Four new species of splanchnotrophid copepods (Poecilostomatoida) parasitic on doridacean nudibranchs (Gastropoda, Opistobranchia) from Japan, with proposition of one new genus

Abstract Four new species of splanchnotrophid copepods are described based on specimens collected from 5 species of doridacean nudibranchs from coastal waters of Japan. They belong to 3 genera, one of which, Majimun gen. n., is new. The parasites and their hosts are as follows: Ceratosomicola japonica sp. n. ex Hypselodoris festiva (A. Adams); Splanchnotrophus helianthus sp. n. ex Thecacera pennigera (Montagu); Splanchnotrophus imagawai sp. n. ex Trapania miltabrancha Gosliner & Fahey; and Majimun shirakawai gen. et sp. n. ex Roboastra luteolineata (Baba) and Roboastra gracilis (Bergh). Ceratosomicola japonica sp. n. is the fifth species of Ceratosomicola and is characterized by the shape and armature of the prosome in females. Both Splanchnotrophus helianthus sp. n. and Splanchnotrophus imagawai sp. n. are differentiated from 4 known congeners by the absence of posterolateral processes or lobes on the prosome in females, and the females of these 2 new species are separated from each other by the shape and armature of the genito-abdomen, the mandible, and the swimming legs. Majimun gen. n. is distinguished from other splanchnotrophid genera by the segmentation of the antennule as well as the combination of the following characters in females: 2 postgenital somites and the shape of the antenna, the mandible and the swimming legs.


introduction
The Splanchnotrophidae is a bizarre copepod family of Poecilostomatoida. Its members parasitize marine opisthobranch gastropods (Huys 2001). It is often difficult to detect their presence because almost all parts of the parasite's body are usually embedded inside the host, and only the distal part of the urosome and the egg sacs are exposed and visible externally. Since the mid-19th century, splanchnotrophids have been reported or described by many malacologists in the course of studies on opisthobranch gastropods (see Huys 2001). The taxonomic studies have often been inadequate at the generic level because the original descriptions include errors or have omissions. In his revision of Splanchnotrophidae, Huys (2001) clarified the validity of 3 genera, Splanchnotrophus Hancock & Norman, 1863, Ismaila Bergh, 1868, and Lomanoticola Scott T. & A., 1895, and established 2 new genera, Arthurius Huys, 2001 andCeratosomicola Huys, 2001. Since then, 1 species of Arthurius, 8 of Ismaila, and 3 of Ceratosomicola have been described, and a total of 23 species belonging to 5 genera are recognized in this family at present (Haumayr and Schrödl 2003;Salmen et al. 2008a, b). In this study, 4 new species of splanchnotrophid copepods collected from Japanese waters are described. Based on one new species, a new genus Majimun gen. n. is herein established.

Material and methods
Doridacean nudiblanchs were collected by SCUBA diving in the Seto Inland Sea off Hiroshima, central Japan and in both the North Pacific Ocean and the East China Sea off Okinawa-jima Island, the Central Ryukyu Islands, southern Japan, from April 2008 to December 2010. Collection data including the numbers of copepods found on the nudibranchs examined are shown in Table 1. Copepods were carefully removed from the body cavities of the hosts via dissections and preserved in 80% ethanol. Specimens were soaked in lactophenol for 2 days before dissection. The appendages of the copepods were then dissected and observed using the method of Humes and Gooding (1964). The drawings were made with the aid of a drawing tube. Morphological terminology follows Huys and Boxshall (1991) and Huys (2001). Measurements in millimeters are shown as ranges in parentheses with means and standard deviations. Type specimens are deposited in the crustacean collection of the National Museum of Nature and Science, Tsukuba (NSMT) and the Ryukyu University Museum, Fujukan (RUMF), Okinawa. The scientific names of nudibranchs follow those listed by Debelius and Kuiter (2007), , and Gosliner and Fahey (2008).
Description of holotype female. Body length from rostrum to posterior margin of anal somite: 4.27. Body (Figure 2A) composed of large prosome with 3 pairs of ventrolateral processes and small 3-segmented urosome. Prosome indistinctly 3-segmented, composed of anterior region, cephalosome, middle region comprising first to second pedigerous somites, and posterior region as third and fourth pedigerous somites. Cephalosome (Figures 2A, B, 3A) ellipsoid bearing rostrum with round margin, wider than long, bearing single apical lobe and 1 paired lateral lobes. Middle region large, bearing two transverse dorsal bulges and 5 ventral protrusions; anterodorsal bulge ornamented by 2 paired anterior and 1 paired lateral protrusions; posterior dorsal bulge carrying 2 pairs of lateral protrusions. Posterior region (Figure 2A-C) bearing two ventral protrusions on third pedigerous somite and constriction at border between third and fourth pedigerous somites. Ventrolateral processes (Figure 2A) long and slender, distinctly longer than body. Urosome ( Figure 2D) onion-like shaped, comprising genital double somite and two free postgenital somites ornamented with pattern of small scales on ventral surface. Genital double somite bearing paired ventral genital apertures. Caudal rami ( Figure 2E) globular bearing two and three spiniform elements on outer margin and tip, respectively; one element on tip serrated. Antennule ( Figure 3A, B) 4-segmented; proximal segment rectangular bearing 4 spines on anterior margin; second segment with 3 anterior spiniform and 1 posterior setiform elements; third segment bearing 2 anterior and 1 posterior elements; terminal segment bearing 6 spiniform and 1 setiform elements. Antenna ( Figure 3A, C) 3-segmented, conical with large sclerite at base, comprising coxobasis and 2-segmented endopod; coxobasis unarmed; proximal endopodal segment bearing 1 seta; terminal endopodal segment claw-like bearing 7 small elements. Labrum ( Figure 3A) bilobate, unarmed. Labium ( Figure 3A) bearing two paired spinulose lobes. Mandible ( Figure  3A  Variation of female morphology. The morphology of female paratypes is as in the holotype, except leg 2 shows variability. Leg 3 is distinctly visible on the paratype females. Paratype female (NSMT-Cr 22243) has the exopod of leg 2 ( Figure 3H) tapering into apical process with constriction and 2 elements. Paratype female (NSMT-Cr 22242) possesses a vestigial leg 3 ( Figure 3I), represented by a blunt element on a protrusion. The specimens from type series (n = 3) range from 3.11-4.27 (3.76 ± 0.59) in body length (BL).
Description of allotype male. Sexual dimorphism present in body form, and swimming legs. Body ( Figure 4A-C) 2.81 long, composed of cephalothorax and 5 cylindrical somites. Cephalothorax large, bulbous, incorporating first and second pedigerous somites, bearing transverse constriction and paired lateral and single dorsal protrusions posterior to mouthparts, paired posterolateral outgrowth, and paired and single ventral protrusions. Genital somite ( Figure 4D) incompletely segmented, bearing transverse dorsal folding and paired apertures; opercula unarmed. Caudal rami ( Figure 4E) globular, 2 and 3 elements along outer margin and on tip, respectively. No marked sexual dimorphism in antennule, antenna, and mouthparts. Tip of maxilla ( Figure 4F) slightly sharper than that of female.
Variation of male morphology. The morphology of male paratypes is as in the allotype. The specimens from type series (n = 6) range from 2.16-2.81 (2.42 ± 0.43) in BL.
Site. Female and male specimens were found in the body cavity of the host nudibranchs. Only the posterior tip of the urosome and the egg sacs were exposed from the host's gill circle ( Figure 1A, B). The mantle around the gill circle of the infected nudibranch was malformed into elongate tubes which obscured the host's gills and the egg sacs of the copepod ( Figure 1B).
Etymology. The specific name of the new species "japonica" refers to Japan, where it was collected. Hypselodoris festiva, the type host of this new species, is widely distributed around the Japanese archipelago and is one of the common nudibranchs of Japan. Ceratosomicola japonica sp. n. is the first species of parasitic copepods to have been described from Japan (Fujita 1895).
Remarks. Ceratosomicola sacculata (O'Donoghue, 1924) was originally described as Splanchnotrophus sacculatus. Huys (2001) redescribed this species based on a female and established a new genus Ceratosomicola. Three species, C. coia Salmen, Wilson & Schrödl, 2008;C. delicata Salmen, Wilson & Schrödl, 2008;C. mammilata Salmen, Wilson & Schrödl, 2008, were subsequently described based on specimens of both sexes, and this genus is now composed of four species (Salmen et al. 2008b). The new species clearly differs from C. coia and C. delicata in having 7 ventral protrusions on the prosome of the female (vs. without ventral protorusions on C. coia and C. delicata; Salmen et al. 2008b). Although the female of C. mammilata shares 7 protrusions, this species can be differentiated from the new species by having 2 pairs of lateral lobes  on the anterior region of the prosome and a posterior pair of ventral protrusions located anterior to the base of third ventrolateral processes, i.e. on the second pedigerous somite (vs. 1 pair of lateral lobes on the anterior region of the prosome and a posterior pair of ventral lobes located posterior to the base of third ventrolateral processes, i.e. on the third pedigerous somite). In Huys' (2001) redescription of C. sacculata, the ventral protrusions on the prosome was not described, while in the original description, O'Donoghue (1924) referred to the presence of at least 2 paired ventral lobes on the prosome. However, C. sacculata is distinguishable from the new species by the following characters in females: the anterior region of the prosome is trilobate (vs. ellipsoidal and bearing a pair of lateral lobes in the new species) and the middle region of the prosome bears 3 transverse dorsal bulges (vs. 2 transverse dorsal bulges in the new species).
In the course of dissection to describe Hypselodoris festiva (as Chromodoris marenzalleri) (Nudibranchia: Chromodorididae) from the western North Pacific Ocean off Misaki, Kanagawa Japan (Fujita 1893), one female specimen of splanchnotrophid was discovered by Fujita (1895) from the body cavity of the host. Although Fujita (1895) recognized some differences between this copepod and other splanchnotrophids, he did not describe it as a new species nor deposit it in any museum because of the incomplete specimen. The species was subsequently recognized as a member of Ceratosomicola by Huys (2001). Ceratosomicola japonica sp. n. was collected from the same host species (H. festiva) in the Seto Inland Sea off Nohmi-jima Island, Hiroshima, Japan and shares important characters as follows: the anterior region of the prosome is wider than long, bearing a pair of lateral lobes (Fujita 1895, figure 2) and middle region of the prosome bears a paired anterior and single posterior ventral protrusions with the latter being larger than the others (Fujita 1895, figure 1). Fujita (1895, figure 2) also described the middle region of the prosome as bearing a cross-shaped concavity. This corresponds to the transverse dorsal bulges with 4 bulbous protrusions on each corner of C. japonica sp. n. Therefore, the Fujita's splanchnotrophid is apparently conspecific with C. japonica sp. n.
Variation of female morphology. The morphology of female paratypes is as in the holotype. The specimens from type series (n = 3) range from 2.81-4.47 (3.57 ± 0.83) BL. Description of allotype male. Sexual dimorphism prominent in body form. Body ( Figure 7A-C) cyclopiform, 0.63 long, composed of cephalothorax and 5 cylindrical somites. Cephalothorax ( Figure 7A-C) large, incorporating first and second pedigerous somites, with constriction posterior to mouthparts. Urosome 3-segmented (Figure 7D); genital somite scarcely discernible in dorsal view, bearing paired apertures; opercula carrying 2 processes along posterior margin. Anal somite ( Figure 7D) nearly completely withdrawn into genital somite. Caudal rami ( Figure 7E) cylindrical, about three times as long as wide, bearing 5 setae, styliform terminal seta bipinnate toward tip, and 2 dorsal spiniform spines. No marked sexual dimorphism in antennule, antenna, and mouth parts, except location of antenna. The base of antenna located anterior to labrum ( Figure 7F).
Variation of male morphology. The morphology of male paratypes is as in the allotype. The specimens from type series (n = 11) range from 0.32-0.63 (0.53 ± 0.12) in BL.
Site. Both female and male specimens were found in the body cavity of host nudibranchs. The females grasped the host's visceral sac by the lateral processes on the prosome ( Figure 1D). Only the posterior tip of the urosome and the egg sacs were exposed from the host's gill circle ( Figure 1C).
Etymology. The specific name "helianthus" is from the Latin meaning sunflower. The live body color of this new species is yellowish, and the egg sacs attached on the host nudibranch look like flowers.
Remarks. Four species of Splanchnotrophus are currently recognized as valid (Huys 2001). Splanchnotrophus helianthus sp. n. differs from S. angulatus Hecht, 1893, S. dellachiajei Delamare Deboutteville, 1950, S. gracilis Hancock & Norman, 1863 in the absence of paired posterolateral processes on the prosome and the genito-abdomen bearing lateral lobes in females (vs. the presence of posterolateral processes on the prosome and the genito-abdomen without paired lateral lobes, Hancock and Norman 1863;Delamare Deboutteville 1950;Huys 2001). Huys (2001) claimed that, in S. angulatus, the shape of the female's genito-abdomen is constant, irrespective of prosome valiability, and the size and shape of the posterolateral lobe of the prosome certainly shows variability. Nevertheless, this species always possesses the posterolateral lobe, which is regarded as a useful identification character. The original description of S. willemi by Canu (1891) has no illustration and includes only a minimum amount of information. However, the presence of pleural wings on the third pedigerous somite in S. willemi is not shared with the new species (Canu 1891).
Variation of female morphology. The morphology of the female paratype is as in the holotype. The specimens from type series (n = 2) range from 0.71-1.86 (1.28 ± 0.81) BL.
Male. Unknown. Site. Female specimens were found in the body cavity of host nudibranchs. They grasped the host's visceral sac by the lateral processes. Only the posterior tip of the urosome and the egg sacs were exposed from the host's gill circle ( Figure 1E, F).  Etymology. The specific name "imagawai" honours the collector of this new species, Mr. Kaoru Imagawa who is a professional diver. The discovery of the new species was brought by his extraordinary ability to find small nudibranch gastropods.
Remarks. The female of the new species differs from S. angulatus, S. dellachiajei, S. gacilis and S. willemi in the absence of posterolateral processes on the prosome (vs. present, see Hancock and Norman 1863;Canu 1891;Delamare Deboutteville 1950;Huys 2001). Splanchnotrophus helianthus sp. n. lacks such processes but differs clearly from the new species in the following characters in females: the anterior region of the prosome is elongate and bent to ventral (vs. not elongate); the middle region of the prosome has a constriction posterior to the base of the first lateral processes (vs. without constriction); the genito-abdomen possesses posterolateral lobes (vs. without lobes); the mandible bears dentiform processes (vs. without processes); the endopodal lobe of leg 1 is adpressed to the exopodal lobe via the small protopod (vs. the endopodal lobe separated from the exopodal lobe); and leg 3 is located on the third pedigerous somite directly (vs. leg 3 located on the apex of paired bulbs).
Legs 1 and 2 composed of protopod largely incorporated into ventral wall of prosome, with exopodal and endopodal lobes; protopod bearing outer basal seta, small protrusion at base of endopodal lobe of leg 1; exopodal lobe indistinctly 2-segmented, tapering into spiniform apical process; endopodal lobe cylindrical bearing apical process. Leg 3 represented by conical process with apical seta.
Egg sacs cylindrical and spiral. Diagnosis of adult male. Body cyclopiform, composed of cephalothorax and 5 cylindrical somites. Cephalothorax large, bulbous, incorporating first and second pedigerous somites, bearing transverse constriction posterior to mouth parts and paired posterolateral outgrowth. Genital somite bearing paired apertures; opercula unarmed. Caudal rami conical, about as long as wide, bearing 6 setae; apical seta styliform. No marked sexual dimorphism in antennule and mouth parts. Shape of antenna as in female except terminal endopodal segment bearing 5 elements; inner margin bearing 2 of 5 elements. Mandible elongate, drawn out into spatulate apical blade with 3 dentiform processes.
Legs 1 and 2 composed of round protopod with outer basal seta, indistinctly 2-segmented exopodal lobe drawn out into pointed process, and non-segmented endopodal segment with apical small process. Leg 3 represented by conical process with apical seta.
Type and only species. Majimun shirakawai sp. n. by the present designation. Etymology. The generic name, "majimun", refers to a dialect in Okinawa, which means demons. The gender is neuter.
Remarks. Females of Lomanoticola and Splanchnotrophus differ from Majimun gen. n. in having a 2-segmented urosome comprising the genital double somite and the anal somite (vs. a 3-segmented urosome and the genital double somite separated from the abdomen) (Huys 2001;present study). Females of Ismaila spp. share a 3-segmented urosome, which includes 1 postgenital somite (vs. 2 somites). There are also differences in the following female characters between Ismaila and the new genus: the antennule is 2-segmented (vs. 3-segmented); the mandible consists of a small rod tipped with a short tooth and a slender spine (vs. drawn out into a blade with dentiform processes); the paragnath is absent (vs. present); the maxillule is made of a lobe with 2 setae (vs. absent); and the maxilla has the allobasis drawn out into a multipinnate endite with 2 accessory elements (vs. with the allobasis drawn out into a curved process with 1 seta) (see Ho 1981;Haumayr and Schrödl 2003). Majimun gen. n. does not show distinct sexual dimorphism in the antennule, the antenna, and the mouth parts. The new genus also possesses a 3-segmented antennule, the mandible, and the paragnath in both sexes and 2 postgenital somites in females. These characters are not shared with Arthurius (see Huys 2001). All of these characters are shared with Ceratosomicola, except the 3-segmented antennule. However, Ceratosomicola differs from the new genus by having the following characters in both sexes: the antennule is composed of 4 distinct segments (vs. 3 segments); the antenna is conical (vs. elongate); the mandible is covered with numerous spinules (vs. spatulate bearing a blade with dentiform processes around apex); and the maxilla possesses a lanceolate process without armature (vs. with process and 1 seta) (see Huys 2001;present study).
Description of adult female. Body length ( Figure 10A) 4.99, elongate, composed of elongate prosome with 3 pairs of lateral processes and 3-segmented urosome. Prosome composed of anterior region (cephalosome), middle region (comprising first and second pedigerous somites), and posterior region (comprising third and fourth pedigerous somites). Cephalosome rectangular ( Figure 10A, B), bearing protruded rostral area ( Figure 10G). Middle region ( Figure 10A, B) elongate, about twice as long as wide, bearing constriction at base of first lateral processes and dorsal posterior lobe, without posterolateral processes. Posterior region ( Figure 10A, B) elongate, third and fourth pedigerous somites covered with anchor-shaped spinules ( Figure 10E) along posterior margin ( Figure 10C, D). Lateral processes ( Figure 10A) long and slender, about 1.3 times as long as body length. Urosome ( Figure 10C, D) small; genital double somite cylindrical, narrower at middle length, bearing paired apertures with slightly prominent posterolateral corners; opercula bearing small shield-like structure with 2 spiniform processes. Caudal ramus ( Figure 10F) small, fusiform, about twice as long as wide, bearing 6 setae; apical seta styliform.
Variation of female morphology. The morphology of body parts of female paratypes is as in the holotype. The specimens from type series (n = 4) range from 3.31-4.99 (3.99 ± 0.77) BL.
Legs 1 and 2 ( Figure 12B, I, J) composed of round protopod with outer basal seta, indistinctly 2-segmented exopodal lobe drawn out into pointed process with 4 and 3 elements on legs 1 and 2, respectively, and non-segmented endopodal segment with apical small process. Leg 3 ( Figure 12D, K) represented by conical process with apical seta.
Variation of male morphology. The morphology of male paratypes is as in the allotype. The specimens from type series (n = 3) range from 0.50-1.02 (0.75 ± 0.26) in BL.
Site. All specimens of both sexes were found in the body cavity of the host nudibranchs. The lateral processes on the prosome of females grasped the host's visceral sac, and their mouthparts were in touch with the host's gonads. The posterior tip of the urosome and the egg sacs were exposed from the posterior region of the host's gill circle ( Figure 1G). Males were attached to the posterior part of the female prosome ( Figure  1H). Both females and males bear patches of hook-like spinules ( Figures 10E, 12E) on the posterior margin of the third and fourth pedigerous somites.
Etymology. The new species is named after Mr. Naoki Shirakawa, an expert diver who finds remarkable animals. He collected the nudibranchs infected by the new species.

Discussion
Despite the fact that Splanchnotrophidae comprises 23 species in 5 genera, only 4 species in 3 genera have been recorded from the North Pacific Ocean: 2 species of Ismaila from the East coast and another 2 species in 2 other genera from the western North Pacific (Huys 2001;Salmen et al. 2008a). One species of Ceratosomicola has been reported from Japanese waters (Fujita 1895; see Huys 2001) and this species is described herein as C. japonica sp. n. The other species, Arthurius bunakenensis Salmen, Kaligis, Mamangkey & Schrödl, 2008 was described from Gangga Island off northern Sulawesi, Indonesia (Salmen et al. 2008a). With the descriptions of 4 new species and 1 new genus in this paper, there are now at least 4 species in 3 genera of splanchnotrophids in Japanese waters and 5 species in 4 genera in the western North Pacific Ocean.
Currently, 4 species of Splanchnotrophus are recognized, and all of them have been described or reported from European waters (Huys 2001). Thus, S. helianthus sp. n. and S. imagawai sp. n. are the first and second species found from North Pacific Ocean. On the other hand, the host nudibranchs of these 2 new species, Thecacera pennigera and Trapania miltabrancha, are widely distributed: T. pennigera is known from the subtropical Atlantic and Pacific region (Debelius and Kuiter 1998), and T. miltabrancha is probably widely distributed in the Indo-West Pacific because this species was originally described from Indonesia (Gosliner and Fahey 2008). We infer that the 2 Splanchnotrophus spp. described in this paper are not distributed only in a limited region. In fact, with the recent spread of SCUBA diving, many opisthobranch gastropods infected by splanchnotrophids have been found and their pictures have been taken from temperate to tropical waters around the world. Because of the shape of the egg sac, some of such splanchnotrophids are surmised to be undescribed species of Splanchnotrophus.
The original descriptions of the 4 known species of Splanchnotrophus lack adequate illustrations of mouthparts and the swimming legs (Hancock and Norman 1863;Canu 1891;Hecht 1893;Delamare Deboutteville 1950). Therefore, the shape of the prosome and the urosome has been used for species identification, and the shape of the genito-abdomen is especially important. Nevertheless, some characters in the mouthparts and the swimming legs are useful to separate species of Splanchnotrophus from each other: the mandibles of S. angulatus, S. gracilis, and S. helianthus sp. n. carry several dentiform processes on the apical parts (Huys 2001;present study) but that of S. imagawai sp. n. lack such armature. The shape of legs 1 and 2 in females also differs between S. angulatus, S. helianthus sp. n., and S. imagawai sp. n. Splanchnotrophus spp. have been described based on female specimens, and male specimens were described only for 2 species, S. angulatus and S. gracilis (Hancock and Norman 1863;Huys 2001). Splanchnotrophus helianthus sp. n. is a third species described based on both sexes, and the male anal somite of this new species nearly completely withdrawn into genital somite is not shared with the other 2 species. It is, therefore, considered that the male morphology can also serve as useful character to distinguish between morphologically similar species of Splanchnotrophus. Majimun gen. n. and Ceratosomicola share 2-segmented postgenital somites, cylindrical egg sacs, and posterolateral lobes on the male's prosome (see Huys 2001;present study). On the other hand, Majimun gen. n. shares the following characters in both sexes with Splanchnotrophus: the antenna has elongate middle and terminal segments; the mandible bears a blade that is not recurved and not covered with spinules; the paragnath is present; the maxilla is 2-segmented with a seta; legs 1 and 2 possess exopodal and endopodal lobes; and leg 3 is represented by a conical projection with 1 apical seta (see Huys 2001;present study). Ceratosomicola has a globular caudal ramus with 5 short elements, but that of Splanchnotrophus is elongate with 5 setae and 1 terminal spiniform seta. The somewhat elongate, fusiform caudal ramus with 5 minute and 1 short spiniform terminal setae of Majimun gen. n. shows just an intermediate type between that of Ceratosomicola and Splanchnotrophus. However, the antennule of Majimun gen. n. is 3-segmented, which differs from that of both genera. Huys (2001) mentioned that the ancestral splanchnotrophid antennule is 5-segmented, and the proximal segment in Ceratosomicola is homologous to the first 2 segments in ancestral one. In Majimun gen. n., the proximal segment is large and bears 4 spines and 3 elements on the proximal and distal parts, respectively, and this segment also corresponds to the first 3 segments in the ancestral antennule. Egg sacs attached at midlength with well-developed anterior and posterior lobes; prosome with lateral processes longer than body ....Splanchnotrophus -Egg sacs attached at subterminally, cylindrical, and slightly curved; prosome with conical lateral processes shorter than or as long as body ....Lomanoticola 5 Antennule 3