Fusceulimoides kohtsukai gen. et sp. nov., a Minute Eulimid Gastropod Parasitic on the Little Brittle Star Ophiactis savignyi in Central Japan.

Gastropods of over a dozen genera in the family Eulimidae have been identified as parasites of brittle stars, and many more remain to be discovered and described for a comprehensive understanding of the evolutionary history of their host-parasite relationships. In this study, we describe Fusceulimoides kohtsukai gen. et sp. nov., parasitic on the little brittle star, Ophiactis savignyi (Ophiactidae), in Kanagawa, central Japan. The new genus is distinguished from other eulimid genera by the combination of following seven conchological characters: (1) a very small size of up to 1.7 mm high, (2) a colorless translucent appearance, (3) a conical pupiform shape with a paucispiral protoconch, (4) slightly convex teleoconch whorls, (5) a remarkably large body whorl occupying 65-70% of the total shell height, (6) a broad, somewhat squarish and laterally expanded aperture with a strongly curved outer lip, and (7) a developed parietal callus without an indentation or depression in the umbilical area. A multi-locus molecular phylogeny revealed its distant relationship to Hemiliostraca + Pyramidelloides, a previously known clade of ophiuroid parasites, thereby suggesting multiple origins of this host-parasite association in Eulimidae.

Reproductive Isolation and a Change in the Development Mode of the Tideland Snail Batillaria flectosiphonata (Gastropoda: Batillariidae).

Microsatellite analyses of sympatric populations of a tideland snail endemic to the Nansei Islands, Japan, Batillaria flectosiphonata, and its sister species, Batillaria multiformis, from a tideland on Amami-Oshima Island, indicated that the two species are reproductively isolated from each other, confirming the validity of B. flectosiphonata, whose monophyly was supported only by a low bootstrap probability in the previous molecular phylogenetic analysis. Egg capsules of B. flectosiphonata from Tokunoshima Island of the Amami insular group and Okinawajima Island of the Okinawa insular group were examined, which revealed that this species is a direct developer. Thus, the direct development has evolved twice within batillariids in Japanese waters. The lower genetic diversity of B. flectosiphonata than that of B. multiformis in the sympatric habitat might be attributed to its long-term isolation within the Amami insular group.

Multigene phylogenetic analysis reveals non-monophyly of Anisakis s.l. and Pseudoterranova (Nematoda: Anisakidae).

The nematode genera Anisakis s.l. and Pseudoterranova (Anisakidae) include causative agents of anisakiasis and pseudoterranovosis, parasitic diseases resulting from eating undercooked or raw fish or squid. Species in both genera have thus attracted considerable attention especially in public health and taxonomic studies. The phylogenetic relationships of these genera within the subfamily Anisakinae, however, remain to be investigated with dense taxonomic sampling. In this study, we collected an anisakid third-stage larva, and identified it morphologically and molecularly as Pseudoterranova ceticola. Phylogeny of 15 anisakine species, including the newly collected specimen of Ps. ceticola, was reconstructed based on sequences of three mitochondrial (cox1, cox2, and 12S rRNA) and two nuclear (ITS and 28S rRNA) regions. The obtained tree suggested the non-monophyly of Anisakis s.l. and Pseudoterranova. Anisakis s.l. was divided into two groups, which are distinguished from each other by the shape of the ventriculus. Based on phylogenetic relationships and morphology, three species with a shorter ventriculus ("A." brevispiculata, "A." paggiae, and "A." physeteris) were assigned to the genus Skrjabinisakis, as recently proposed. Pseudoterranova ceticola was distantly related to the monophyletic Ps. decipiens species complex. Although the phylogenetic position of the type species Ps. kogiae has not been investigated due to a lack of sequence data, this species may morphologically and ecologically resemble Ps. ceticola, inferring a close kinship between the two species.

Stasis and diversity in living fossils: Species delimitation and evolution of lingulid brachiopods.

The Lingulidae are often considered living fossils, because they have shown little morphological change since the Paleozoic. Limited morphological variation has also made the taxonomic study of living lingulids challenging. We investigated species diversity and phylogenetic relationships of extant lingulids and show that they are substantially more diverse than realized, demonstrating that morphological stasis was commonly accompanied by speciation. Species delimitation based on cytochrome c oxidase subunit I (COI) gene sequences from 194 specimens sampled from East Asia, Australia, Oceania, and the Americas suggested 14-22 species in the lingulids (9-17 species in Lingula and 4-5 species in Glottidia), in contrast to the 11-12 species currently recognized globally in the family. Four-gene phylogenetic analyses supported the sister relationship between Lingula and Glottidia. Within Lingula, L. adamsi, which possesses large, brownish shells, was recovered as sister to all remaining Lingula species, which have more or less greenish shells. Within the greenish Lingula clade, the 'L. anatina' complex was sister to the clade that includes the 'L. reevei' complex. The 'L. anatina' complex was further separated into two major clades with partly separate ranges centered on (i) temperate East Asia, and (ii) the tropical west-central Pacific. Within Glottidia, Pacific species were nested within Atlantic species. Time-calibrated phylogenetic analyses suggested that Lingula likely originated in the early Cretaceous contrary to a previously proposed hypothesis advocating a Cenozoic origin. The separation of Lingula and Glottidia appears to date from the Mesozoic, not from the Carboniferous, contrary to a previous hypothesis. Overall, our results uncovered substantial cryptic diversity in lingulids, which will form the basis for conservation and further taxonomic revision.

Species composition and infection levels of Anisakis (Nematoda: Anisakidae) in the skipjack tuna Katsuwonus pelamis (Linnaeus) in the Northwest Pacific.

Parasites can be used as biological tags to assess stock structures in various marine fish species. In the present study, the species composition and infection levels of parasitic nematodes of the genus Anisakis in the skipjack tuna Katsuwonus pelamis were examined in the Northwest Pacific and adjacent seas. A total of 867 third-stage larvae of Anisakis were collected from 112 skipjack tunas captured around Japan and in other subtropical localities. All larvae were identified as A. berlandi, A. pegreffii, A. simplex (s.s.), A. typica, and A. physeteris (s.l.) by the direct sequencing of the mitochondrial cox2 gene and real-time PCR assays targeting the nuclear ITS region. Anisakis species composition differed among northeastern Japan, the Sea of Japan, and other areas (central Japan, the Nansei Islands, and subtropical region), which is largely concordant with previous stock discrimination of skipjack tuna. Molecular phylogenetic analysis resulted in two intraspecific genetic groups in A. simplex (s.s.), one of which occurred almost exclusively in northeastern Japan. This could be a useful indicator for stock discrimination. Skipjack tunas from northeastern Japan were also characterized by a remarkable variety in the intensity of A. simplex (s.s.), suggesting the commingling of individuals with different migration patterns. This idea might be further justified by the geographic distribution of two genetically distinct groups of A. physeteris (s.l.).

Snails riding mantis shrimps: Ectoparasites evolved from ancestors living as commensals on the host's burrow wall.

The molluscan class Gastropoda includes over 5,000 parasitic species whose evolutionary origins remain poorly understood. Marine snails of the genus Caledoniella (Caledoniellidae) are obligate parasites that live on the abdominal surface of the gonodactylid mantis shrimps. They have highly modified morphological characteristics specialized to the ectoparasitic lifestyle that make it difficult to infer their close relatives, thereby posing a question about their current systematic position in the superfamily Vanikoroidea. In the present study, we performed molecular phylogenetic analyses using three nuclear and three mitochondrial gene sequences to unveil the phylogenetic position of these enigmatic snails. The resulting trees recovered Caledoniella in the superfamily Truncatelloidea and within a subclade of commensal species that live on the burrow wall of marine benthic invertebrates. More specifically, Caledoniella formed the sister clade to a commensal snail species living in mantis-shrimp burrows and they collectively were sister to Sigaretornus planus (formerly in the family Tornidae or Vitrinellidae), a commensal living in echiuran burrows. This topology suggests that the species of Caledoniella achieved their ectoparasitic mode of life through the following evolutionary pathway: (1) invasion into the burrows of benthic invertebrates, (2) specialization to mantis shrimps, and (3) colonization of the host body surface from the host burrow wall with the evolution of the parasitic nature. The final step is likely to have been accompanied by the acquisition of a sucker on the metapodium, the loss of the radula and operculum, and the formation of monogamous pair bonds. The present molecular phylogeny also suggested parallel evolution of planispiral shells in a subclade of Truncatelloidea and enabled us to newly redefine the families Caledoniellidae, Elachisinidae, Teinostomatidae, Tornidae and Vitrinellidae.

Probable association between Anisakis infection in the muscle of skipjack tuna (Katsuwonus pelamis) and human anisakiasis in Tokyo, Japan.

Anisakiasis is a gastrointestinal disease caused by parasitic anisakid nematodes, mainly Anisakis simplex sensu stricto (A. simplex). Anisakiasis is prevalent in Japan and approximately 40% of anisakiasis cases in Tokyo occur through the consumption of raw or marinated mackerel. However, in 2018, there was a sudden increase in the number of the food poisoning cases in Tokyo caused by consumption of skipjack tuna (Katsuwonus pelamis). Therefore, we investigated anisakiasis cases resulting from ingestion of skipjack tuna in Tokyo, and surveyed the presence of Anisakis larvae in skipjack tuna in 2018 and 2019. Nineteen samples from 15 patients (13 in 2018 and 2 in 2019) with anisakiasis surely caused by ingestion of skipjack tuna were all identified as A. simplex. The higher mean abundance of Anisakis simplex larvae in skipjack tuna muscle in May 2018 (1.30; 13 larvae/10 fishes) compared to that in the other periods was regarded as a contributing factor in the increase in anisakiasis cases by ingesting skipjack tuna in 2018. To verify whether Anisakis larvae migrate from the visceral organs to the muscle during the period from fishing on the boat until processing for sale, the number of Anisakis larvae in skipjack tuna caught from August to November 2018 was investigated by removing the visceral organs at three different timings, i.e., immediately after catching, after landing, and after transport to the laboratory. Anisakis larvae were detected in the muscle irrespective of the timings at which visceral organs were removed. All larvae from the muscle were detected only from the ventral part and were identified as A. simplex. We thus consider that avoiding raw consumption of the ventral muscle should be an effective measure to prevent anisakiasis.

Host identification for the deep-sea snail genus Haliella with description of a new species (Caenogastropoda, Eulimidae).

A new parasitic species of eulimid gastropod, Haliella seisuimaruae sp. nov., is described from bathyal (728-978 m) waters off the Pacific coasts of Japan. It shows the closest resemblance to the type species H. stenostoma from the North Atlantic and Barents Sea in having a tall shell with an almost straight outer lip, but differs in having a junction of the parietal wall and columellar lip at 38% of the aperture height from the suture (33% in H. stenostoma), a slightly wider aperture and a more curved and extended columellar lip. The holotype of H. seisuimaruae sp. nov. was found attached to an irregular sea urchin, Brissopsis sp. cf. luzonica (Spatangoida: Brissidae). This represents the first direct observation of parasitic ecology and echinoderm host for the genus Haliella. A new replacement name, Eulima tsushimensis nom. nov., is proposed here for Eulima stenostoma A. Adams, 1861, which is preoccupied by Eulima stenostoma Jeffreys, 1858 (type of Haliella).

Molecular phylogeny of Maldanidae (Annelida): Multiple losses of tube-capping plates and evolutionary shifts in habitat depth.

Inter-familial relationships of the phylum Annelida have been widely studied using molecular phylogenetic/genomic approaches; however, intra-familial relationships remain scarcely investigated in most annelid families. The Maldanidae (bamboo worms) comprise more than 280 species of 40 genera and six subfamilies that occur in various environments from intertidal to hadal zones. Within this family, the taxon Maldanoplaca, which consists of four subfamilies (Maldaninae, Notoproctinae, Nicomachinae, and Euclymeninae), was proposed based on the presence of cephalic and anal plates. Phylogenetic relationships within the family remain largely undetermined based on molecular data. In this study, we reconstructed a molecular phylogeny using 52 maldanid species from six subfamilies based on two nuclear genes (18S rDNA and 28S rDNA) and two mitochondrial genes (16S rDNA and COI). Our analysis confirmed the monophyly of the subfamilies Rhodininae, Maldaninae, Lumbriclymeninae, and Nicomachinae, but neither Maldanoplaca nor the subfamily Euclymeninae were recovered as monophyletic. Nicomachinae was clustered within Euclymeninae. Ancestral state reconstruction suggested that cephalic plates were lost at least three times, despite the functional importance of capping tubes, and that anal plates were lost once. Mapping habitat depth on the phylogenetic tree suggested that habitat shifts among depth zones frequently occurred in distinct maldanid lineages.

The complete mitochondrial genome of the longfin dragonfish Tactostoma macropus (Stomiiformes: Stomiidae).

The complete mitochondrial genome (mitogenome) was determined for the longfin dragonfish Tactostoma macropus, which is the first for the genus and the third within the family Stomiidae. The mitogenome sequence is 17,690 bp in length containing 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes, and a control region, as in most fishes. The gene order of T. macropus showed an unreported deviation from the typical vertebrate one. Phylogenetic reconstruction using the maximum likelihood method placed T. macropus in the monophyletic Stomiiformes. Three stomiid species were recovered as a moderately supported clade in the phylogenetic tree.

Megadenus atrae n. sp., an endoparasitic eulimid gastropod (Mollusca) from the black sea cucumber Holothuria atra Jaeger (Aspidochirotida: Holothuriidae) in the Indo-West Pacific.

An eulimid gastropod, Megadenus atrae n. sp., endoparasitic in the cloacal chamber of the black sea cucumber Holothuria atra Jaeger is described from Okinawa, Japan, as the fifth species of the genus. Conspecific specimens have also been found from southeast India, northeast Australia and New Caledonia. The generic assignment is justified by the presence of (i) a thick, long proboscis that bears a large fold (pseudopallium) near the base and a collar-like structure at the middle, (ii) a thin, globose shell that is covered by the pseudopallium, and (iii) sexual dimorphism with the female generally larger than the male. The new species is distinguishable from the four previously described congeners by its cauldron-shaped pseudopallium, moderately-developed collar of the proboscis and rounded basal lip of the shell. The comparisons of the size and sex of solitary and paired individuals support a previous hypothesis that the species of Megadenus Rosén, 1910 are protandrous with environmental sex determination. The present species occurs mostly as monogamous pairs despite its very low population density, implying that the presence of a conspecific individual acts as a cue for larval settlement. Both mechanisms would increase individual reproductive success in such permanent parasites with low prevalence and abundance as the species of Megadenus.

Molecular phylogenetic investigations of the relationships of the echinoderm-parasite family Eulimidae within Hypsogastropoda (Mollusca).

The gastropod family Eulimidae has attracted considerable attention as one of the most diverse groups of parasitic molluscs in terms of number of species and ranges of body plans and parasitic strategies. However, the phylogenetic position of the family has not been established within the Hypsogastropoda and this has hampered the inference of ancestral states in the evolution of the morphology and parasitic strategies. Here we present Bayesian and maximum likelihood phylograms of Hypsogastropoda based on nuclear and mitochondrial loci (18S and 28S rRNA, Histone H3, COI and 16S rRNA) and a better taxonomic sampling than in previous molecular analyses, to determine the position of Eulimidae. The resulting trees suggest Vanikoridae as the sister group of Eulimidae; the two families are collectively placed in the newly redefined superfamily Vanikoroidea, with Truncatelloidea and (potentially paraphyletic) Rissooidea as closest relatives. Vanikorids are protandrous hermaphrodites as are many eulimids and are essentially carnivorous, differing from the mostly gonochoristic and herbivorous/detritivorous Truncatelloidea and Rissooidea. The mode of feeding may have a phylogenetic signal also within Eulimidae, where radula-less species constitute a robust clade. Other new findings include a close affinity of the submarine-cave Pickworthiidae to Cerithioidea and a terminal position of Nystiellidae within the paraphyletic Epitoniidae.