Developmental Stages of an Invasive, Non-indigenous Sea Louce Caligus sclerotinosus Roubal, Armitage and Rhode, 1983 (Copepoda: Siphonostomatoida: Caligidae) Parasitic on Red Sea Bream Pagrus major (Temminck and Schlegel, 1843) from Japan

© 2025 Academia Sinica, Taiwan Open Access Developmental Stages of an Invasive, Nonindigenous Sea Louce Caligus sclerotinosus Roubal, Armitage and Rhode, 1983 (Copepoda: Siphonostomatoida: Caligidae) Parasitic on Red Sea Bream Pagrus major (Temminck and Schlegel, 1843) from Japan Yusuke Nishida1, Panakkool Thamban Aneesh1,2,3,*, Yusuke Kondo2, Ione Madinabeitia4, Hirofumi Yamashita5, Sho Shirakashi6, Kazuo Ogawa7, Kaori Wakabayashi1, Kenta Adachi1,8 , and Susumu Ohtsuka1,2,3,* 1Graduate School of Integrated Sciences for Life, Hiroshima University, 5–8–1 Minato-machi, Takehara, Hiroshima, 725–0024, Japan. *Correspondence: E-mail: [email protected] (Aneesh); [email protected] (Ohtsuka) E-mail: [email protected] (Nishida); [email protected] (Wakabayashi); [email protected] (Adachi) 2Fisheries Laboratory, Blue Innovation Division, Seto Inland Sea Carbon-neutral Research Center, Hiroshima University, 5–8–1 Minato-machi, Takehara, Hiroshima, 725–0024, Japan. E-mail: [email protected] (Kondo) 3Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Terengganu, Terengganu, Malaysia 4Graduate School of Biosphere Science, Hiroshima University, 5–8–1 Minato-machi, Takehara, Hiroshima, 725–0024, Japan (formerly). E-mail: [email protected] (Madinabeitia) 5Ehime Prefecture Aquaculture Research Group Promotion Office of Agriculture, Forestry and Fisheries Research Institute, Fisheries Research Center, Uwajima, Ehime 798-0104, Japan. E-mail: [email protected] (Yamashita) 6Aquaculture Research Institute, Kindai University, 3153 Shirahama, Nishimuro, Wakayama, 649-2211, Japan. E-mail: [email protected] (Shirakashi) 7Meguro Parasititological Museum, 4-1-1 Shimomeguro, Meguro-ku, Tokyo 153-0064, Japan. E-mail: [email protected] (Ogawa) 8Japan Fisheries Research and Education Agency, Fisheries Technology Institute, Kamiura, Saeki, Oita, 879-2602, Japan Received 4 August 2023 / Accepted 23 April 2025 / Published 20 August 2025 Communicated by Benny K.K. Chan The present study describes and illustrates the morphology of naupliar and copepodid stages of Caligus sclerotinosus Roubal, Armitage and Rohde, 1983 (Copepoda: Siphonostomatoida: Caligoida), an invasive, non-indigenous parasite collected from commercially important red sea bream Pagrus major (Temminck and Schlegel, 1843) (Sparidae) cultured as well as wild populations in western Japan. The life cycle of C. sclerotinosus consists of 8 stages: two nauplii, five copepodids (copepodid I to V) and one adult, which exhibited the general pattern of other congeners. Sexual dimorphism was first observed in the urosome and antenna of copepodid IV. The body lengths of the developmental stages of 14 Caligus spp. are compared to those of C. sclerotinosus. The oral cone of copepodid I is highly unique for its suckerlike shape, widely expanded terminally. All post-naupliar stages were found on the same host fish P. major, rejecting the previous hypothesis for the presence of intermediate hosts for the development of C. sclerotinosus. Key words: Aquaculture, Caligus sclerotinosus, Intermediate host, Japan, Life cycle, Non-indigenous, Pagrus major Citation: Nishida Y, Aneesh PT, Kondo Y, Madinabeitia I, Yamashita H, Shirakashi S, Ogawa K, Wakabayashi K, Adachi K, Ohtsuka S. 2025. Developmental stages of an invasive, non-indigenous sea louce Caligus sclerotinosus Roubal, Armitage and Rhode, 1983 (Copepoda: Siphonostomatoida: Caligidae) parasitic on red sea bream Pagrus major (Temminck and Schlegel, 1843) from Japan. Zool Stud 64:25. doi:10.6620/ZS.2025.64-25. Zoological Studies 64:25 (2025) doi:10.6620/ZS.2025.64-25 1 © 2025 Academia Sinica, Taiwan BACKGROUND Caligus sclerotinosus was originally described by Roubal (1981) based on a single specimen found on the body surface of a surf bream Acanthopagrus australis (Günther, 1859) from northern New South Wales, Australia. Since the specimen was damaged, Roubal (1981) reported this ectoparasite as an unidentified species of Caligus. Two years later, with the additional finding of more specimens from silver seabream Chrysophrys (= Pagrus) auratus (Forster, 1801) caught off Coffs Harbour, New South Wales, Roubal et al. (1983) amended the description and named the parasite ‘Caligus sclerotinosus, sp. nov.’. Since the late 1990s, this sea louse species has been observed infecting the body surface of the extensively cultured red seabream Pagrus major (Temminck and Schlegel, 1843) in coastal areas of western Japan. Ho et al. (2004) identified and described C. scleronitosus based on specimens collected from red seabream cultured in Oita Prefecture. Likewise, the parasite was also reported infecting the external surface of red seabream cultured in Ehime Prefecture (Ohtsuka 2010; Ohtsuka et al. 2018). According to Ho et al. (2004), C. sclerotinosus might have been introduced from Australia to Japan together with its hosts due to anthropogenic causes. In 2013, Venmathi Maran et al. recorded the parasite on cultured red seabream from the southern coastal waters of South Korea. The prevalence of C. sclerotinosus on farmed sparids has been reported to be high during the warm seasons in both Japan and Korea, causing dermal caligosis to it host (Tanaka et al. 2013; Venmathi Maran et al. 2013; Ohtsuka et al. 2018). Sea lice infestations in fish farms can be controlled only through the successful tracking and understanding of their life cycle. To date, information on the complete life cycle of the members of the family Caligidae is known only for quiet few species; 21 out of 518 valid species. Those 21 species are divided into four genera, such as Caligus (14 species), Lepeophtheirus (5 species), Alebion Krøyer, 1863 (1 species) and Paralebion Wilson C.B., 1911 (1 species) (see González and Carvajal 2003; Benz et al. 1992; Venmathi Maran et al. 2013; Khoa et al. 2019; Piasecki et al. 2023). The life cycle of Caligus rogercresseyi was successfully tracked by González and Carvajal (2003) from the farmed salmon. Recently the influence of temperature and the duration of life cycle of another important pathogen of sea floating cage culture, Caligus minimus, infesting the seabass, Lates calcarifer (Bloch, 1790) from, Malaysia was illustrated by Khoa et al. (2019). Despite the frequent occurrence of C. sclerotinosus on cultured red seabream, its biology and life cycle are little known. The adult of this caligid species, which is usually found parasitic on the body surface of its host, was also reported free-swimming in marine plankton samples (González and Carvajal 2003; Venmathi Maran and Ohtsuka 2008; Venmathi Maran et al. 2012a b). Previously, only two naupliar, one copepodid and the adult stages of C. sclerotinosus have been described, leaving its life cycle incomplete. Therefore, the objective of the present study is to describe the morphology of all developmental stages of C. sclerotinosus ectoparasitic on both wild and cultured red seabream from Japanese waters. A morphological comparison of the postnaupliar stages of C. sclerotinosus to those of 14 caligid species is included herein. Furthermore, a traditional method of studying the life cycle of parasitic copepods is to rear nauplii hatching from the egg strings of an ovigerous female (see González and Carvajal 2003). In the present study, we used the same methods; the nauplii I hatching out from the egg strings of ovigerous females recovered from the wild host, are reared to obtain the naupli II and copepodid I. Subsequent copepodid stages were obtained from the host. We also confirmed the identity by comparing the morphological description, which was supported by the genetic data. MATERIALS AND METHODS Post-naupliar stages obtained from farm Five and ten individuals of P. major were obtained from a farm of Kindai University at Shirahama, Wakayama Prefecture, Japan on December 10, 2019 and August 3/4, 2020, respectively. The body surface of the hosts was carefully washed with tap water and gently stroked by hands to remove ectoparasitic copepods. In our preliminary surveys, we have already confirmed the adherence of second to fifth copepodid stages with a frontal filament adhesive to scales of the host body surface. Washing waters were filtered with a small net of 0.2 mm in mesh size to collect copepods and then contaminated blood and mucus of the hosts were removed by relatively strong pouring of tap waters. All copepod specimens were picked up with fine forceps in filtered seawater and finally moved to 99.5% ethanol. Methods to rear copepodid stages on the host Egg strings of Caligus sclerotinosus, infecting the wild red sea bream collected by fishing from Takehara City, Hiroshima Prefecture during July to October 2021, were incubated at 22°C to obtain nauplii I and II and copepodid I. Cultures from copepodid stages I to VI were conducted using parasite-free red sea bream (standard page 2 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan length: 61.2–150.4 mm) artificially produced by the Hiroshima Prefectural Farming Fisheries Center. Copepodid I stage hatched and cultured from egg strings of adult females were exposed for 3 hours to red sea bream reared in a tank containing 20 liters of seawater. During the exposure, the seawater was kept at a standstill. After 3 hours, the volume of seawater in the tank was raised to 80 liters and the sea bream were reared under pouring conditions. The growth of Caligus screlotinosus was observed daily, with the time when the copepodid I was introduced as 0 hour. Genetic identification of copepodid stages Copepodids of C. sclerotinosus obtained from Kindai University on December 10, 2019 were genetically identified as well as one adult female collected off Takehara City, Hiroshima on June 25, 2020. Specimens of the third to sixth copepodid stages after infection (see Piasecki et al. 2023) were preserved with 99% ethanol, respectively. Total DNA of copepodids III–V and adult samples were extracted from whole body using DNeasy Blood & Tissue kits (Qiagen, Venlo, Netherlands). For small sized samples, DNA was extracted from whole body according to the method described by Suyama (2011). DNA was quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and then adjusted to 1 ng μL-1 with sterilized water for PCR amplification. The 28S nuclear ribosomal DNA region (28S) for specimens of the above-mentioned development stages after infection were amplified using a Taq PCR master mix kit and primer sets: 28S-F1a (5'-GCG GAG GAA AAG AAA CTA AC-3') and 28S-R1a (5'- GCA TAG TTT CAC CAT CTT TCG GG-3'; BlancoBercial et al. 2011). Thermocycling conditions for 28S were 94°C for 7 min; 35 cycles at 94°C for 45 s, 50°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 5 min. Amplification results were verified using 2% (w/v) agarose electrophoresis. Excess primers and dNTPs were removed with ExoSAP-IT (Thermo Fisher Scientific, MA, USA), and the sequencing was outsourced commercially (Macrogen Japan, Kyoto, Japan). No difference between sequences (801 bp) were confirmed by the sequences alignment that was performed using CLUSTAL W (Thompson et al. 1994) in MEGA 7 (Kumar et al. 2016). The sequence for 28S (Accession number; LC596096) was deposited in the DDBJ/EMBL/NCBI GenBank databases Bayesian inference (BI) analysis was computed using MrBayes v. 3.2.7 (Ronquist et al. 2012) under the GTR+Γ model for each data subset. The model was selected based on Hierarchical likelihood ratio tests (hLRTs) using MrModeltest v. 2.4 (Nylander 2004). Two parallel analyses of Metropolis-Coupled Markov Chain Monte Carlo (MC3) were conducted for 1,000,000 generations, and topologies were sampled every 100 generations. The convergence of MCMC was checked with the value of the average standard deviation of split frequencies (ASDSF) in MrBayes. The phylogenetic tree of BI analysis was visualized by Figtree v. 1.4.4 (Rambaut 2012). Descriptions and measurement of all developmental stages The post-naupliar stages of Caligus sclerotinosus were collected from the body surface of 15 specimens of cultured red seabream captured on December 10, 2019 and August 3/4, 2020 from a farm of Kindai University, Wakayama Prefecture, Japan. Preliminary surveys during this study have confirmed the adherence of copepodids I–V with a frontal filament to the scales of red seabream. The body surface of the hosts was carefully washed with tap water and gently stroked by hand to remove ectoparasitic copepods. The dislodged debris was filtered with a 0.2 mm mesh size net to recover the detached copepods. All copepod specimens were picked up with a pair of forceps in filtered seawater, preserved in 99.5% ethanol, cleared in lactophenol, and observed with a microscope following the methods of Humes and Gooding (1964). Copepods were counted and classified into different stages. The naupliar (NI, II) and first copepodid stages (CI) of C. sclerotinosus were obtained via incubation. Ovigerous adult females collected from wild P. major caught off Takehara City, Hiroshima Prefecture, Japan, on September 16, 2020 were incubated in conical beaker (water volume: ca. 100 mL) at a temperature of ca. 22.5°C in an incubator (CR-14C, Hitachi, Ltd.). The nauplii and CI first appeared ca. 6 and 48 hours after incubation, respectively. They were preserved in 10% neutralized formalin/seawater for descriptions and measurement. The first copepodid stage (CI) was observed with a scanning electron microscope (JSM-6510LV, Jeol Co., Ltd.). Before electron microscopy, specimens were fixed in 10% neutralized formalin/seawater for 10 days and soaked in distilled water for ca. 3 hrs, dehydrated through a graded ethanol series from 70% to 100% (twice), critical-pointed fried, and coated with gold. Intact specimens of second to fifth copepodid stages (CII–CV) were illustrated with the aid of a drawing tube attached to a differential interference microscope (BX50, Olympus Co., Ltd.). Body lengths of nauplii and copepodids were measured from the anterior to the posterior tips of bodies, and from the anterior margin of the cephalothorax to the terminal ends of caudal rami page 3 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan excluding caudal setae, respectively. This study follows the terminology of the developmental stages of caligids recently been proposed by Piaseck et al. (2023). According to Piasecki et al. (2023), since chalimi and pre-adults of caligids were not strictly defined, these should be replaced by standardized terms used in free-living copepods: copepodids. Instead, the post-naupliar stages of caligids should be simply called first to fifth copepodid stages (CI–V) and adults (CVI). RESULTS Genetic identification All copepodids (CIII 3 indiv.; CIV 6 indiv.; CV 6 indiv.) showed the exactly same nucleoride sequence of 28S in 801 bp as that of the adult female, which leads to a conclusion that these are developmental stages of C. sclerotinosus (see Fig. 12). Although the first and second copepodid stages (CI, CII) were not analyzed by the molecular technique, they were morphologically identified. The morphology of CI was directly confirmed through nauplii via incubation of live ovigerous adult females distinctly identified as C. sclerotinosus. CII was morphologically determined in comparison between CI and CIII. Description of developmental stages Nauplius I (Figs. 1 and 10) Shape of newly hatched larva with very short cylinder, resembles shape of egg. Larva eventually attaining definite elongated, oval body (Fig. 1A), average length 0.35 ± 0.02 mm (range: 0.32–0.37 mm, N = 4) (Fig. 10). Anterior end of body wider with three pairs of appendages antero-ventrally. Posterior end, narrower and blunt, paired balancers on posterolateral margin curved outwards; and visible nauplius eye dorsally. Antennule (Fig. 1B) uniramous, two-segmented: proximal segment short with 2 unequal naked setae on outer surface; distal segment 1.5 times longer than proximal segment, distinctly separated from proximal by ridge articulation; distal segment armed apically with 2 large serrated setae, 1 small aesthetasc arising between setae and 3 subapical prominences on sides. Antenna (Fig. 1C) biramous, with protopod indistinctly divided into coxa and basis; basis not separated from proximal segment of rami; endopod two-segmented, distal segment with one subterminal seta and two long terminal setae ornamented with serrated and plumose margins, third element short and blunt; exopod foursegmented, segments 2–4 carrying a total of 4 long setae, ornamented with serrated, outer and plumose inner margins. Mandible (Fig. 1D) biramous, with unFig. 1. Caligus sclerotinosus, first nauplius. A, Habitus, dorsal view; B, Antennule; C, Antenna; D, Mandible. page 4 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan segmented protopod, not separated from proximal-most exopodal segment; endopod with two long terminal setae and one subterminal short spiniform seta; exopod four-segmented, each segment with one long seta setae, ornamented with serrated outer and plumose inner margins. Nauplius II (Figs. 2 and 10) Resembling previous stage, with body (Fig. 2A) longer than first nauplius. Average size 0.40 ± 0.02 mm (range: 0.37–0.42 mm, N = 5) (Fig. 10), with three appendages antero-ventrally as seen in nauplius I; paired balancers on posterolateral margin curved outwards; nauplius eye present. In older specimens, next stage (copepodid) visible inside. Antennule (Fig. 2B) as in preceding stage; uniramous, two-segmented, segments of equal length, with additional small element terminally and additional minute spinule on distal margin; surface of both segments with 2 or 3 patches of minute spinules. Antenna (Fig. 2C) as in preceding stage with patches of minute spinules on surface of sympod and endopod. Mandible (Fig. 2D) as in preceding stage. Anlage of paired maxillipeds [sensu Piasecki (1996)] (Fig. 2E) first appearing in this stage, consisting of pair of slender, posteriorly directed processes. Copepodid I (Figs. 3 and 10) Body (Fig. 3A, B) elongated with average length of 0.55 ± 0.03 mm (range: 0.47–0.60 mm, N = 22) (Fig. 10). Body with indistinct segmentation, consisting of cephalothorax, free thoracic leg bearing somites, genital complex and caudal rami. Cephalothorax incorporating first pedigerous somite, longer than wide. Second and third pedigerous somites much narrower than cephalothorax. Genital complex indistinctly separated from fourth somite and abdomen. Anal region bearing short caudal rami, each armed with 5 unequal plumose Fig. 2. Caligus sclerotinosus, second nauplius. A, Habitus, dorsal view; B, Antennule; C, Antenna; D, Mandible; E, Buds of maxilliped. page 5 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan setae and one short aesthetasc (Fig. 3N). Mouth cone large, forming trumpet-shape (sucker-like structure), comprising flat labrum and posterior expanded labium (Fig. 3C). In younger specimens coiled frontal filament (Fig. 3A) seen inside of anterior part of cephalothorax. After attaching to their host, older copepodids show frontal filament extruded from anterior margin, with rounded frontal plate at anterior corners (Fig. 3B, D). Antennule (Fig. 3E) two-segmented; proximal segment armed with 3 setae; distal segment bearing 2 aesthetascs and 11 setae, of which 4 setae are terminally bifurcated. Antenna (Fig. 3F) three-segmented; basal segment smallest, unarmed; second segment largest, unarmed; terminal segment recurved claw armed with minute spiniform element at inner base; post-antennal process (Fig. 3G-a) represented by a simple, blunt knob. Mandible (Fig. 3H) rod-shaped gnathobase, foursectioned; proximal section with vestige of naupliar palp; third section longest; terminal section serrated on medial margin of flatten blade, equipped with 11 teeth. Oral cone (Figs. 3C, 4) widely expanded terminally, forming sucker-like structure. Maxillule (Fig. 3Gb) with dentiform process and papilla armed with 3 setae. Maxilla (Fig. 3J) two-segmented; proximal segment (lacertus) robust, unarmed; distal segment (brachium) slender with terminal claw-like calamus and sub-terminal canna possessing serrated membrane. Maxilliped (Fig. 3I) sub-chelate, two-segmented; proximal protopodal segment (corpus) with a pointy process; shaft long and slender, with sub-terminal bifid element and terminal claw. Post-oral process (Fig. 3Gc) represented by simple, pointed knob. Legs 1 (Fig. 3K) and 2 (Fig. 3L) biramous, with indistinctly two-segmented sympod. Distal segment with medium-sized pinnate seta on lateral margin. Both rami unsegmented, equal in size. Exopod flat and oval; endopod sub-rectangular having unarmed lateral margin. Leg 3 (Fig. 3M) rudimentary, with two unequal setae on postero-lateral corner of second free somite. Armature formula of legs 1 and 2 as follows (Roman numerals indicating spines and Arabic numerals, setae): Sympod Exopod Endopod Leg 1 0–0; 1–0 I, III, 4 6 Leg 2 0–0; 1–0 I, II, 4 6 Copepodid II (Figs. 5 and 10) Body (Fig. 5A) with average length of 0.67 ± 0.05 mm (range: 0.58–0.80 mm, N = 21) (Fig. 10). Frontal filament present for attachment to the host. Base of frontal filament (Fig. 5B) with signs of transverse subdivisions. Cephalothorax protruded anteriorly and emarginated posteriorly; about 1.5 times longer than posterior tagma. Second and third pedigerous somites much narrower than cephalothorax; fourth pedigerous somite unarmed. Genital complex distinctly separated from fouth pedigerous somite. Anal somite bearing small caudal rami (Fig. 5L) each armed with 6 unequal naked setae. Antennule (Fig. 5C) two-segmented; proximal segment with 3 naked setae; distal segment with 11 simple naked setae and 2 aesthetascs. Antenna (Fig. 5D) non-prehensile, with robust base and small distal segment weakly sclerotized and carrying 4 marginal processes. Mandible (Fig. 5E) four-segmented, bearing 12 teeth on distal blade. Maxillule (Fig. 5F) comprising blunt dentiform process and basal papilla bearing 3 unequal setae. Maxilla (Fig. 5G) two-segmented, terminal segment lacking flabellum, canna and calamus bearing serrated membrane. Maxilliped (Fig. 5H) indistinctly three-segmented; first segment robust; distal endopodal segment as subchela in form of large, curved claw and short inner seta. Sternal furca absent. Legs 1 (Fig. 5I), 2 (Fig. 5J) and 3 (Fig. 5K) biramous, with segmented rami; legs 1 and 2 bearing bulbous processes at inner proximal corner of coxa. Leg 3 (Fig. 5K) unsegmented and rudimentary, but with anlagen of rami distinct. Setae on legs 1–3 simple. Armature formula of legs 1–3 as follows: Sympod Exopod Endopod Leg 1 1–0 1, 7 1 Leg 2 1–0 1, 6 4, 1 Leg 3 0–0 4 rudiment Copepodid III (Figs. 6, 7 and 10) Body (Figs. 6, 7A) average length of 0.92 ± 0.06 mm (range: 0.88–1.03 mm, N = 6) (Fig. 10). Body with cephalothorax laterally expanded incorporating both first and second pedigerous somites without visible suture dorsally; anterior margin with frontal filament (Fig. 7B) bearing 2 basal lobes; cephalothorax about 3 times longer than indistinctly three-segmented posterior tagma; attenuated distally, bearing anlagen of leg 4 (Fig. 7L) ventro-laterally; caudal rami broader than previous stage, with 3 plumose setae and 3 naked setae. Antennule (Fig. 7C) two-segmented; proximal segment bearing 7 simple setae; distal segment armed with 11 setae and 2 terminal aesthetascs. Antenna (Fig. 7D) indistinctly two-segmented; proximal segment small; distal tip slender, with 2 small spiniform elements page 6 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan Fig. 3. Caligus sclerotinosus, first copepodid. A, Habitus, before ejection of frontal filament, dorsal view; B, Habitus, after ejection of frontal filament, dorsal view; C, Antennule; C, Oral cone and mouthpart appendages; D, Ejected frontal filament; E, Antennule; F, Antenna; G, Postantennal process (a), Maxillule (b), postoral process (c): H, Mandible; I, Maxilla; J, Maxilliped; K, Leg 1; L, Leg 2; M, Leg 3; N, Caudal ramus. page 7 of 21 Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan Fig. 4. SEM micrographs of oral cone of first copepodid of Caligus sclerotinosus. A, Ventral view; B, Lateral view. Abbreviations: A1. Antennule; A2. Antenna; MX. Maxillule; OC. Oral cone. page 8 of 21 Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan Fig. 5. Caligus sclerotinosus, second copepodid. A, Habitus, dorsal view; B, Extension lobes at base of frontal filament; C, Antennule; D, Antenna; E, Mandible; F, Maxillule; G, Maxilla; H, Maxilliped; I, Leg 1; J, Leg 2; K, Leg 3; L, Caudal ramus. page 9 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan within 0.3–0.7 mm and 0.4–0.8 mm range, respectively (Table 1). This narrow range seems to be related to lecithotrophy during the naupliar and the first copepodid stages (Kearn 2004), which feed solely on the yolk originally contained within the egg. The body lengths of the second to sixth copepodid stages (CII–VI) gradually increased molt by molt at specific and sexual rates. A comparison of the body lengths of two consecutive stages, CIV–VI, revealed that the body becomes approximately 1.3–1.8 times and 1.1–2.0 times larger at each molt in females and males, respectively. The most useful features to differentiate each developmental stage of caligid copepodids were the number of extension lobes at the base of the frontal filament (Piasecki 1996) and the number of setae present on the first segment of the antennule (Kim 1993; Ohtsuka et al. 2009; Piasecki et al. 2023). To date, the number of setae on the first antennulary segment increases following two basic patterns as caligid copepodids develop into adults (CI–VI): 3-3-7-1826/27/29-27/28/29 and 3-7-13/14-20-27-27 (Ohtsuka et al. 2009). The case of C. sclerotinosus was similar to Table 1. Comparison in body lengths of developmental stages among Caligus spp. in the previous and present studies. Generally sexual dimorphism appears in the fourth copepodid stage but in some papers, sexes were not distinguished. -: not available Stages Species N I N II C I C II C III Reference Caligus sclerotinosus 0.37 ± 0.02 0.40 ± 0.02 0.55 ± 0.03 0.67 ± 0.05 0.92 ± 0.06 present study C. centrodonti 0.57 - 0.63–0.67 0.7–0.8 1.1–1.2 Gurney (1934) C. clemensi 0.46 0.53 0.66 0.91 1.31 Kabata (1972) C. curtus 0.5 0.55 0.7 1.5 - Heegaard (1947) C. elongatus 0.448 ± 0.005 0.487 ± 0.02 0.661 ± 0.03 0.82 ± 0.1 1.34 ± 0.13 Piasecki (1996) C. epidemicus 0.284 0.295 0.428 - - Lin et al. (1996) C. fugu 0.34 ± 0.02 0.37 ± 0.02 0.49 ± 0.03 0.61 ± 0.05 0.95 ± 0.11 Ohtsuka et al. (2009) C. latigenitalis - - 0.55 0.77 ± 0.05 1.06 ± 0.06 Madinabeitia and Nagasawa (2011) C. minimus 0.44 0.74 0.77 1.64 1.29 Caillet (1979); Madinabeitia and Nagasawa (2011) C. orientalis 0.270–0.405 0.342–0.475 0.57–0.72 0.81–0.93 1.18–1.41 Hwa(1965) C. pageti 0.38 ± 0.014 0.43 ± 0.016 0.67 ± 0.008 0.74 ± 0.08 1.23 ± 0.30 Madinabeitia and Nagasawa (2011); Ben Hassine (1983) C. punctatus 0.385 0.416 0.565 0.734 1.05 Kim (1993) C. rotundigenitalis 0.33 0.37 0.5 0.71 0.99 Ho and Lin (2004) C. spinosus 0.38 0.49 0.7 0.73 0.94 Izawa (1969) C. undulatus 0.31 ± 0.01 0.42 ± 0.02 0.64 ± 0.01 0.77 ± 0.09 1.30 ± 0.22 Nawata et al. in preparation Stages Species C IV C V C VI Reference FMFMFM Caligus sclerotinosus 1.38 ± 0.09 1.24 ± 0.16 1.90 ± 0.2 1.84 ± 0.18 2.77 ± 0.21 2.56 ± 0.30 present study C. centrodonti 1.7–1.9 2.77 3.2 - - Gurney (1934) C. clemensi 1.31 2.94 3.15 - - Kabata (1972) C. curtus 2.5 - - - - Heegaard (1947) C. elongatus 2.34 ± 0.22 3.79 ± 0.29 3.36 ± 0.28 5.38 ± 0.21 4.33 ± 0.25 Piasecki (1996) C. epidemicus 1.158 1.052 1.462 1.26 1.877 1.453 Lin et al. (1996) C. fugu 1.44 ± 0.13 1.41 ± 0.06 2.20 ± 0.11 2.13 ± 0.16 3.65 ± 0.30 3.05 ± 0.20 Ohtsuka et al. (2009) C. latigenitalis 1.77 ± 0.05 2.15 ± 0.06 2.48 ± 0.07 3.26 ± 0.25 3.36 ± 0.14 3.91 ± 0.12 Madinabeitia and Nagasawa (2011) C. minimus 1.83 - 2.24 - - - Caillet (1979); Madinabeitia and Nagasawa (2011) C. orientalis 1.37–2.25 2.35–2.51 2.51–2.75 2.22–3.39 3.72–4.56 Hwa(1965) C. pageti 2.17 ± 0.04 - 3.82 ± 0.04 - - - Madinabeitia and Nagasawa (2011); Ben Hassine (1983) C. punctatus 1.64 1.62 2.51 2.38 2.96 2.81 Kim (1993) C. rotundigenitalis 1.42 1.2 1.85 1.5 2.49 1.88 Ho and Lin (2004) C. spinosus 1.16 - 1.63 - - - Izawa (1969) C. undulatus 1.94 ± 0.21 1.88 ± 0.25 2.68 ± 0.28 2.61 ± 0.32 - - Nawata et al. in preparation page 16 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan the former pattern but slightly different at CIII: 3-3-720-29-29. In the genus Caligus, seven species groups are distinguished: bonito-, confusus-, diaphanus-, macarovi-, productus-, pseudorhambi-, and undulatusspecies groups (Boxshall and El-Rashidy 2009; Boxshall 2018; Ohtsuka and Boxshall 2019; Ohtsuka et al. 2020). The complete life cycle of the macarovi-species group is only known for two species, C. orientalis (Hwa 1965) and C. sclerotinosus (present study). Even though the description of the former species is not accurate enough, the approximate number of setae on the first antennulary segments of CI–VI can be barely counted from the illustrations as follows: 3-3-6-14(?)-24(?)-28(?). This pattern, in particular, that of the early copepodid stages, resembles that of C. sclerotinosus rather than the other type. Therefore, it is suggestive that each species group may exhibit its own developmental pattern based in the number of setae on the first segment of antennule. Members belonging to some species groups, such as the macarovi-species group, mainly defined on the basis of the segmentation and setation of leg 4 may be convergently assigned. On the other hand, the bonito-, Fig. 11. The life cycle of Caligus sclertotinosus, duration and temperature in each stage is also provided. NI: first nauplius; NII: second nauplius; CI: copepodid 1; CII: copepodid 2; CIII: copepodid 3; CIV: copepodid 4; CV: copepodid 5. page 17 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan confususand productusspecies groups seem to be the robust ones with distinct synapomorphies. These species groups should be redefined based not only on genetics but also on developmental biology. The anlagen of the post-antennary process in C. sclerotinosus is visible for the first time at the first copepodid stage (CI) (Figs. 3C, 4). In comparison to other caligid species, the anlagen of the postantennary process appears for the first time at the third copepodid stage of C. centrodonti, C. elongatus, C. fugu, C. latigenitalis, C. pagetti, C. punctatus, C. rotundigenitalis, and C. spinosus Yamaguti, 1939. In C. clemensi, C. curtus Müller, 1785, C. epidemicus Hewitt, 1971, the third copepodid stage lacks the post-antennary process, but such structure will appear in later stages. In C. orientalis, the process is retained throughout all the copepodid stages (CII–V), except for the first copepodid stage. The sternal furca located between the right and left maxillipeds is found in most of Caligus spp. but absent in C. absens and other caligid species (Ho and Lin 2004; Ohtsuka personal observation). The sternal furca is suggested to function as brakes to prevent the caligid copepod from slipping backwards on the host (Kabata and Hewitt 1971; Ho and Lin 2004; Dojiri and Ho 2013). Generally, it first appears at the final moult without the appearance of “bud” in the preceding copepodid stages (Kim 1993; Ho and Lin 2004; Ohtsuka et al. 2009; Madinabeitia and Nagasawa 2011; present study). However, the bud of the sternal furca appears in the fifth copepodid stage in the following species: C. centrodonti, C. clemensi, C. elongatus and C. orientalis (Gurney 1934; Hwa 1965; Kabata 1972; Piasecki 1996). Although the adhesive behaviour of the adult caligids to the substrata with the cephalo-thoracic sucker and the lunules has been revealed with a highspeed camera (Ohtsuka et al. 2021), the function of the sternal furca is still unknown. When caligid adults adhere to the substrata, the attachment is accomplished at first with the frontal plate pressing the paired lunules to the substrata, and subsequently, with the cephalothoracic sucker (Ohtsuka et al. 2021). Since the tines of the sternal furca are directed backwards, it may be related to the attachment and detachment of the lunules serving as a fulcrum rather than brakes to avoid slipping backwards. The lunules seem to function as supplementary suckers when a small amount of water within a concavity of the lunule enters or leaves via a small anterior pit (Kaji et al. 2012). This seems to be controlled by the muscular movement of the frontal plate (Ohtsuka et al. 2021). The wide oral cone (Fig. 4) observed on the infective copepodid stage (CI) is found exclusively in C. sclerotinosus (Ohtsuka et al. 2018; present study). The oral cone of CI of other congeners has been illustrated in several manuscripts but it has never appeared expanded (e.g., Gurney 1933 1934; Izawa 1969; Kim 1993; Piasecki 1996). When CI of Caligus spp. attaches to the fish host for the first time, it grasps the settlement site with the antennae and maxillipeds at first, which Fig. 12. Bayesian inference (BI) tree of the copepodid III, IV and adults of Caligus sclerotinosus based on 28S sequence. Numbers at nodes indicate bootstrap support values. The accession numbers in GenBank are shown after each scientific name. page 18 of 21Zoological Studies 64:25 (2025) © 2025 Academia Sinica, Taiwan is subsequently replaced by the attachment with the frontal filament as shown in Lepeophtheirus spp. (Kearn 2004; Ohtsuka personal observation). Although the function of the expanded oral cone of CI of C. sclerotinosus is totally unknown, it is thought to be utilized for supplementary attachment to the host due to its sucker-like shape. This hypothesis may be enhanced by the fact that the second to fifth copepodids (CI–V) of C. sclerotinosus, which attach by means of the frontal filament, lack such a specialized oral cone. Except for the oral cone and the antenna at the first copepodid stage, the oral appendages, including the maxilliped, show few changes in development throughout the copepodid stages (CI–VI) of C. sclerotinosus. In contrast, both exoand endopods of legs 1–4 show an increase in the number of segments and setae ornamentation throughout the developmental stages of CI–VI. Leg 4 firstly appears at the third copepodid stage as in C. clemensi, C. elongatus, C. epidemicus, C. fugu, C. latigenitalis, C. punctatus, and C. rotundigenitalis. Meanwhile, in C. centrodonti, C. curtus, C. minimus, and C. pagetti leg 4 is visible at the second copepodid stage (see Khoa et al. 2019). Leg 5 in C. sclerotinosus appears at the fourth copepodid stage as it does in C. elongatus, C. fugu, C. punctatus, and C. rotundigenitalis. In C. centrodonti, C. curtus, C. latigenitalis, C. minimus, and C. pagetti leg 5 appears at the third copepodid stage. Leg 5 appears at the fifth copepodid stage in C. epidemicus, and at the sixth copepodid stage (= adult) in C. clemensi. In the present study copepodid I of C. sclerotinosus took four to five days at 25–26°C and seven days at 18.6°C to become copepodids VI (adults) under laboratory conditions (see Fig. 11). The influence of temperature on the development of Caligus minimus was also previously reported by Khoa et al. (2019). The successful tracking of the duration and the information on the influence of temperature on the development of C. sclerotinosus, will be the key to develop further control for this inversive species in aquaculture. CONCLUSIONS This study is pioneer in adopting the alternative terminology for the developmental stages of caligid copepods proposed by Piasecki et al. (2023). According to Piasecki et al. (2023), since chalimi and pre-adults of caligids were not strictly defined, these should be replaced by standardized terms used in free-living copepods: copepodids. Instead, the post-naupliar stages of caligids should be simply called first to fifth copepodid stages (CI–V) and adults (CVI). Thus, the life cycle of Caligus sclerotinosus consists on the following eight stages separated by molts: two naupliar stages, five copepodid stages (CI–V) and one adult (CVI). To avoid further terminological problems, future works on the life cycle of other siphonostomatoid copepods should follow the nomenclature proposed by Piasecki et al. (2023), as done in this study. Acknowledgments: Thanks are due to Ms N. Yumura and Mr. S. Tanaka who supported our genetic analysis of the developmental stages of C. sclerotinosus. This study was partially supported by grants from the Japan Society of the Promotion of Science (KAKEN, No. 19H03032, awarded to SO; JSPS Bilateral Partnership Program, No. JPJSBP120209924, awarded to SO). Authors’ contributions: YN, YK, HY, SS, KO and SO conducted the fieldwork. YN worked on illustrations. 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