Does the frontal sensory organ in adults of the hoplonemertean Quasitetrastemma stimpsoni originate from the larval apical organ?

BACKGROUND: The apical organ is the most prominent neural structure in spiralian larvae. Although it has been thoroughly investigated in larvae of the class Pilidiophora in phylum Nemertea, studies on its structure in other nemertean larvae are limited. Most adult hoplonemertean worms have a frontal organ located in a position corresponding to that of the larval apical organ. The development and sensory function of the frontal organ has not been thoroughly characterized to date. RESULTS: The apical organ in the early rudiment stage of Quasitetrastemma stimpsoni larvae consists of an apical plate enclosed by ducts of frontal gland cells and eight apical neurons. The apical plate is abundantly innervated by neurites of apical neurons. During the late rudiment stage, the larval apical organ has external innervation from below by two subapical-plate neurons, along with 11 apical neurons, and its plate contains serotonin-like immunoreactive (5-HT-lir) cells. In the vermicular stage (free-swimming juvenile), the number of apical neurons is reduced, and their processes are resorbed. Serotonin is detected in the apical plate with no visible connection to apical neurons. In adult worms, the frontal organ has a small apical pit with openings for the frontal gland ducts. The organ consists of 8 to 10 densely packed 5-HT-lir cells that form the roundish pit. CONCLUSIONS: Although the ultrastructure of the Q. stimpsoni larval apical organ closely resembles that of the apical organ of Polycladida larvae, the former differs in the presence of flask-shaped neurons typical of Spiralia. Significant differences in the structure of the apical organs of hoplonemertean and pilidia larvae point to two different paths in the evolutionary transformation of the ancestral apical organ. Ultrastructural and immunoreactive analyses of the apical organ of a hoplonemertean larva in the late rudiment and vermicular stages and the frontal organ of the adult worms identified common morphological and functional features. Thus, we hypothesize that the larval apical organ is modified during morphogenesis to form the adult frontal organ, which fulfills a sensory function in the hoplonemertean worm. This unique developmental trait distinguishes the Hoplonemertea from other nemertean groups.

Pseudocnidae of archinemerteans (Nemertea, Palaeonemertea) and their implications for nemertean systematics.

The structure of pseudocnidae of 16 species of Palaeonemertea clade Archinemertea (= Cephalotrichida s.l.) was investigated with confocal laser, scanning, and transmission electron microscopy (TEM). All species of the genus Cephalothrix possess two kinds of pseudocnidae, large and small. Only one type of pseudocnida is present in Balionemertes and Cephalotrichella. TEM revealed variation in the ultrastructure of large and small pseudocnidae of four species of Cephalothrix. Pseudocnidae of Balionemertes, Cephalotrichella, and Cephalothrix differ in substructure: in Balionemertes and Cephalotrichella the medulla is located in the basal half of the pseudocnidae with а precore layer situated in the apical half, whereas in Cephalothrix spp. and other palaeonemerteans the medulla surrounds a precore layer. Our results confirm the division of archinemerteans into Cephalotrichidae (with genus Cephalothrix) and Cephalotrichellidae (with genera Cephalotrichella and Balionemertes). The synapomorphy of Cephalotrichidae is pseudocnida dimorphism and the synapomorphies of Cephalotrichellidae are the position of the pseudocnidae on epithelial ridges and the distinct organization of pseudocnida layers, specifically the relative position of the medulla and precore layers. The pseudocnida lateral process, one or more of which is present in most species observed, is a probable synapomorphy of the clade Archinemertea. This is the first application of pseudocnida features to distinguish super-generic nemertean taxa and the results suggest that pseudocnidae provide a useful source of characters for nemertean systematics.

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.

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.

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.

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 Hubrechtella's 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).

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.