Life cycle of tetrodotoxin-producing <Emphasis Type="Italic">Bacillus</Emphasis> sp. on solid and liquid medium: Light and electron microscopy studies

The lifecycle of the Bacillus sp. 1839 cultivated during a long period on solid and liquid Youschimizu-Kimura medium was investigated, and then bacteria and spores were studied by light and transmission electron microscopy. Sporulation in this strain is distinguished by engulfment of forespore by mother cell. In the liquid medium, bacteria have the decondensed nucleoid and the loose granular component of cytoplasm; bacteria and spores are generally smaller; the outer coat of spores includes 2 concentric rings. On the solid substratum, the nucleoid is condensed, and the cytoplasmic region is extensive and dense; a longer cultivation stimulates transition of vegetative cells into the spore form; spores have a thicker outer coat with 3–5 rings. On the solid substratum, sporulation in Bacillus sp. 1839 is spontaneous, without additional stimulation; spores have a larger diameter and thicker layers than those in the liquid medium. This research contributes to the current understanding of biotechnological tetrodotoxin production from a bacterial raw material.     

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). J. Morphol. 274:1397–1414, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructure of larval epidermis of the nemertean <Emphasis Type="Italic">Quasitetrastemma stimpsoni</Emphasis> (Chernyshev, 1992) (Hoplonemertea)

The ultrastructure of the epidermis of free-swimming larvae of the nemertean Quasitetrastemma stimpsoni was examined. At about 24 hours after hatching, the provisional epithelium of larva is 28–35 μm thick and consists of two layers of cells—peripheral and basal. The peripheral layer consists of multiciliated cells and two kinds of gland cells. The “basal cup” zone is formed from the vacuoles of basal cells. At about 50 hours after hatching, the definitive epithelium is 14–17 μm thick and exhibits a typical hoplonemertean structure. However, it has numerous yolk vesicles, few processes of granular basal cells, and a weakly developed dermis. Thus, the replacement of the provisional epithelium by the definitive one occurs in Q. stimpsoni at an earlier stage, compared to the hidden larva of Tetrastemma candidum.     

Physiological and Biochemical Characteristics of the Halophilic Bacteria Vibrio parahaemolyticus and V. alginolyticus Isolated from Marine Invertebrates of Peter the Great Bay, Sea of Japan

One hundred strains of halophilic vibrios were isolated from 16 species of marine invertebrates of Peter the Great Bay. Based on their morphological and biochemical characteristics, the bacteria were identified as Vibrio parahaemolyticus and Vibrio alginolyticus. Bacterial isolates possessed virulence enzymes (DNAase, lecithinase, catalase) and were characterized by a high enterotoxigenicity. It was determined that 76% of the V. parahaemolyticus strains and 43% of the V. alginolyticus strains were Kanagawa-positive. The isolates showed a high adhesive capability, the average adhesion index was 18.06 cells per erythrocyte for V. parahaemolyticus and 12.55 for V. alginolyticus. The results of this study suggest a high pathogenic potential of the isolated halophilic vibrios, which are an epidemic hazard to marine invertebrates and to humans.     

Rapid production of tetrodotoxin-like compounds during sporulation in a marine isolate <Emphasis Type="Italic">Bacillus</Emphasis> sp. 1839

The marine Bacillus sp. strain 1839 produces tetrodotoxin-like (TTX-like) compounds during sporulation. In this study, the environmental signals that regulate spore formation and the synthesis of TTX-like substances were examined. The main sporulation trigger for Bacillus sp. 1839 was changes in environmental osmolality. Confocal laser scanning (CLSM) and transmission electron microscopy with anti-TTX anti-bodies were used to locate TTX-like compounds in a sporulated culture of Bacillus sp. 1839. Based on CLSM studies, the toxin was synthesized within a few minutes after the induction of sporulation, and the fluorescence signal reached the maximum intensity in 60 min. Immunoelectron microscopy enabled the detection of TTX-positive structures in spores with unformed cores. The results obtained in this study provide an important basis for the development of biotechnological production methods for the highly effective anesthetic drug.     

The taxonomic composition,characteristics, and neurotoxic activities of ribbon worm-associated bacteria from the Sea of Japan

The taxonomic composition of bacteria associated with two species of tetrodotoxin-bearing (TTX-bearing) (Hubrechtella juliae and Lineus alborostratus) and two species of non-TTX-bearing (Quasitetrastemma stimpsoni and Malacobdella grossa) ribbon worms collected from the Peter the Great Bay of Sea of Japan was studied. Bacterial isolates were identified using 16S rRNA gene sequencing and phenotypic characteristics. Thirty-eight strains of heterotrophic bacteria from the eight genera: Pseudoalteromonas, Shewanella, Ruegeria, Pseudomonas, Defluviicoccus, Vibrio, Alteromonas, and Bacillus, were isolated and characterized. γ-Proteobacteria dominated among the associated microflora of nemerteans (76.3% of the total number of isolates). Sensitivity analysis of 38 strains to antibiotics of various classes revealed multiple resistance to three or more antibiotics in all of the studied isolates. The 15 bacterial strains isolated in the study exhibited antimicrobial activities against at least one of five indicator microorganisms, most of which corresponded to the Pseudoalteromonas genus. Screening of the TTX-producing bacteria was performed using confocal laserscanning microscopy and polyclonal antibodies. A TTX-producing strain, Pseudoalteromonas sp., was found in the nemertean H. juliae. A correlation between the presence of TTX-positive microflora and the toxicity of nemerteans was determined.     

The structure of the proboscis musculature in <Emphasis Type="Italic">Baseodiscus delineatus</Emphasis> (Delle Chiaje, 1825) (Heteronemertea) and the comparative analysis of the proboscis musculature in heteronemerteans

The proboscis musculature was studied in the nemertean Baseodiscus delineatus using confocal laser scanning and electron transmission microscopy. Three muscle layers were differentiated in the proboscis wall: the outer-longitudinal, the diagonal, and the inner-circular layer. The endothelium consists of two cell types: apical supportive cells with rudimentary cilia and subapical myocytes making up the inner-circular musculature of the proboscis. The supportive cells have thin processes attached to the basal extracellular matrix and their perikarya are spread over the apical surfaces of myocytes. The endothelium of B. delineatus is characterized by a folded basal layer of the extracellular matrix and by different heights of myocyte processes, giving an impression that the inner-circular musculature is multilayered. Comparative analysis shows that the diagonal musculature of Baseodiscus is not homologous to that of other heteronemerteans. An assumption is made that the inner-circular muscles have endothelial origin in all heteronemerteans.     

Coelomocyte Morphology in the Holothurians Apostichopus japonicus (Aspidochirota: Stichopodidae) and Cucumaria japonica (Dendrochirota: Cucumariidae)

The cellular composition of the coelomic fluid of the Far Eastern holothurinans Apostichopus japonicus and Cucumaria japonica was studied using light and transmission electron microscopy and histochemistry. In the coelomic fluid of A. japonicus, the following types of coelomocytes were distinguished: progenitor cells; amoebocytes; vacuolated cells; small (or young) morula cells; morula cells of type I, type II, and type III; crystal cells; and vibratile cells. In the coelomic fluid of C. japonicawere found progenitor cells, amoebocytes, vacuolated cells, morula cells of type I and type II, crystal cells, and hemocytes containing a respiratory pigment. The issue of stem cell type, which gives rise to coelomocytes, is discussed.     

Characterization,identification, and screening for tetrodotoxin production by bacteria associated with the ribbon worm (Nemertea) Cephalotrix simula (Ivata, 1952)

The taxonomic composition of bacteria associated with the proboscis worm Cephalothrix simula was studied and screening of the tetrodotoxin (TTX)-producing bacteria was carried out using confocal laser scanning microscopy and polyclonal antibodies. Bacterial isolates were identified using the 16S rRNA gene sequencing and phenotypic characteristics. A Bacillus species was found to be responsible for tetrodotoxin production in proboscis worm C. simula. Vibrio spp. dominated the associated microflora (68.18% of the total number of isolates). The analysis of sensitivity of 16 strains to antibiotics of various classes revealed multiple resistance to three or more antibiotics in all the studied isolates. Poor growth of most of the isolates on all the laboratory media was an indirect confirmation of the symbiotic relationships between the micro- and macroorganisms.     

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.