Intrabody Tetrodotoxin Distribution and Possible Hypothesis for Its Migration in Ribbon Worms Cephalothrix cf. simula (Palaeonemertea, Nemertea).

Tetrodotoxin (TTX) is a potent neurotoxin found in many marine and terrestrial animals, but only a few species, such as the ribbon worms of the genus Cephalothrix, accumulate it in extremely high concentrations. The intrabody distribution of TTX in highly toxic organisms is of great interest because it helps researchers to understand the pathways by which the toxin migrates, accumulates, and functions in tissues. Using immunohistochemistry with anti-TTX antibodies, the authors of this study investigated the toxin's distribution inside the organs, tissues, and cells of Cephalothrix cf. simula. The cell types of TTX-positive tissues were identified by light microscopy. The main sites of TTX accumulation occurred in the secretory cells of the integuments, the microvilli of the epidermal ciliary cells, cephalic glands, the glandular epithelia of the proboscises, the enterocytes of the digestive systems, and nephridia. Obtained data suggest the toxin migrates from the digestive system through blood vessels to target organs. TTX is excreted from the body through the nephridia and mucus of epidermal cells.

CLSM Analysis of the Phalloidin-Stained Muscle System of the Nemertean Proboscis and Rhynchocoel.

The proboscis and rhynchocoel musculature of 56 nemertean species was studied using phalloidin labelling and confocal laser scanning microscopy. Six types of muscle layers are found in the anterior proboscis of the nemerteans: inner circular, inner diagonal, inner longitudinal, outer diagonal, outer circular, and outer longitudinal. Only the inner circular and inner longitudinal muscle layers are present in all the nemerteans studied. Ten types of arrangement of the proboscis musculature are described. Three primary types ('palaeotype', 'heterotype', and 'hoplotype') correspond to the three nemertean supergroups (Palaeonemertea, Heteronemertea, and Hoplonemertea). The evolutionary transformations of the initial 'palaeotype' proboscis proceeded in two primary ways: increasing bilateral symmetry (Callinera, Hubrechtella, and most of Heteronemertea) and increasing polyradial symmetry (Baseodiscidae, Oxypolellinae, and Hoplonemertea). The musculature of the middle portion of the proboscis differs among the three groups with armature: Palaeonemertea (genus Callinera), Polystilifera, and Monostilifera. The musculature of the stylet apparatus of the monostiliferous nemerteans is more complicated than that of the polystiliferous nemerteans, and consists of four muscle components--basal and anterior sphincters, radial and longitudinal musculature. Among the studied monostiliferans, the different components of the stylet musculature are developed to varying degrees. In addition, data on the structure of the rhynchocoel with interwoven musculature are provided. The taxonomic significance and phylogenetic interpretation of the proboscis and rhynchocoel musculature is discussed.

Structure of the Proboscis Endothelium in Nemertea.

We studied the ultrastructure of the proboscis endothelium of 14 nemertean species. In all nemerteans examined, the endothelium is organized as a pseudostratified myoepithelium consisting of two types of cells resting on the basal extracellular matrix: apically situated supportive cells and subapical myocytes covered by cytoplasmic sheets of the supportive cells. Myocytes form the inner circular musculature of the proboscis; the endothelium in the bulb of monostiliferous nemerteans lacks myocytes. The endothelium of the studied species differs in the number of rows of muscle fibres (one vs. several rows), the number of myofibrils in myocytes (one vs. two to five), the number of processes of myocytes covered by one supportive cell (one vs. two to 23), and in the number of processes in supportive cells (one vs. two to five). In some of the species, rudimentary cilia of supportive cells were revealed by using cLSM and an antibody against tubulin. The data obtained indicate that the proboscis endothelium in nemerteans is in fact a coelothelium, but recognition of the ancestral state of the coelomic lining in Nemertea is problematic, as the rhynchocoel peritoneum lacks myocytes.

DNA Taxonomy of Paranemertes (Nemertea: Hoplonemertea) with Spirally Fluted Stylets.

Of the 14 nominal species that are now or have ever been assigned to the genus Paranemertes Coe, 1901 , four have been reported to have stylets with a spirally fluted or braided appearance. Although differentiation in color patterns has been documented among species/populations, these nemerteans share similar external characters. Using the sequence datasets of mitochondrial cytochrome c oxidase subunit I (COI), 16S rRNA, and nuclear 28S rRNA genes of specimens from 14 localities of Canada, USA, Russia, Japan, and China, we analyzed the genetic differentiation and reconstructed the phylogenetic trees for these nemerteans. In conjunction with the external characters, we discuss their taxonomy and species delimitation. An analysis based on COI dataset showed high genetic variations among populations and even among worms from the same geographic area. The analyzed 111 individuals were assigned into seven networks by statistical parsimony analysis. The inter-network uncorrected p-distances ranged from 0.044 to 0.172 and the mean intra-network uncorrected p-distances varied from 0.001 to 0.005. With the exception of two networks that contain specimens from the East China Sea, all networks were well-supported by the results of Bayesian and neighbor-joining analyses on the COI data. Phylogenetic trees based on 16S rRNA and 28S rRNA datasets were basically similar to the COI trees, but specimens in some networks were merged into larger clades. Present molecular analyses support the validity of P. sanjuanensis and the synonymization of P. cylindracea with P. peregrina. Nemerteans previously recorded as P. peregrina may contain several species and sympatric speciation might have been occurred in this nemertean group.

Taxonomic identity of a tetrodotoxin-accumulating ribbon-worm Cephalothrix simula (Nemertea: Palaeonemertea): a species artificially introduced from the Pacific to Europe.

We compared the anatomy of the holotype of the palaeonemertean Cephalothrix simula ( Iwata, 1952 ) with that of the holotypes of Cephalothrix hongkongiensis Sundberg, Gibson and Olsson, 2003 and Cephalothrix fasciculus ( Iwata, 1952 ), as well as additional specimens from Fukue (type locality of C. simula) and Hiroshima, Japan. While there was no major morphological discordance between these specimens, we found discrepancies between the actual morphology and some statements in the original description of C. simula with respect to supposedly species-specific characters. Our observation indicates that these three species cannot be discriminated by the anatomical characters so far used to distinguish congeners. For objectivity of scientific names, topogenetypes of the mitochondrial cytochrome c oxidase subunit I (COI) sequences are designated for C. simula, C. hongkongiensis, and C. fasciculus. Analysis of COI sequence showed that the Hiroshima population can be identified as C. simula, which has been found in previous studies from Trieste, Italy, and also from both the Mediterranean and Atlantic coasts of the Iberian Peninsula, indicating an artificial introduction via (1) ballast water, (2) ship-fouling communities, or (3) the commercially cultured oyster Crassostrea gigas ( Thunberg, 1793 ) brought from Japan to France in 1970s. Cephalothrix simula is known to be toxic, as it contains large amounts of tetrodotoxin (TTX). We report here that the grass puffer Takifugu niphobles ( Jordan and Snyder, 1901 )-also known to contain TTX- consumes C. simula. We suggest that the puffer may be able to accumulate TTX by eating C. simula.

Microscopic anatomy of the integument in the ribbon worm Micrura bella (Stimpson, 1857) (Pilidiophora, Nemertea).

The integument of ribbon worms in the order Heteronemertea is distinct from the integuments in the other taxa of nemerteans due to the presence of a special subepidermal glandular layer, the cutis. Among heteronemerteans, the ultrastructure of the cutis has been studied only in the Lineus ruber species complex. In the current study, ultrastructural (transmission electron microscopy) and histochemical studies of the epidermis and the cutis of Micrura bella from the basal Lineage A of the family Lineidae were performed. The epidermis consisted of ciliated and serous gland cells and is separated from the cutis by a layer of the subepidermal extracellular matrix; the basal lamina was not detected. The cutis comprised musculature, two types of mucous and four types of granular gland cells, and pigment cells with four types of granules. In the cutis of juvenile worms, type II granular gland cells and type II mucous cells were not observed. The integument of the caudal cirrus consisted of ciliated and serous gland cells and two intraepidermal lateral nerve cords; the cutis was absent. The compositions of the integument glands of M. bella and the L. ruber species complex are similar, except for the presence of type IV granular gland cells with narrow rod-shaped and lamellated granules exhibiting an alternating dark and light transverse layers and type II mucous cells found only in M. bella.

Phylogenetic Relationships and Taxonomic Position of the Ribbon Worms of the Genus Parahubrechtia (Nemertea, Palaeonemertea) with Descriptions of Two New Species.

The genus Parahubrechtia Gibson and Sundberg, 1999 was first described within the family Hubrechtiidae (class Pilidiophora) and subsequently transferred to the family Callineridae (class Palaeonemertea). Here we describe two new species, Parahubrechtia rayi sp. nov. from the Sea of Japan (Russia) and P. peri sp. nov. from the South China Sea (China). A phylogenetic analysis based on partial sequences of five nuclear and mitochondrial gene regions, 18S rRNA, 28S rRNA, histone H3, 16S rRNA, and COI, has confirmed the monophyly of the genus Parahubrechtia, and indicated a close relationship to Callinera Bergendal, 1900, whose monophyly is not confirmed. Both genera belong to the family Tubulanidae, with its junior synonym being Callineridae. Three major subclades are distinguished within the Tubulanidae: subclade Tubulanus s. str., subclade Tubulanus punctatus, and subclade Parahubrechtia + Callinera. The further status of Parahubrechtia depends on whether the paraphyly of Callinera is confirmed or not and how the problem of paraphyly of the genus Tubulanus Renier, 1804 is resolved.

A new species of Metaruncina (Gastropoda: Heterobranchia: Runcinida) from Vietnam with a discussion on the so-called rudimentary radula in runcinids.

Marine heterobranch slugs of the order Runcinida comprise 61 species, most of which inhabit the Atlantic Ocean and are referred to the genus Runcina. The Runcinida of the Pacific Ocean have been much less studied to date: only 12 species, from the genera Metaruncina Baba, 1967, Rfemsia Chernyshev, 1999, Runcinida Burn, 1963, Runcinella Odhner, 1924, Pseudoilbia M. C. Miller Rudman, 1968, and Runcina Forbes [in Forbes and Hanley], 1851, have been described from the region. Metaruncina, Rfemsia, and Runcinida are found in the Pacific Ocean only (Baba 1954, 1967; Burn 1963; Gosliner 1991; Chernyshev 1998, 1999, 2005, 2006). The genus Metaruncina, endemic from the Pacific coast of Asia, includes two species: M. setoensis (Baba, 1954) and M. nhatrangensis Chernyshev, 2005. The latter is the only runcinid described from the coastal waters of the South China Sea, where it was found in the bays of Nha Trang and Van Phong (Chernyshev 2005). During subsequent expeditions to the coastal waters of Vietnam, this species was also recorded from off Tho Chu Island (09°19'N, 103°30'E) and off Nam Du Island (09°43' N, 104°23' E). However, in addition to this common species, an undescribed species of Metaruncina was collected. A description of this new species is provided in the present paper. Type material was deposited at the Museum of the A.V. Zhirmunsky National Scientific Center of Marine Biology FEB RAS, Vladivostok, Russia (MIMB).

An integrative description of a new Cephalothrix species (Nemertea: Palaeonemertea) from the South China Sea.

A new nemertean species of the genus Cephalothrix from intertidal calcareous red algae off the Vietnam coast is described based on histological sections, confocal laser scanning microscopy, and nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, H3) and two mitochondrial (COI, 16S rRNA) DNA fragments. Cephalothrix suni sp. nov. is characterized by a unique body colour pattern consisting of transverse brown on ventral body side. A molecular phylogenetic analysis of the family Cephalotrichidae using a multigene approach has identified three clades for Cephalothrix: Cephalothrix, Procephalothrix, and interstitial cephalotrichids. The new species belongs to clade Procephalothrix sensu Chernyshev Kajihara 2019 and a subclade of species with red, orange, or dark yellow anterior tip.

New species of latest Eocene/earliest Oligocene microgastropods (Heterobranchia: Orbitestellidae and Omalogyridae) from the Gries Ranch Formation, Lewis County, Washington State, USA.

Continued sampling of the latest Eocene to earliest Oligocene Gries Ranch Formation in Lewis County, Washington State, has yielded new heterobranch microgastropod species. Orbitestella kieli sp. nov., is the third fossil species of this genus and family Orbitestellidae from western North America. Two new species of Ammonicera, A. rolani sp. nov. and A. danieli sp. nov., are together only the second fossil record of this genus and the family Omalogyridae from the northeastern Pacific Ocean. New specimens of two previously recorded species, O. palaiopacifica Squires Goedert and A. benhami Squires Goedert, from early Eocene rocks of the Crescent Formation provide new data regarding shell morphology. The fossil record of both Ammonicera and Orbitestella in western North America is restricted to early Eocene to earliest Oligocene age rocks in Washington State.

Pseudocnidae of ribbon worms (Nemertea): ultrastructure, maturation, and functional morphology.

The fine structure of mature pseudocnidae of 32 species of nemerteans, representatives of 20 genera, six families, and two classes was investigated with scanning and transmission electron microscopy. Pseudocnidae are composed of four layers (cortex, medulla, precore layer, and core) in most species investigated, but the degree of development and position of each layer can vary between different species. The secretion products comprising immature pseudocnidae segregate into separate layers: a thin envelope, which subsequently separates into the cortex and medulla and an extensive internal layer. We distinguish two pseudocnida types: type I is characterized by a two-layered core and type II by a three-layered core. Type I pseudocnidae are present in archinemertean species, Carinoma mutabilis, and in all pilidiophoran species, except Heteronemertea sp. 5DS; type II pseudocnidae occur in all studied species of Tubulanidae and the basal Heteronemertea sp. 5DS. Based on the structure of the discharged pseudocnidae observed in eleven species of palaeonemerteans and in eight species of pilidiophorans, we distinguish three different mechanisms (1-3) of core extrusion/discharge with the following characteristics and distribution: (1) the outer core layer is everted simultaneously with the tube-like layer and occurs in type I pseudocnidae of most species; (2) the extruded core is formed by both eversion of the outer core layer and medullar layer, and occurs in type I pseudocnidae of Micrura cf. bella; (3) the eversion of the outer core layer begins together with the core rod and core rod lamina and occurs in type II pseudocnidae. Morpho-functional comparison with other extrusomes (cnidae, sagittocysts, rhabdtites, and paracnids) confirm that pseudocnidae are homologous structures that are unique to nemerteans.

Spermatozoa ultrastructure of two basal pilidiophoran nemerteans, Hubrechtella juliae and Sonnenemertes cantelli (Nemertea, Pilidiophora).

Nemertea is a phylum of worms with a simple internal morphology; nemerteans' spermatozoon morphology can be used for their classification and phylogenetic analyses. The aim of the present study was to describe spermatozoa of the nemerteans Hubrechtella juliae and Sonnenemertes cantelli from the basal groups of the class Pilidiophora at the ultrastructure level. Both species have primitive ('compact-head' sensu Stricker and Folsom, 1998) spermatozoa with ovoid head and five mitochondria in the midpiece, but differ in the structure of acrosomal complex: in Hubrechtella juliae, the single lens-shaped acrosomal vesicle contains an area of moderate electron density not enclosed by a separate membrane; in Sonnenemertes cantelli, the acrosome shows a unique morphology and contains a few electron-dense vesicles with irregular shapes and positions and one more electron-lucent elongated vesicle. Such a pattern of the acrosomal complex organization is described for Nemertea for the first time. An assumption is made that the states "two or more mitochondria" and "posterior acrosomal ring component" may be synapomorphies of Hubrechtiiformes+Heteronemertea (class Pilidiophora), whereas "the posterior margin of the acrosomes forms an acrosomal ring component" is presumably an autapomorphy of the family Lineidae s.l. The results suggest that spermatozoa provide a useful source of characters for nemertean systematics.

A histology-free description of a new species of the genus Tetrastemma (Nemertea: Hoplonemertea: Monostilifera) from Hawaii and India.

A new species of the genus Tetrastemma Ehrenberg, 1831, T. freyae sp. nov., is described and illustrated from Hawaii and India. The description is based on light microscopy examination of the external and internal morphology, as well as on two gene markers (cytochrome c oxidase subunit I and histone H3 DNA).

Meloscaphander grandis (Heterobranchia: Cephalaspidea), a deep-water species from the North Pacific: Redescription and taxonomic remarks.

Meloscaphander grandis is a little-known species missing from databases and papers on taxonomic revision and phylogenetic analysis of Scaphandridae. This species is redescribed herein, based on the type specimen and specimens from the abyssal plain adjacent to the Kuril-Kamchatka Trench. A phylogenetic analysis of COI, 16S, and 28S markers show M. grandis to nest within the Scaphander clade. Additionally, Scaphander lignarius and S. bathymophilus are suggested to be a complex of cryptic species. Morphological differences between the genera Meloscaphander and Scaphander are of dubious significance and, when coupled with molecular data, give a strong reason for reconsidering Meloscaphander as a junior synonym of Scaphander. Thus, according to an integrative taxonomic analysis, Meloscaphander grandis has been transferred to the genus Scaphander. The diagnosis of the genus Scaphander is expanded. We propose new combinations as follows: Scaphander grandis (Minichev, 1967) comb. n. for Meloscaphander grandis, Scaphander sibogae (Schepman, 1913) comb. n. for Meloscaphander sibogae, and Scaphander imperceptus (Bouchet, 1975) comb. n. for Meloscaphander imperceptus. Due to the homonymy of Scaphander sibogae Schepman, 1913 (with a sunken spire) and Scaphander sibogae (Schepman, 1913) comb. n. (with an elevated spire), the name S. attenuatus Schepman, 1913 becomes valid for the former species (with a sunken spire).

Genetic diversity and phylogeny of limpets of the genus Nipponacmea (Patellogastropoda: Lottiidae) based on mitochondrial DNA sequences.

Species of the genus Nipponacmea inhabit only the Pacific coast of Asia, including the Russian Far East. Their external morphological characters are highly variable and often lead to misidentifications of species. So far, little research has been conducted using molecular markers. We used sequences from three mitochondrial genes (fragments of cytochrome c oxidase subunit I gene (COI), 12S and 16S rDNA). For comparison, additional genetic and taxonomic data on other species belonging to this genus were derived from GenBank. The molecular phylogenetic trees suggest that the species N. fuscoviridis and N. nigrans are species complexes. N. fuscoviridis is divided into three subgroups with high support and relatively large distances between them (N. fuscoviridis A, B and C); N. nigrans fall into two subgroups and one of them (N. nigrans A) is more closely related to N. moskalevi than to the other subgroup of N. nigrans (B).