Reference transcriptome assembly of a protogynous sex change fish, harlequin sandsmelt (Parapercis pulchella).

The harlequin sandsmelt (Parapercis pulchella) is a female-to-male sex change fish in which functional females possess ovotestes that consist of both ovarian and testicular tissues. These features indicate that this species could be an excellent model for studying the flexibility of sex differentiation in vertebrates. However, genetic resources in this species have so far been limited. Therefore, in this study, the reference transcriptome of this fish was constructed through RNA-sequencing, de novo transcriptome assembly, superTranscripts construction, and functional annotations. To obtain as many genes as possible, RNA was extracted from various tissues (brains, gills, hearts, livers, guts, and gonads) and various sexual stages (females, individuals during sex change, and males) and then subjected to sequencing and downstream analyses. As a result, 91,884 representative transcripts with 32,627 protein-coding sequences were generated. 72.2% of protein-coding sequences (23,566 sequences) were functionally annotated. Also, our analysis shows that the superTranscripts method effectively removes redundant sequences from raw-assembled data compared with other strategies. The resultant dataset is a valuable resource for future molecular developmental studies on sex change in P. pulchella.

Differences of Sucker Formation Processes Depending on Benthic or Pelagic Posthatching Lifestyles in Two Octopus Species.

AbstractMorphologies of animal appendages are highly diversified depending on animal lifestyles. In cephalopods (Mollusca, Cephalopoda), an individual possesses multiple arms that contribute to elaborate behaviors, and suckers on them enable various arm functions. In octopus hatchlings, arm and sucker morphologies can be divided into two different types due to alternative posthatching lifestyles, that is, pelagic or benthic lifestyles, although the underlying developmental differences have yet to be elucidated. In this study, therefore, detailed developmental processes of arms and suckers were observed during embryogenesis in two different octopus species, Octopus parvus and Amphioctopus fangsiao, showing pelagic and benthic posthatching lifestyles, respectively. In O. parvus, sucker formation stopped at a relatively early stage in which three suckers on an arm were produced. In addition, at late embryonic stages, cell proliferation was hardly detected in whole arms, while in A. fangsiao, sucker production continued throughout embryogenesis and cell proliferation also remained active in whole arms even in the late stages. Therefore, although further investigations in other octopus species are required, it is suggested that in octopus evolution, the developmental program of suckers has been modified in accordance with the acquisition of a novel lifestyle.

Gonadal and cellular dynamics during protogynous sex change in the harlequin sandsmelt Parapercis pulchella.

Some teleost fishes change their sex, and some of these fishes have specific gonads known as "ovotestes," that is, gonads containing both ovarian and testicular tissues. In this study, we revealed the gonadal transformation process and cell dynamics during the female-to-male sex change in the harlequin sandsmelt, Parapercis pulchella (Pinguipetidae), in which females possess ovotestes. Histological observations revealed that although female ovotestes were composed of oocytes, a few cysts of male germ cells were observed among them. At the initial phase of sex change, male germ cells increased, and spermatogenesis proceeded. After that, oocytes decreased and finally disappeared, and the gonads became functional testes. Immunohistochemistry using antibodies against Pcna (proliferating cell nuclear antigen) as a cell proliferation marker revealed that spermatogonia were Pcna positive, whereas spermatocytes were negative, in female ovotestes. This suggests that spermatogenesis is arrested at the spermatocyte stage. In addition, some somatic cells surrounding oocytes, which were thought to be the female follicle cells, were Pcna positive during sex change, indicating that these cells proliferate during sex change and are reused in male testes after sex change. Also, immunostaining using antibodies against active cleaved-Caspase3a as an apoptosis marker demonstrated that oocytes degenerated through apoptotic cell death at the late transition stage. Together with previous findings in other fishes, these findings suggested that the histological processes in gonads during sex change, such as the order of developmental events, developmental fates of ovarian cavities, and ovotestis structures, are diversified among fish species. In contrast, cellular dynamics of female germ and somatic cells during sex change are common among protogynous species.

Two new species of the bathyal holothurian genus Pannychia (Elasipodida, Laetmogonidae) from Japanese waters.

Sea cucumbers are one of the most common large animals in the deep-sea benthic communities, and those of the genus Pannychia are particularly abundant in the bathyal North Pacific Ocean. In Japanese waters, three species of Pannychia have been reported, mainly from the northern and eastern parts of the country. Here, we describe two new species from the western part of Japan. These two new species were placed in Pannychia by the presence of dorsal papillae on the ventrolateral radii and the presence of large wheel ossicles with rim teeth. They are distinguished from all other nominal species of Pannychia on the basis of the morphological characteristics of their body wheel ossicles. Pannychia nagasakimaruae sp. nov. has a unique ossicle composition in its dorsal and ventral body walls: four forms of wheel ossicles. Pannychia rinkaimaruae sp. nov. differs from other congeners in its small wheel ossicles in the dorsal and ventral body walls, which have conical rim teeth. We barcoded partial COI gene sequences from holotypes and paratypes of two new Japanese species. Our molecular phylogenetic analysis showed that these two new morphologically recognized Japanese species form distinct clades separated from other Pacific Pannychia species, so that the two new Japanese species were also supported by genetic results. In addition, we describe and visualize the poorly calcified calcareous rings of the two new species using X-ray µCT system, a useful method for observing such structures, which are important structures in holothurian anatomy.

Three new species in Tetrastemma Ehrenberg, 1828 (Nemertea, Monostilifera) from sublittoral to upper bathyal zones of the northwestern Pacific.

Monostiliferous nemerteans in the genus Tetrastemma Ehrenberg, 1828 are generally characterized as having four eyes, and they occur worldwide, from the intertidal zone to the deep-sea bottom. Recent extensive sampling of Tetrastemma has explored the high species diversity, including many undescribed forms, but phylogenic analysis has revealed non-monophyly of the genus. We herein describe three new species of the genus (T.albumsp. nov., T.personasp. nov., and T.shohoensesp. nov.) from northwestern Pacific waters based on specimens collected by dredging or by use of a remotely operated vehicle at depths of 116-455 m. Since anatomical and histological characters traditionally used in systematics of the genus are sometimes interspecifically uniform, a histology-free approach is applied for the species descriptions in this study. To confirm the generic affiliation of the new species, a molecular phylogenetic analysis based on partial sequences of cytochrome c oxidase subunit I, 16S rRNA, 18S rRNA, 28S rRNA, and histone H3 genes was performed. Our result shows that all three new species are nested in a subclade formed by species from the North Pacific and American Atlantic, inferring that geographic distribution does not reflect the cladogenesis of Tetrastemma. Furthermore, two Tetrastemma species with a cylindrical stylet basis, T.freyaeChernyshev et al., 2020 from off the coast of India and Hawaii and T.shohoensesp. nov. from Shoho Seamount, Japan, constitute a clade in the resulting tree.

Ontogenetic Expressions of Sexually Dimorphic Traits in the Skeleton Shrimp Caprella scaura (Crustacea: Amphipoda).

In sexual dimorphism, males often exhibit exaggerated characters as weapons or ornaments. Among the numerous amphipod species (Amphipoda, Crustacea) showing sexual dimorphism, caprellids (Caprellidae) are characterized by considerably larger males that possess weapons, although the developmental processes underlying these sex-related differences are largely unknown. Therefore, to clarify the process of sexual differentiation during postembryonic development in caprellids, morphometric analyses of Caprella scaura were conducted. Principal component analysis using 31 morphometric traits showed drastic allometric changes occurring at two ontogenetic body length (BL) points (i.e., 3.8 and 8.8 mm). In individuals larger than 3 mm, head spines appeared in both sexes, and penises did only in males, allowing the discrimination of juveniles from larvae. Moreover, in larger males (BL > 8.8 mm), traits used in reproductive behavior, i.e., the first antenna, second gnathopod, and first to fifth pereonites, were extremely exaggerated. Observations of pre-copulatory behavior along with morphological assays revealed that sexually mature males could be identified by the size ratio between the triangular projection and palmar spine on the propodus of the second gnathopod. In contrast, female maturation could be determined by the marginal setae of oostegites forming a brood pouch. The body size distribution of sexually mature females was concentrated within a narrow range of BLs (6-9 mm), whereas that of sexually mature males showed a broader range (BL 9-18 mm), suggesting that, in C. scaura, males continue to molt and grow even after sexual maturation via indeterminate growth, to increase their lifetime reproductive success.

Three new species of the family Aoridae collected from Sagami Bay, central Japan (Crustacea: Amphipoda).

Three new species of the amphipod family Aoridae Stebbing, 1899 were collected from the bottom of a 40400 m depth in Sagami Bay, central Japan. Aora biarticulata sp. nov. is characterized by the accessory flagellum with 2 articles, the weakly setose male gnathopod 1 and the male gnathopod 2 with setose carpus and propodus. Aoroides sagamiensis sp. nov. has a distinctive male gnathopod 1 with poorly setose basis and heavily setose merus. Grandidierella gracilis sp. nov. has stridulating ridges on the carpus of the male gnathopod 1. This new species can be distinguished from its related congeners by the teeth arrangement on the male gnathopod 1 carpus, the poorly setose antennae, the sparsely setose posterior margins of the pereopods 6, 7 bases, and the ordinary form of the male gnathopod 2.

Partial redescriptions of three holothurians with hook papillae (Apodida: Chiridotidae): Taeniogyrus japonicus (Marenzeller, 1882), T. dendyi (Mortensen, 1925), Scoliorhapis theelii (Heding, 1928).

New specimens of Taeniogyrus japonicus (Marenzeller) were collected from Iwami coast and Sado island of the Sea of Japan. According to detailed observations of external and internal organs, we transferred T. japonicus, T. dendyi (Mortensen), and Scoliorhapis theelii (Heding) to the newly revived genus Scoliodota, with a large retractor organ and hook papillae as major diagnostic characters. We also concluded that Scoliorhapis dianthus Solis-marin et al. is a synonym of T. japonicus. Our molecular phylogeny and genetic distance analysis showed that specimens from Iwami and specimens from Sado of T. japonicus form a monophyletic clade, indicating that they are most likely the same species despite their morphological variation. The phylogenetic analysis also indicated that T. japonicus forms a sister group relationship with Taeniogyrus verruculosus Yamana Tanaka and Scoliorhapis sesokoensis Yamana Tanaka, suggesting that Taeniogyrus is not monophyletic. Taken together, these results brought new insights to sea cucumber diversity in Japanese waters.

Exploring the trends of adaptation and evolution of sclerites with regards to habitat depth in sea pens.

Octocorals possess sclerites, small elements comprised of calcium carbonate (CaCO3) that are important diagnostic characters in octocoral taxonomy. Among octocorals, sea pens comprise a unique order (Pennatulacea) that live in a wide range of depths. Habitat depth is considered to be important in the diversification of octocoral species, but a lack of information on sea pens has limited studies on their adaptation and evolution across depth. Here, we aimed to reveal trends of adaptation and evolution of sclerite shapes in sea pens with regards to habitat depth via phylogenetic analyses and ancestral reconstruction analyses. Colony form of sea pens is suggested to have undergone convergent evolution and the loss of axis has occurred independently across the evolution of sea pens. Divergences of sea pen taxa and of sclerite forms are suggested to depend on habitat depths. In addition, their sclerite forms may be related to evolutionary history of the sclerite and the surrounding chemical environment as well as water temperature. Three-flanged sclerites may possess the tolerance towards the environment of the deep sea, while plate sclerites are suggested to be adapted towards shallower waters, and have evolved independently multiple times. The common ancestor form of sea pens was predicted to be deep-sea and similar to family Pseudumbellulidae in form, possessing sclerites intermediate in form to those of alcyonaceans and modern sea pens such as spindles, rods with spines, and three-flanged sclerites with serrated edges sclerites, as well as having an axis and bilateral traits.

Hydrocoel morphogenesis forming the pentaradial body plan in a sea cucumber, Apostichopus japonicus.

Echinoderms constitute an animal phylum characterized by the pentaradial body plan. During the development from bilateral larvae to pentaradial adults, the formation of the multiple of five hydrocoel lobes, i.e., the buddings from the mesodermal coelom, is the firstly emerging pentameral character. The developmental mechanism underlying the hydrocoel-lobe formation should be revealed to understand the evolutionary process of this unique and highly derived body plan of echinoderms, although the morphogenetic mechanisms of hydrocoel lobes are largely uninvestigated. In this study, using the sea cucumber Apostichopus japonicus, in which hydrocoel is easily observable, the developmental process of hydrocoel lobes was described in detail, focusing on cell proliferation and rearrangement. Cell proliferation was not specifically distributed in the growing tips of the hydrocoel lobes, and inhibition of cell proliferation did not affect lobe formation. During lobe formation, the epithelium of the hydrocoel lobes was firstly thickened and then transformed into a simple epithelium, suggesting that tissue expansion via tissue remodeling contributes to the hydrocoel-lobe formation.

Lineid Heteronemerteans (Nemertea: Pilidiophora) from Sagami Bay, Japan, with Some Proposals for the Family-Level Classification System.

We performed molecular phylogenetic analyses using nucleotide sequence data from five genes (16S rRNA, cytochrome c oxidase subunit I, 18S rRNA, 28S rRNA, histone H3) determined from 36 specimens representing 25 (10 named, 15 unnamed) species of lineid heteronemerteans collected in Sagami Bay, Japan, along with other sequences obtained from public databases. External features of the 25 species are briefly described and illustrated. Lineus fuscoviridis Takakura, 1898 is transferred to Notospermus Huschke, 1830. Our molecular analysis indicated that one of our Notospermus specimens from Sagami Bay and material previously collected from the Seto Inland Sea are conspecific with Notospermus geniculatus (Delle Chiaje, 1822) s. str. [the year of publication is not 1828 as previously regarded], originally described from Naples, Italy. The new species Siphonenteron nakanoi is established; our tree shows it as the sister taxon to Siphonenteron bilineatum Renier in Meneghini, 1847; it differs from the latter in having more diffuse, scattered yellow pigmentation at the anterolateral margin of the head on each side. Pros and cons of lumping and splitting Lineidae in future studies are discussed.

Ceratosomicola oki n. sp., a New Species of Copepod (Cyclopoida: Splanchnotrophidae) Parasitic on the Chromodoridid Nudibranch Glossodoris misakinosibogae Baba, 1988 Off the Oki Islands, Japan, with Microanatomical Observation Using Micro-CT.

A new species of the family Splanchnotrophidae Norman and Scott, 1906 (Cyclopoida) is described based on both sexes collected from off the Oki Islands, the Sea of Japan. Specimens of both sexes of Ceratosomicola oki n. sp. were found in the body cavities of Glossodoris misakinosibogae Baba, 1988 (Nudibranchia: Chromodorididae). The copepod is characterized by the following female characters: the cephalosome with a pair of dorsolateral horn-like processes; the prosome with hemispherical posterolateral lobes on the middle region. Non-destructive, micro-computed tomography (micro-CT) imaging performed on a single specimen of the nudibranch revealed a heavy infection by a total 17 specimens of C. oki n. sp. Almost all individuals of the copepod were attached on the surface of the middle to posterior parts of the visceral sac, forming a dense cluster. The four females bearing developed lateral processes on the prosome faced the anterior end of the visceral sac and positioned the posterior tip of the body under the secondary gills of the host. The males fitted in the gaps between the females' bodies. Further, the distribution and shape of the reproductive organs of both sexes were partially clarified by micro-CT imaging.

Sucker formation in a bigfin reef squid: Comparison between arms and tentacles.

Cephalopods have acquired numerous novelties and expanded their habitats to various marine environments as highly agile predators. Among cephalopod novelties, multiple arms are used for complex behaviors, including prey capture. Suckers on arms are innovative features for realizing these arm functions. In addition, tentacles in Decapodiformes (squids and cuttlefishes) are arms specialized in prey capture and tentacular suckers show unique morphologies. However, little is known about the developmental process of sucker formation that should differ between tentacles and other arms. In this study, therefore, sucker formation processes on second arms and tentacles were observed and compared in a bigfin reef squid, Sepioteuthis lessoniana, to reveal the developmental processes forming the unique sucker morphologies, especially in tentacles. Morphological and histological observations of suckers during embryogenesis showed that, in second arms, the sucker-producing area appeared at the most distal part. At the most proximal side of the sucker-producing area, new sucker buds were isolated by invagination of the epithelial tissue. At the proximal arm parts, suckers with functional structures were observed. In tentacles, although the basic sucker formation pattern was similar to that in second arms, sucker formation started at earlier embryonic stages and the number of suckers was drastically increased compared to that in second arms. In addition, although four sucker rows were observed at the tentacular club, that is, the thickest part of a tentacle, our observations suggested that two sets of two sucker rows are compressed to form the four rows. Therefore, the sucker-formation processes are temporally and spatially different between arms and tentacles. In addition, S. lessoniana shows conserved and unique patterns of sucker formation in comparison with previously described species, suggesting that sucker formation patterns were diversified among Decapodiformes lineages.

Metarhachotropis parva, a new genus and species of Eusiridae (Crustacea: Amphipoda) from Sagami Bay, central Japan.

The monotypic genus Metarhachotropis (Crustacea: Amphipoda: Eusiridae) is erected with M. parva sp. nov. from Sagami Bay, central Japan, as its type species. This new genus is characterized by the massive head with large rostrum, the very short coxae and the strongly produced coxa 1. Metarhachotropis resembles Eusirella Chevreux, 1908 and Rhachotropis Smith, 1883; however, it can be distinguished from Eusirella by the ordinary length of the maxilla 1 palp, and from Rhachotropis by the smooth body.

Description of a New Species of the Marine Flatworm Prosthiostomum (Platyhelminthes: Polycladida) and its Three Known Congeners from Misaki, Japan, with Inference of Their Phylogenetic Positions within Prosthiostomidae.

The present study provides morphological descriptions of four species of Prosthiostomum (Polycladida, Prosthiostomidae)-P. auratum Kato, 1937; P. hibana sp. n.; P. cf. ostreae Kato, 1937; and P. vulgare Kato, 1938-based on specimens collected among branching coralline algae and kelp holdfasts in Misaki, Japan. The new species P. hibana sp. n. is characterized by i) the dorsal surface of the body covered with numerous orange maculae, some of which coalesce together to form larger ones; ii) a pair of linear cerebral-eyespot clusters, each consisting of relatively few (7-9) cerebral eyespots; iii) 3-4 pairs of ventral eyespots embedded in parenchyma: iv) the inner wall of the male atrium deeply ruffled; v) the lumen of the seminal vesicle being narrow and elongated in shape; and vi) a large sucker situated in the center of the body. We remark on some morphological characters that were not mentioned in the original description of P. auratum. We infer the phylogenetic positions of these four species within Prosthiostomidae using the maximum-likelihood analysis based on partial 28S rRNA and COI gene sequences determined de novo, in addition to those that are currently available in public databases. In the resulting tree, the four species-P. auratum, P. hibana sp. n., P. cf. ostreae, and P. vulgare-were nested in a clade that was composed of all the other Prosthiostomum species included in the analysis.

Description of a New Brooding Species of Ophiodelos (Echinodermata: Ophiuroidea) from Japan.

A new species of brittle star, Ophiodelos okayoshitakai, is described from two specimens collected in Sagami Bay, central-eastern Japan. Photographic examination of the holotype specimen of the sole other congener, Ophiodelos insignis Koehler, 1930, indicates that Ophiodelos okayoshitakai sp. nov. is distinguished from O. insignis by i) the disc stumps covering on the dorsal side of the disc, ii) the dorsal and ventral arm plates being separated from each other on the proximal arm regions, iii) the dorsal arm plate being smooth, iv) the arm spines at proximal portion of the arm being six in number and smooth in shape, and v) the number and shape of the tentacle scales at proximal portion of the arm being up to two and spine-shaped adradially and oval abradially. Detailed morphological observations of this new species suggest the inclusion of Ophiodelos, whose familial affiliation remains unclear, in the suborder Ophiacanthina. More than 10 juveniles of various sizes were found in the disc of Ophiodelos okayoshitakai sp. nov., indicating a brooding reproduction. This is the first report of the genus Ophiodelos from Japanese waters. We also provided a nucleotide sequence for part of the cytochrome c oxidase subunit I (COI) gene in O. okayoshitakai sp. nov. for future studies of DNA barcoding and phylogeny.

Developmental Process of a Heterozooid: Avicularium Formation in a Bryozoan, Bugulina californica.

In bryozoans (phylum Bryozoa), representative colonial animals mostly found in marine environments, some species possess different types of individuals (heterozooids) specialized in different functions such as defense or structural support for their colonies. Among them, the best-known heterozooids are the avicularia, known to function as defenders. The differentiation processes of heterozooids, including avicularia, should be important keys to understand the evolutionary significance of bryozoans. However, the developmental process of avicularium formation remains to be fully understood. In this study, therefore, in order to understand the detailed developmental process and timing of avicularium formation, extensive observations were carried out in a bryozoan species, Bugulina californica (Cheilostomata, Bugulidae), that possesses adventitious avicularia, by performing stereomicroscopy on live materials, in addition to scanning electron microscopy and histological observations. The whole process can be divided into seven stages based on developmental events. Especially notably, at the earlier stages, there are three major budding events that produce proliferating cell masses corresponding to primordial tissues: (1) budding of the peduncle cushion at the outer margin of the distal part of a young autozooid, (2) budding of the head-part primordium from the peduncle cushion, and (3) budding of the polypide inside the head part. Experimental control of temperature showed that 20°C would be the best to maintain B. californica colonies.

Two new deep-sea species of the genus Henricia (Asteroidea: Spinulosida: Echinasteridae) from Japanese waters.

Two new deep-sea Henricia species, Henricia margarethae n. sp. and Henricia fragilis n. sp. are described from Sagami Bay and the Ogasawara Islands in Japan. The two new species show an affinity with eight congeners in having multiple furrow spines and abactinal papular areas which are larger than surrounding plates. The two species described herein are characterized by the arrangement of abactinal pseudopaxillae, the arrangement of abactinal papulae, the shape and arrangement of abactinal spines, the length of intermarginal and ventrolateral series, and the armament of superomarginal, inferomarginal, and adambulacral plates.

Morphogenesis and histogenesis during the arm regeneration in a basket star Astrocladus dofleini (Euryalida, Ophiuroidea, Echinodermata).

Basket stars, that is, Ophiuroidea in Echinodermata, exhibit distinctive morphological characteristics with their complicatedly branched arms that can regenerate immediately after mutilation. Although, in brittle stars, that is, ophiuroids with nonbranched arms, the arm regeneration process following accidental trauma or autotomy have been morphologically and histologically observed in several species, few studies have so far been carried out on the regeneration of branched arms in basket stars. In this study, the developmental and morphological features of arm regeneration in Astrocladus dofleini (Gorgonocephalidae, Euryalida, Euryophiurida), one of the most common basket star species in Japanese waters, was anatomically and histologically investigated. Results clearly showed the following phases during the arm regeneration: (a) repair phase, (b) early regenerative phase, (c) intermediate regenerative phase, (d) advanced regenerative Phase I, and (e) advanced regenerative Phase II. The morphogenetic process during the arm regeneration in the basket star showed similar patterns to those of nonbranched arms observed in other ophiuroids. However, differences were also seen between the two ophiuroid types, that is, there were some developmental features specific to the basket star. In the early regenerative phase, branching of coelomic cavities was observed prior to the formation of other tissues, probably inducing the later morphogenesis of branched arms. In addition, hard skeletal ossicles form rapidly at the advanced regenerative Phase II. These developmental features may have led the evolution of bizarre morphologies seen in basket stars, probably contributing to the adaptation to shallow waters from deep-sea habitats.

A taxonomic review of the genus Astrocladus (Echinodermata, Ophiuroidea, Euryalida, Gorgonocephalidae) from Japanese coastal waters.

Japanese species of the genus Astrocladus (Echinodermata, Ophiuroidea, Euryalida, Gorgonocephalidae) are reviewed. Astrocladus coniferus recently has two junior synonyms, A. dofleini Döderlein and A. pardalis Döderlein, however, status of these species has long been questioned. These species concepts have not been reviewed in recent years and no molecular phylogenetic analyses have been performed. Observations of the lectotype of A. coniferus, as well as the lectotype and four paralectotypes of A. dofleini and the holotype of A. pardalis have revealed that A. coniferus and A. pardalis are conspecific and morphologically distinguishable from A. dofleini. Astrocladus coniferus and A. dofleini are supported as distinct species by our molecular data. Additionally, we re-describe A. exiguus and A. annulatus, based on recently collected specimens and the holotype. We conclude that four species, A. annulatus, A. coniferus, A. dofleini, and A. exiguus are present in Japanese waters.