Alexey V. Chernyshev

Statistical Parsimony Networks and Species Assemblages in Cephalotrichid Nemerteans (Nemertea)

Background It has been suggested that statistical parsimony network analysis could be used to get an indication of species represented in a set of nucleotide data, and the approach has been used to discuss species boundaries in some taxa. Methodology/Principal Findings Based on 635 base pairs of the mitochondrial protein-coding gene cytochrome c oxidase I (COI), we analyzed 152 nemertean specimens using statistical parsimony network analysis with the connection probability set to 95%. The analysis revealed 15 distinct networks together with seven singletons. Statistical parsimony yielded three networks supporting the species status of Cephalothrix rufifrons, C. major and C. spiralis as they currently have been delineated by morphological characters and geographical location. Many other networks contained haplotypes from nearby geographical locations. Cladistic structure by maximum likelihood analysis overall supported the network analysis, but indicated a false positive result where subnetworks should have been connected into one network/species. This probably is caused by undersampling of the intraspecific haplotype diversity. Conclusions/Significance Statistical parsimony network analysis provides a rapid and useful tool for detecting possible undescribed/cryptic species among cephalotrichid nemerteans based on COI gene. It should be combined with phylogenetic analysis to get indications of false positive results, i.e., subnetworks that would have been connected with more extensive haplotype sampling.

Confocal laser scanning microscopy analysis of the phalloidin-labelled musculature in nemerteans

The musculature of 31 species was studied using phalloidin labelling and confocal laser scanning microscopy. New data are presented on the patterns of the body wall, proboscis and gonadal musculature. Two kinds of epidermal muscles are described in the palaeonemerteans and heteronemerteans – radial fibres (common in anoplans) and a muscular meshwork of longitudinal and diagonal fibres found in Carinoma mutabilis. All palaeonemerteans and hoplonemerteans studied here were found to possess diagonal muscles between the outer circular and the inner longitudinal body wall musculature; however, seven studied heteronemerteans had diagonal muscles only in the cutis. The proboscis musculature of all nemerteans studied here includes inner circular muscles. In addition, palaeonemerteans and heteronemerteans have a layer of outer diagonal muscles in the proboscis. The gonadal musculature of the hoplonemerteans consists of muscle meshwork or regularly arranged longitudinal fibres. Confocal studies of nemertean musculature may provide useful new characters for phylogenetic analysis of this phylum.

Tetrodotoxin-producing Bacillus sp. from the ribbon worm (Nemertea) Cephalothrix simula (Iwata, 1952)

Specimens of the toxic ribbon worm Cephalothrix simula from the Sea of Japan were screened for tetrodotoxin-producing bacteria. A single TTX-producing bacterial strain (No 1839) was isolated from tissues of C. simula and studied by immunohistochemical methods (including immunoelectron and immunofluorescent microscopies) with anti-TTX antibodies. Sequencing of 16S rRNA gene of the strain 1839 showed that it is most likely Bacillus sp. CU040510-015 and Bacillus asahii. Based on its morphological and biochemical properties, however we suppose that the isolated Bacillus sp. 1839 should be classified as representing a new species. Microdistribution of TTX in bacterial cell was investigated under electron microscope by immunoenzymatic methods. TTX was concentrated in the forespore and free spores, but it was not detected in the vegetative cells of Bacillus sp. 1839. We suggest that release of free mature spores from sporangium of Bacillus sp. 1839 leads to appearance of toxin in tissues of C. simula. Confocal laser-scanning microscopy (CLSM) method with anti-TTX antibodies can be recommended for preliminary detection of apparent TTX accumulation.

Ultrastructural study of the proboscis of malacobdella grossa (Nemertea: Hoplonemertea)

The proboscis of the symbiotic nemertean Malacobdella grossa was examined with transmission electron microscopy to reveal more features of bdellonemerteans for phylogenetic analysis. General morphology of the proboscis of Malacobdella corresponds to that of a typical hoplonemertean: the middle chamber is present; the nervous system of the proboscis is embedded in the connective tissue between the inner and outer longitudinal muscle layers; the radially arranged nerve trunks are clearly seen over a considerable portion of their extent. Unique features (i.e. autapomorphies) of Malacobdella’s proboscis include: interwoven longitudinal muscle fibres; nerve plexus in the anterior part of the proboscis; absence of mucus glands; presence of subepithelial glands; reduced musculature in the middle chamber; and absence of glandular epithelium in the posterior chamber.

Tetrodotoxin-Producing Bacteria: Detection, Distribution and Migration of the Toxin in Aquatic Systems

This review is devoted to the marine bacterial producers of tetrodotoxin (TTX), a potent non-protein neuroparalytic toxin. In addition to the issues of the ecology and distribution of TTX-producing bacteria, this review examines issues relating to toxin migration from bacteria to TTX-bearing animals. It is shown that the mechanism of TTX extraction from toxin-producing bacteria to the environment occur through cell death, passive/active toxin excretion, or spore germination of spore-forming bacteria. Data on TTX microdistribution in toxic organs of TTX-bearing animals indicate toxin migration from the digestive system to target organs through the transport system of the organism. The role of symbiotic microflora in animal toxicity is also discussed: despite low toxin production by bacterial strains in laboratory conditions, even minimal amounts of TTX produced by intestinal microflora of an animal can contribute to its toxicity. Special attention is paid to methods of TTX detection applicable to bacteria. Due to the complexity of toxin detection in TTX-producing bacteria, it is necessary to use several methods based on different methodological approaches. Issues crucial for further progress in detecting natural sources of TTX investigation are also considered.

Morphology of the proboscis of Hubrechtella Juliae (Nemertea, Pilidiophora): implications for pilidiophoran monophyly.

The proboscis of Hubrechtella juliae was examined using transmission electron microscopy, scanning electron microscopy, and confocal laser scanning microscopy to reveal more features of basal pilidiophoran nemerteans for morphological and phylogenetic analysis. The proboscis glandular epithelium consists of sensory cells and four types of gland cells (granular, bacillary, mucoid, and pseudocnidae‐containing cells) that are not associated with any glandular systems; rod‐shaped pseudocnidae are 15–25 μm in length; the central cilium of the sensory cells is enclosed by two rings of microvilli. The nervous plexus lies in the basal part of glandular epithelium and includes 26–33 (11–12 in juvenile) irregularly anastomosing nerve trunks. The proboscis musculature includes four layers: endothelial circular, inner diagonal, longitudinal, and outer diagonal; inner and outer diagonal muscles consist of noncrossing fibers; in juvenile specimen, the proboscis longitudinal musculature is divided into 7–8 bands. The endothelium consists of apically situated support cells with rudimentary cilia and subapical myocytes. Unique features of Hubrechtellas proboscis include: acentric filaments of the pseudocnidae; absence of tonofilament‐containing support cells; two rings of microvilli around the central cilium of sensory cells; the occurrence of subendothelial diagonal muscles and the lack of an outer diagonal musculature (both states were known only in Baseodiscus species). The significance of these characters for nemertean taxonomy and phylogeny is discussed. The proboscis musculature in H. juliae and most heteronemerteans is bilaterally arranged, which can be considered a possible synapomorphy of Hubrechtellidae + Heteronemertea (= Pilidiophora). J. Morphol. 274:1397–1414, 2013.

Distribution of tetrodotoxin in the ribbon worm Lineus alborostratus (Takakura, 1898) (nemertea): Immunoelectron and immunofluorescence studies

Transmission electron and confocal laser scanning (CLSM) microscopies with monoclonal anti-tetrodotoxin antibodies were used to locate tetrodotoxin (TTX) in tissues and gland cells of the ribbon worm Lineus alborostratus. CLSM studies have shown that the toxin is primarily localized in the cutis (special subepidermal layer) of the body wall and in the glandular epithelium of the proboscis. Immunoelectron micrographs have shown that only subepidermal bacillary gland cells type I in cutis and pseudocnidae-containing and mucoid gland cells manifested TTX-gold labeling. TTX was associated with the nuclear envelope, endoplasmic reticulum membrane, and secretory granules of TTX-positive gland cells. These studies indicate that ТТХ is brought into the cytoplasm of the glandular cells of the cutis and proboscis epithelium, where it is associated with membrane-enclosed organelles involved in protein secretion and then concentrated in glandular granules.

Runcinida valentinae Chernyshev, 2006 (Opisthobranchia: Runcinacea), a new opisthobranch species for the sea of Japan

Runcinida valentinae, an opisthobranch mollusk that was previously known from the waters off Iturup Island (Kuril Islands), has been found off Rudnaya Bay and in Oprichnik Bay in the Sea of Japan. New data on the radula morphology in this species are presented. Partial sequences of nuclear 28S rRNA and mitochondrial COI genes of the genus Runcinida have been obtained for the first time. Their analysis shows the distinctiveness of R. valentinae, which is morphologically close to R. marisae Chernyshev, 1998 from Peter the Great Bay. The strong nucleotide differences between these species confirm importance of body coloration patterns as species-specific traits in runcinid systematics.

The species composition of the nemertean genus Oerstedia (Nemertea: Hoplonemertea) in the Far Eastern seas of Russia

According to the literature, six species of nemerteans of the Oerstedia genus occur in the Far Eastern seas of Russia. The results of genetic (28S) and allozyme (11 loci) analysis revealed that O. oculata (Kulikova, 1987), O. zebra (Chernyshev, 1993) and O. valentinae (Chernyshev, 1993) are junior synonyms of O. polyorbis Iwata, 1954, while O. phoresiae (Kulikova, 1987) is a valid species. The species validity of O. dorsalis sensu Iwata, 1954 was also corroborated. New data on the variability and distribution of certain nemerteans are furnished.

Germinal granules in interstitial cells of the colonial hydroids Obelia longissima pallas, 1766 and Ectopleura crocea Agassiz, 1862

Electron-dense germinal granules, which are usually regarded as markers and key organelles of germline cells, were revealed in the interstitial (stem) cells of the colonial hydroids Obelia longissima and Ectopleura crocea. The interstitial cells of O. longissima displayed intense alkaline phosphatase activity, a histochemical marker for vertebrate embryonic stem and primary germ cells, as well as positive reaction to proliferating cell nuclear antigen (PCNA), which is an immunochemical marker for cell reproduction. Our findings and the literature data suggest the evolutionary conservation and similarity of the morphological and functional organization of potentially gametogenic stem cells in asexually reproducing invertebrates and germ cells in all studied Metazoa. The self-renewing pool of such stem cells provides the cellular source for blastogenesis and gametogenesis and the cellular basis for life functions, including both asexual and sexual reproduction.