental equipment. We are deeply grateful to four reviewers for their helpful comments and suggestions.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. A longspined porcupinefish, Diodon holocanthus Linnaeus, 1758 (Fish ID 2, SMBL-V0852) infested by Cymothoa pulchrum Lanchester, 1902, collected from Kashiwajima port, Kashiwajima Island, Otsuki, Kochi Prefecture (32°46′08.7″ N 132°37′36.0″ E) on 5 July 2024, and then raised in the laboratory. (A,B) Alive D. holocanthus raised from 5 July to 30 September 2024. (C,D) D. holocanthus preserved in ethanol after being raised from 5 July to 17 October 2024. Allows indicate C. pulchrum. Scale bars: 30 mm.
Figure 1. A longspined porcupinefish, Diodon holocanthus Linnaeus, 1758 (Fish ID 2, SMBL-V0852) infested by Cymothoa pulchrum Lanchester, 1902, collected from Kashiwajima port, Kashiwajima Island, Otsuki, Kochi Prefecture (32°46′08.7″ N 132°37′36.0″ E) on 5 July 2024, and then raised in the laboratory. (A,B) Alive D. holocanthus raised from 5 July to 30 September 2024. (C,D) D. holocanthus preserved in ethanol after being raised from 5 July to 17 October 2024. Allows indicate C. pulchrum. Scale bars: 30 mm.
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Figure 2. Dorsal views of Cymothoa pulchrum Lanchester, 1902 collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) SMBL-V0841, non-ovigerous female (TL 29.6 mm); (B) SMBL-V0846, transitional (TL 23.2 mm); (C) SMBL-V0844, male (TL 19.0 mm); (D,E) SMBL-V0845, V0847, juveniles (TL 6.0 mm, 4.0 mm); (F) SMBL-V0849, manca (TL 2.63 mm). Scale bars: (A–C) 3 mm; (D–F) 1 mm.
Figure 2. Dorsal views of Cymothoa pulchrum Lanchester, 1902 collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) SMBL-V0841, non-ovigerous female (TL 29.6 mm); (B) SMBL-V0846, transitional (TL 23.2 mm); (C) SMBL-V0844, male (TL 19.0 mm); (D,E) SMBL-V0845, V0847, juveniles (TL 6.0 mm, 4.0 mm); (F) SMBL-V0849, manca (TL 2.63 mm). Scale bars: (A–C) 3 mm; (D–F) 1 mm.
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Figure 3. Ventral views of Cymothoa pulchrum Lanchester, 1902, collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) SMBL-V0841, non-ovigerous female (TL 29.6 mm); (B) SMBL-V0846, transitional (TL 23.2 mm); (C) SMBL-V0844, male (TL 19.0 mm); (D,E) SMBL-V0845, V0847, juveniles (TL 6.0 mm, 4.0 mm); (F) SMBL-V0849, manca (TL 2.63 mm). Scale bars: (A–C) 3 mm; (D–F) 1 mm.
Figure 3. Ventral views of Cymothoa pulchrum Lanchester, 1902, collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) SMBL-V0841, non-ovigerous female (TL 29.6 mm); (B) SMBL-V0846, transitional (TL 23.2 mm); (C) SMBL-V0844, male (TL 19.0 mm); (D,E) SMBL-V0845, V0847, juveniles (TL 6.0 mm, 4.0 mm); (F) SMBL-V0849, manca (TL 2.63 mm). Scale bars: (A–C) 3 mm; (D–F) 1 mm.
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Figure 4. Non-ovigerous female (TL 29.6 mm, SMBL-V0841) of Cymothoa pulchrum Lanchester, 1902 collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) ventral view of cephalon; (B) pleotelson and uropods; (C) pereopod 1; (D) pereopod 7; (E) pleopod 1; (F) pleopod 2. Scale bars: (A,B) 3 mm; (C–E) 1 mm; (F) 2 mm.
Figure 4. Non-ovigerous female (TL 29.6 mm, SMBL-V0841) of Cymothoa pulchrum Lanchester, 1902 collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) ventral view of cephalon; (B) pleotelson and uropods; (C) pereopod 1; (D) pereopod 7; (E) pleopod 1; (F) pleopod 2. Scale bars: (A,B) 3 mm; (C–E) 1 mm; (F) 2 mm.
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Figure 5. Transitional (TL 29.6 mm, SMBL-V0846) of Cymothoa pulchrum Lanchester, 1902, collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) ventral view of cephalon; (B) pleotelson and uropods; (C) pereopod 1; (D) pereopod 7; (E) pleopod 1; (F) pleopod 2. Scale bars: (A) 2 mm; (B) 3 mm; (C–F) 1 mm.
Figure 5. Transitional (TL 29.6 mm, SMBL-V0846) of Cymothoa pulchrum Lanchester, 1902, collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) ventral view of cephalon; (B) pleotelson and uropods; (C) pereopod 1; (D) pereopod 7; (E) pleopod 1; (F) pleopod 2. Scale bars: (A) 2 mm; (B) 3 mm; (C–F) 1 mm.
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Figure 6. Male (TL 19.0 mm, SMBL-V0844) of Cymothoa pulchrum Lanchester, 1902, collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) ventral view of cephalon; (B) pleotelson and uropods; (C) pereopod 1; (D) pereopod 7; (E) pleopod 1; (F) pleopod 2. Scale bars: 1 mm.
Figure 6. Male (TL 19.0 mm, SMBL-V0844) of Cymothoa pulchrum Lanchester, 1902, collected from longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A) ventral view of cephalon; (B) pleotelson and uropods; (C) pereopod 1; (D) pereopod 7; (E) pleopod 1; (F) pleopod 2. Scale bars: 1 mm.
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Figure 7. Juveniles of Cymothoa pulchrum Lanchester, 1902, collected from the longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A,C–H,J) SMBL-V0847 (TL 4.0 mm); (B,I) SMBL-V0845 (TL 6.0 mm). (A,B) dorsal view; (C) ventral view of cephalon; (D) pleotelson; (E) pereopod 1; (F) pereopod 7; (G) pleopod 1; (H,I) pleopod 2; (J) uropod. Scale bars: (A,B) 1 mm; (C,D,I) 0.3 mm; (E–H,J) 0.2 mm.
Figure 7. Juveniles of Cymothoa pulchrum Lanchester, 1902, collected from the longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A,C–H,J) SMBL-V0847 (TL 4.0 mm); (B,I) SMBL-V0845 (TL 6.0 mm). (A,B) dorsal view; (C) ventral view of cephalon; (D) pleotelson; (E) pereopod 1; (F) pereopod 7; (G) pleopod 1; (H,I) pleopod 2; (J) uropod. Scale bars: (A,B) 1 mm; (C,D,I) 0.3 mm; (E–H,J) 0.2 mm.
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Figure 8. Manca of Cymothoa pulchrum Lanchester, 1902, collected from the longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A–E,G,H) TL 2.6 mm, SMBL-V0849; (F) TL 2.9 mm. (A) dorsal view; (B) ventral view of cephalon; (C) pleotelson and uropod; (D) pereopod 1; (E) pereopod 6; (F) pereopod 7; (G) pleopod 1; (H) pleopod 2. Scale bars: (A) 0.5 mm; (B,C) 0.3 mm; (D–H) 0.1 mm.
Figure 8. Manca of Cymothoa pulchrum Lanchester, 1902, collected from the longspined porcupinefish, Diodon holocanthus Linnaeus, 1758. (A–E,G,H) TL 2.6 mm, SMBL-V0849; (F) TL 2.9 mm. (A) dorsal view; (B) ventral view of cephalon; (C) pleotelson and uropod; (D) pereopod 1; (E) pereopod 6; (F) pereopod 7; (G) pleopod 1; (H) pleopod 2. Scale bars: (A) 0.5 mm; (B,C) 0.3 mm; (D–H) 0.1 mm.
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Figure 9. Neighbor-joining trees based on the cytochrome c oxidase subunit I (COI) and 16S rRNA genes, detected from the Cymothoa pulchrum Lanchester, 1902 collected in this study along with Cymothoa sequences downloaded from GenBank. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap values (1000 replicates) are shown next to the branches. Cymothoa pulchrum and Cymothoa eremita (Brünnich, 1783) diverged into groups 1–5 in COI tree. Bootstrap supports of lower than 80% are abbreviated.
Figure 9. Neighbor-joining trees based on the cytochrome c oxidase subunit I (COI) and 16S rRNA genes, detected from the Cymothoa pulchrum Lanchester, 1902 collected in this study along with Cymothoa sequences downloaded from GenBank. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap values (1000 replicates) are shown next to the branches. Cymothoa pulchrum and Cymothoa eremita (Brünnich, 1783) diverged into groups 1–5 in COI tree. Bootstrap supports of lower than 80% are abbreviated.
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Table 1. List of samples of Cymothoa pulchrum Lanchester, 1902 collected from the buccal cavity of Diodon holocanthus Linnaeus, 1758.
Table 1. List of samples of Cymothoa pulchrum Lanchester, 1902 collected from the buccal cavity of Diodon holocanthus Linnaeus, 1758.
DateFish IDNumber of Parasites (Specimen ID)Note
FemaleTransitionalMaleJuvenileManca
5 July 2024a1 (SMBL-V0841)
5 July 2024b 1 (SMBL-V0842)
5 July 2024c1 (SMBL-V0843)
10 July 202411 1 (SMBL-V0844)1 (SMBL-V0845) reared from 5 July 2024
30 September 20242, SMBL-V0852 6 reared from 5 July 2024
17 October 20243 1 (SMBL-V0846) 2 (SMBL-V0847, V0848) +1363 (SMBL-V0849–V0851) +8reared from 5 July 2024
Table 2. List of the presence or absence of morphological information on the mancae and juveniles of Cymothoa.
Table 2. List of the presence or absence of morphological information on the mancae and juveniles of Cymothoa.
MancaJuvenileReference
Cymothoa asymmetrica Pillai, 1954--
Cymothoa borbonica Schioedte and Meinert, 1884--
Cymothoa brasiliensis Schioedte and Meinert, 1884--
Cymothoa bychowskyi Avdeev, 1979--
Cymothoa carangii Avdeev, 1979--
Cymothoa catarinensis Thatcher, Loyola e Silva, Jost and Souza-Conceiçao, 2003〇-Thatcher et al. [60]
Cymothoa cinerea Bal and Joshi, 1959--
Cymothoa curta Schiödte and Meinert, 1884--
Cymothoa dufresni Leach, 1818--
Cymothoa elegans Bovallius, 1885--
Cymothoa epimerica Avdeev, 1979--
Cymothoa eremita (Brünnich, 1783)--
Cymothoa excisa Perty, 1833--
Cymothoa exigua Schioedte and Meinert, 1884--
Cymothoa eximia Schioedte and Meinert, 1884--
Cymothoa frontalis H. Milne Edwards, 1840〇〇Aneesh et al. [61]
Cymothoa gadorum Brocchi, 1875--
Cymothoa gerris Schioedte and Meinert, 1884--
Cymothoa gibbosa Gourret, 1891--
Cymothoa globosa Schiödte and Meinert, 1884--
Cymothoa guadeloupensis Fabricius, 1793--
Cymothoa hermani Hadfield, Bruce and Smit, 2011--
Cymothoa ianuarii Schioedte and Meinert, 1884--
Cymothoa ichtiola Bosc, 1801--
Cymothoa indica Schioedte and Meinert, 1884-〇Jones et al. [58]; Fujita [57]
Cymothoa liannae Sartor and Pires, 1988〇-Thatcher et al. [60]
Cymothoa limbata Schioedte and Meinert, 1884--
Cymothoa nigropunctata Risso, 1816--
Cymothoa oestrum (Linnaeus, 1758)〇-Thatcher et al. [60]
Cymothoa parupenei Avdeev, 1979--
Cymothoa plebeia Schioedte and Meinert, 1884--
Cymothoa propria Avdeev, 1979--
Cymothoa pulchrum Lanchester, 1902This studyThis study
Cymothoa recifea Thatcher and Fonseca, 2005--
Cymothoa recta Dana, 1853--
Cymothoa rhina Schioedte and Meinert, 1884--
Cymothoa rotunda Avdeev, 1979--
Cymothoa scopulorum (Linnaeus, 1758)--
Cymothoa selari Avdeev, 1978--
Cymothoa slusarskii Rokicki, 1986--
Cymothoa sodwana Hadfield, Bruce and Smit, 2013--
Cymothoa spinipalpa Thatcher, de Arujo, de Lima and Chellapa, 2007〇-Thatcher et al. [62]; Abd El Aal and El Ashrum [63]
Cymothoa truncata Schioedte and Meinert, 1884--
Cymothoa vicina Hale, 1926 --
〇 indicates that there is morphological information.
Table 3. Pairwise intra- and interspecific genetic distances calculated based on the cytochrome c oxidase subunit I using Kimura-2-parameter model in genus Cymothoa. The first line was calculated excluding the sequences labeled as “C. pulchrum” and “C. eremita”. The second line was calculated treating “group 1”, “group 2”, “group 3”, and “group 4 + group 5” in Figure 9 as separate species. The third line was calculated treating “group 1”, “group 2”, “group 3”, “group 4”, and “group 5” in Figure 9 as separate species.
Table 3. Pairwise intra- and interspecific genetic distances calculated based on the cytochrome c oxidase subunit I using Kimura-2-parameter model in genus Cymothoa. The first line was calculated excluding the sequences labeled as “C. pulchrum” and “C. eremita”. The second line was calculated treating “group 1”, “group 2”, “group 3”, and “group 4 + group 5” in Figure 9 as separate species. The third line was calculated treating “group 1”, “group 2”, “group 3”, “group 4”, and “group 5” in Figure 9 as separate species.
Intraspecific (%)Interspecific (%)
MinMaxMinMax
Except for C. pulchrum and C. eremita (4 species)0.001.275.4021.36
“Group 1”, “2”, “3”, “4 + 5” + 4 species0.002.332.0121.36
“Group 1”, “2”, “3”, “4”, “5” + 4 species0.001.271.6921.36
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Fujita, H.; Shinoda, H.; Okumura, Y. Description of Life Cycle Stages of Fish Parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA Barcode Linked to Morphological Details. Fishes 2025, 10, 155. https://doi.org/10.3390/fishes10040155

AMA Style

Fujita H, Shinoda H, Okumura Y. Description of Life Cycle Stages of Fish Parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA Barcode Linked to Morphological Details. Fishes. 2025; 10(4):155. https://doi.org/10.3390/fishes10040155

Chicago/Turabian Style

Fujita, Hiroki, Haruki Shinoda, and Yuzumi Okumura. 2025. "Description of Life Cycle Stages of Fish Parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA Barcode Linked to Morphological Details" Fishes 10, no. 4: 155. https://doi.org/10.3390/fishes10040155

APA Style

Fujita, H., Shinoda, H., & Okumura, Y. (2025). Description of Life Cycle Stages of Fish Parasite Cymothoa pulchrum (Isopoda: Cymothoidae), with DNA Barcode Linked to Morphological Details. Fishes, 10(4), 155. https://doi.org/10.3390/fishes10040155

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