Milky hemolymph syndrome (MHS) associated with a virus in tanner crab Chionoecetes bairdi off the Pacific coast of Kamchatka.

Milky hemolymph syndrome (MHS) caused by a bacilliform virus (BV) was found in tanner crab Chionoecetes bairdi in the shelf zone of Kamchatka. The prevalence of the disease varied from 0.18 to 1.02%. A total lesion of the cells of the interstitial connective tissue and the connective tissue component of all internal organs was noted, which was expressed in the hypertrophy of their nuclei. In addition, hypertrophy of fixed phagocytes and circulating hemocytes was noted. Ultrastructural analysis of the tissues confirmed that in the interstitial connective tissue of pathologically altered organs, virus particles of two morphotypes were found - rod-shaped and globular. In the cytoplasm of infected cells, bands of microtubules formed near where viral particles were concentrated. In the area of contacts at the poles of microtubules, successive stages of the transformation of rod-shaped viruses into globular viruses was observed. The bacilliform virus that infects C. bairdi is structurally very similar to CoBV found in Chionoecetes opilio. Structural features are characteristic of representatives of fam. Nimaviridae. The molecular data obtained suggest that the virus causing MHS in C. bairdi is systematically very close, if not identical, to CoBV.

First detection of Hematodinium sp. In spiny king crab Paralithodes brevipes, and new geographic areas for the parasite in tanner crab Chionoecetes bairdi, and red king crab Paralithodes camtschaticus.

A parasitic dinoflagellate of the genus Hematodinium was found off the Pacific coast of Kamchatka in three species of crabs: red king crab Paralithodes camtschaticus, tanner crab Chionoecetes bairdi, and spiny king crab Paralithodes brevipes. This is the first detection of Hematodinium in spiny king crab. The results of the genetic analysis showed that the pathogen found in P. brevipes, P. camtschaticus, and C bairdi from the Avacha and Kronotsky bays off the Pacific coast of Kamchatka was the same or very close to the Hematodinium sp., which infects many species of crustaceans in the Northern Hemisphere. The prevalence of infection was 0.2% for tanner crabs and 2.7% for red king crabs. Due to a limited sample size, we were unable to calculate the prevalence for spiny king crabs and female red king crabs. Both the macroscopic and microscopic signs of the pathology were similar in all diseased crabs. The differences in the micromorphology of the Hematodinium cells we found in the three crab species, including the presence or absence of trichocysts, the shape of the plasmodia, and the structure of pore complexes, are most likely related to the life cycle and the physiology of the parasite. The results of the genetic analysis showed that the pathogen found in P. brevipes, P. camtschaticus, and C. bairdi from the Avacha and Kronotsky bays of the Pacific coast of Kamchatka was the same or very close to the Hematodinium sp., which infects many species of crustaceans in the Northern Hemisphere.

First report of bacterial kidney disease in coho salmon Oncorhynchus kisutch in Russia.

This paper describes the first case of bacterial kidney disease (BKD) to be identified in coho salmon Oncorhynchus kisutch in Russia. The fish in question was caught in Lake Bolshoi Vilyui on the Kamchatka Peninsula. The diseased fish had foci of granulomatous inflammation in the kidneys. The diagnosis was confirmed by isolating the bacterium Renibacterium salmoninarum from kidney tissue in pure culture, and by determining the partial 16S RNA gene sequence of the isolate. This is the first detection of this pathogen in the genus Oncorhynchus in Russia, and detection of BKD in coho salmon indicates that the pathogen is present in the natural fish populations of Kamchatka. Therefore, it will be necessary to conduct screening studies of mature salmon selected for artificial reproduction, for the presence of BKD signs and asymptomatic infection with R. salmoninarum, which will allow us to estimate the prevalence of the pathogen.

First record of new rickettsia-like organism in the blue king crab Paralithodes platypus from the Sea of Okhotsk: Distribution, morphological evidence and genetic analysis.

An infection caused by a rickettsia-like organism (RLO) was detected in the blue king crab Paralithodes platypus from the eastern Sea of Okhotsk. The external signs of the disease are lethargy and an empty gastrointestinal tract. Dissection of infected individuals revealed that their hepatopancreas was light yellow in color. The causative agent of infection is Gram-negative rod-shaped bacterium targeted exclusively at hepatopancreas tissues. In the cytoplasm of infected cells, the bacteria are enclosed in parasite vacuoles or located immediately in cytosol. An ultrastructural analysis showed two main morphological types corresponding to the life cycle stages in the RLO: the vegetative stage of intermediate bodies, characterized by growth and division processes, and the infection stage of elementary bodies, which are spore-like non-dividing short rods surrounded by a multilayered membrane and having an osmiophilic inclusion body. At the terminal stage of infection, as a result of lysis of the infected cells, the RLO enters the lumen of the hepatopancreatic tubules which contributes to the spread of infection. According to genetic analysis based on 16S rRNA gene sequences, the RLO from P. platypus is most closely related to the Candidatus Hepatobacter penaei, NCBI #JX981946 (94.7% similarity) and NCBI #KY363553 (94.1% similarity). The high level of genetic differences (more than 5%) of the studied pathogen, along with the structural features, allows characterizing the RLO isolated from P. platypus as a new species of the genus Candidatus Hepatobacter paralithodi nov. sp., NCBI #MK928971.

Metalloproteinase inhibitor GM6001 delays regeneration in holothurians.

The effect of the GM6001 metalloproteinase inhibitor on the regeneration of ambulacral structures in Eupentacta fraudatrix has been investigated. Inhibition of proteinase activity exerts a marked effect on regeneration, being dependent on the time when GM6001 is injected. When administration of the inhibitor begins on day 3 post-injury, regeneration is completely abolished, and the animals die. This means that early activation of proteinases is crucial for triggering the regenerative process in holothurians. When GM6001 in first injected on day 7 post-injury, the regeneration rate decreases. However, this effect has proven to be reversible: when inhibition ceases, the regeneration resumes. The effect of the inhibitor is manifested as a retarded degradation of the extracellular matrix, the lack of cell dedifferentiation, and, probably, a slower cell migration. The gelatinase activity is detected in all the regenerating organs of E. fraudatrix. In the holothurian Cucumaria japonica, which is not capable of healing skin wounds and ambulacrum reparation, no gelatinase activity was observed at the site of damage. A suggestion is made that proteinases play an important role in regeneration in holothurians. The most probable morphogenesis regulators are matrix metalloproteinases with gelatinase activity.

The ultrastructural features of embryonic and early larval development in Yesso scallop, Mizuhopecten yessoensis.

Fine structural features of Mizuhopecten yessoensis in different embryonic and larval developmental stages from oocyte to early veliger have been studied. Spermatozoa have a conical head, consisting of a short acrosome, nucleus, and 4 mitochondria in the middle piece. The flagellum is approximately 40 µm in length. Oocyte cytoplasm contains numerous yolk granules, which are gradually expended during the larval development and disappear at the early veliger stage. Gastrula has two invaginations: shell gland anlage on the dorsal side and ventral side archenteron. Prototroch of a trochophore consists of two ciliary rings. Telotroch is absent. Myoblasts containing bundles of myofilaments are found in the blastocoel of trochophore. Velum and shell begin forming at the late trochophore stage. Cells of gut epithelium form numerous microvilli, organized into a brush border structure on the surface of enterocytes. A suggestion is made that larvae at the late trochophore stage are capable of digesting food and feeding. The structure of a M. yessoensis veliger is typical for pectinids. The velar retractor muscles have cross striation with a period of 1 µm. The digestive system of an 8-day-old veliger consists of the esophagus, stomach (with subdivided gastric shield and style sac), and short intestine. The digestive gland (with no discernible loops at this stage) is a part of the stomach epithelium with two types of digestive enzyme-producing cells.

A new host for Hematodinium infection among lithodid crabs from the Sea of Okhotsk.

A disease caused by parasitic dinoflagellates of the genus Hematodinium has been found in the soft crab Hapalogaster grebnitzkii from the Sea of Okhotsk, which is considered a new host for this agent among lithodid crabs. This report provides macro- and micromorphological descriptions (using light and transmission electron microscopy) of the disease, as well as molecular identification of Hematodinium sp. from soft crabs, based on 18S RNA sequence data.

Assessment of the toxic effect exerted by fluorescent pseudomonads on embryos and larvae of the sea urchin Strongylocentrotus nudus.

Strains of bacteria capable of growing on artificial culture media were isolated from the fouling of brass plates submerged in Nha Trang Bay, South China Sea, and from tissues of the seastar Distolasterias nipon, caught in Peter the Great Bay, Sea of Japan. According to the complex of data of genetic and physiological/biochemical analyzes, two strains of cultivated bacteria were identified by us as the species Pseudomonas aeruginosa, two strains as Pseudomonas fluorescens, and one strain as Ruegeria sp. It was shown that the cultivated strains of P. aeruginosa released exotoxins, particularly phenazine pigments, into the environment. Production of the toxins did not depend on presence of a target organism in the system and was aimed at regulation of interactions in the microbial community. The toxicity of the studied natural isolates of fluorescent pseudomonads was analyzed by using embryos and larvae of the sea urchin Strongylocentrotus nudus, which are the sensitive and dynamic toxicological sea-urchin embryo test (SET) system. As was established, exotoxins produced by the strains of P. aeruginosa inhibit activity of cilia in sea urchin larvae, as well as disturb processes of cell differentiation in embryos and larvae. Their toxic influence is accompanied by disturbances of protein synthesis and the disruptions of cytoskeleton in the course of zygote cleavage and larval development. Unlike P. aeruginosa, the strains of P. fluorescens and Ruegeria sp. did not exert the toxic effect on SET. The obtained data allow considering objects of the environment as the natural reservoir of opportunistic microorganisms posing a potential threat to human, whereas the use of SET for determination of toxicity of isolated bacteria provides an opportunity to study the mechanisms of their interactions with organisms in marine ecosystems.

A herpes-like virus in king crabs: Characterization and transmission under laboratory conditions.

A herpes-like virus was found infecting the antennal gland and bladder epithelium in the blue king crab Paralithodes platypus from the eastern area of the Sea of Okhotsk. Electron microscopic analysis of antennal gland samples from blue king crabs with histologically confirmed signs of disease revealed virus particles, which were mostly hexagonal in shape and located primarily in the nucleus; these particles were rarely observed in the cytoplasm of infected cells. Most virus particles ranged in size from 115 to 125nm. Hemocytes of the red king crab Paralithodes camtschaticus in cell culture could be experimentally infected with virus from thawed antennal gland samples of the blue king crabs with histologically confirmed signs of viral infection. Clear signs of infection were observed in hemocyte cultures at 3-4days post-inoculation as small foci of highly vacuolated formations. These formations included several nuclei and were surrounded by a halo of small cytoplasmic bubbles containing actin and tubulin. As demonstrated by electron microscopic studies, no virus-like particles were found in the cells 1day post-inoculation, but particles become abundant at 7days post-inoculation. We developed a consensus primer PCR method for amplification of a region of the herpesviral DNA-directed DNA polymerase. Primers were designed to target sequences encoding highly conserved amino acid motifs covering a region of approximately 800bp. Thus, macroscopic, histological and ultra-structural examinations of blue king crabs infected with a virus and the molecular identification of the pathogen revealed the presence of herpesviruses. The frequency of the herpes-like viral infection in natural populations of blue king crabs in the Sea of Okhotsk ranged from 0% to 3% in different years.

PUMA protein in p53 regulatory molecule pattern determines the prognosis for patients with lymphoproliferative diseases.

The pool of apoptosis regulator proteins (p53, PUMA, p21, and MDM2) in the bone marrow, lymph node, and other tumor substrate cells were studied by immunocytochemical analysis in patients with chronic lymphoproliferative diseases. Two groups of patients were distinguished, with the disease course differing by activities of the studied molecules. Activity of p53 was the minimum in the group with benign course of the disease, and protein p21 was not detected; MDM2 protein was present in moderate amounts in the presence of high PUMA activity. High activities of p53, p21, and MDM2 and low PUMA activity were found in tumor cells of patients with malignant disease. These data can serve as a prognostic sign determining the aggressiveness of chronic lymphoproliferative disease course and for determining the strategy of chemotherapy and its correction.

Apoptosis regulator proteins: basis for the development of innovation strategies for the treatment of rheumatoid arthritis in patients of different age.

Apoptosis markers (p53, PUMA, p21, and Mdm2) were studied in patients with rheumatoid arthritis at the early and late stages of disease on bone marrow smears and frozen sections of the articular synovial membrane. The target molecules were found in all patients. The early stage was characterized by maximum expression of antiapoptotic (Mdm2) molecule and the minimum expression of proapoptotic molecules (p53, PUMA, and p21). The proapoptotic proteins predominated in patients with the late stage of rheumatoid arthritis, while the antiapoptotic factor (Mdm2) was depressed significantly. These data suggested total suppression of apoptosis at the early stage of rheumatoid arthritis and recommended the Mdm2 molecule as a prospective target for the development of new drugs.

Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia.

A disease caused by a parasitic dinoflagellate of the genus Hematodinium was identified in red, Paralithodes camtschaticus, and blue, Paralithodes platypus, king crabs from the north-east region of the Sea of Okhotsk, Russia, during annual stock surveys. No carapace color change was observed even in heavily infected crabs, but diseased crabs possessed creamy-yellow hemolymph, which was visible through the arthrodial membranes of the abdomen and appendages. Several stages of the parasite's life history, including trophonts, plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. Numerous parasite cells were observed in the lumina of the myocardium, the gills, the connective tissue of antennal glands and the sinuses of nerve ganglia, eyestalks and gastrointestinal tract of king crabs with gross signs of infection. Based on sequencing of the 18S rDNA, it appears that the Hematodinium sp. found in red and blue king crabs is identical or closely related to Hematodinium sp. isolated from crabs of the genera Chionoecetes and Lithodes. Observed prevalences were 0.33% in sublegal male red king crabs, 0.18% in female red king crabs, 0.34% in sublegal male blue king crabs and 0.31% in female blue king crabs.

[Collation of data on the ploidy levels and mitochondrial DNA phylogenetic lineages in the silver crucian carp Carassius auratus gibelio from Far Eastern and Central Asian populations].

The distribution of the diploid and triploid forms and the correspondence between ploidy and mitochondrial DNA (mtDNA) phylogenetic lineages of the silver crucian carp have been studied in Far Eastern water bodies and the Syr Darya River. Both diploid and triploid forms have been found in large river systems (the Amur, Suifun, Tumangan, and Syr Darya river basins). Only the diploid form has been detected in lakes of Bol'shoi Pelis Island (Peter the Great Bay of the Sea of Japan), Sakhalin Island, and the Kamchatka River basin (the Kamchatka Peninsula). It has been confirmed that there are two mtDNA phylogroups in the silver crucian carp in the area studied. Both mtDNA phylogenetic lineages are present in the Suifun and Tumangan river basins. Only one mtDNA phylogroup (characteristic of the gynogenetic form) has been detected in two samples from the Amur River and in the Syr Darya population. The other mtDNA phylogroup is predominant in insular populations and in Kamchatka. The gynogenetic form carries only mtDNA phylogroup I, whereas both phylogroups have been found in diploid bisexual fish. The existence of only two mtDNA phylogroups substantially differing from each other indicates that the gynogenetic form has emerged from the diploid form only once and evolved independently for a long time after that. The absence of haplotypes transitional between the two mtDNA phylogroups suggests that the secondary contact between the gynogenetic and bisexual forms in continental populations occurred within recent historical time. The obtained data confirm that genetic (though asymmetric) exchange between the two forms is possible, which explains the high morphological and, probably, genetic similarity between them.

[A study of quantitative dynamics of F-actin during oocyte maturation in the starfish Asterias amurensis].

We studied the actin cytoskeleton state in Asterias amurensis oocytes within 30 min after the 1-methyladenine-induced maturation until the germinal vesicle breakdown. The total amount of actin remained unchanged during oocyte maturation. In immature oocytes, the major part of actin is not a part of filaments, but in the presence of 1-methyladenine massive actin polymerization began already within 20 min. Electron immunocytochemistry methods demonstrated joint localization of actin and alpha-protein in the cytoplasm. They were redistributed from the cortex to the cytoplasm in the presence of 1-methyladenine. A possible involvement of actin cytoskeleton in transmembrane transduction of the hormonal signal at the postreceptor stages is discussed.

[Mitochondrial DNA variation in diploid and triploid forms of silver crucian carp Carassius auratus gibelio].

Variation of two mtDNA fragments amplified in polymerase chain reaction was compared by the RFLP method in a population of the sympatric bisexual (diploid) and gynogenetic (triploid) forms of silver crucian carp. The mtDNA haplotypes of all individuals differed in at least 2.5% of nucleotide substitutions and fall into two phylogroups. All gynogenetic individuals have haplotypes of a single phylogroup, whereas haplotypes of diploid fish belonged to both mtDNA phylogroups. This testifies to a possibility of transformation of gynogenetic individuals into a bisexual form, whereas the reverse process is either extremely rare or impossible.

[Mitochondrial DNA variation in goldfish Carassius auratus gibelio from water reservoirs in the Far East]. / Izmenchivost' mitochondrial'noi DNK u serebrianogo karasia (Carassius auratus gibelio) v vodoemakh Dal'nego Vostoka.

MtDNA variation of goldfish samples from several water bodies of Southern Primorye was examined by RFLP analysis. High mtDNA polymorphism was found in the river populations but not in the lake ones. Considerable among-haplotype divergence was found within samples, which suggests periodic gene exchange between populations having long histories of independent evolution. The absence of substantial differences between clusters of mtDNA haplotypes indicates recurrent transfer from bisexual to gynogenetic reproduction mode and vice versa.

[Effect of 1-methyladenine on localization of Galpha1-protein in starfish oocytes]. / Vliianie 1-metiladenina na lokalizatsiiu Galpha1-belka v ootsitakh morskoi zvezdy.

Localization and quantitative dynamics of alpha i subunit of G protein was studied by electron immunocytochemistry and immunoenzyme assay after hormonal induction of maturation in Asterias amurensis starfish oocytes. G alpha i protein was chiefly localized in the plasma membrane of immature oocytes; 1-methyladenine induced redistribution of the alpha i protein from the plasma membrane to intracellular structure up to the germinal physical breakdown.

[Myogenic differentiation of Mytilus larval cells in vitro]. / Miogennaia differentsirovka lichinochnykh kletok midii in vitro.

A myogenic differentiation program can be realized during the cultivation of Mytilus trossulus cells derived from larvae in premyogenic developmental stages. About 10-15% of cells in such cultures showed that they are capable of contracting actively. The shape of such cells and the high concentration of actin microfilaments indicate a similarity with smooth muscle cells. However, the pattern of contractile activity and the protein composition of these cells differ significantly from the corresponding characteristics of differentiated smooth muscle cells. The proportion between the main proteins of the thick fiber, paramyosin, and myosin in cultivated cells is far lower than in the muscles of larvae or adult molluscs. We also found that substrates with different adhesional characteristics may determine cell development towards one or the other phenotype. Cells attached to the collagen substrate, but not spread on it, had high proliferative potential; the collagen substrate, however, inhibited myogenic differentiation.

Isolation and properties of a mannan-binding lectin from the coelomic fluid of the holothurian Cucumaria japonica.

A new 44-kD, C-type mannan-binding lectin (MBL-C) consisting of two identical subunits was isolated from the coelomic fluid of the holothurian Cucumaria japonica. In the direct hemagglutination assay, the lectin was effectively inhibited by highly branched mannans similar in structure to yeast mannans and composed of alpha-(1-->2)- and alpha-(1-->6)-bound D-mannopyranose residues. Hemagglutination was not inhibited by mannosaccharides, common constituents of the hydrocarbon chains of "normal" glycoproteins. The lectin reaction depends on Ca2+ concentration: maximum activity of MBL-C is observed at 10 mM Ca2+. The activity of MBL-C increases in the pH range from 5 to 7 and reaches maximum at pH 7.0. The lectin is sensitive to temperature. Heating of the lectin solution at temperatures above 40 degrees C decreases activity, while incubation at 90 degrees C for 1 h leads to complete irreversible inactivation. Carbohydrate specificity, Ca2+-dependence, and amino acid composition indicate that MBL-C belongs to the C-type mannan-binding lectins. Polyclonal antibodies against MBL-C revealed its immunochemical similarity to a mannan-binding lectin from another holothurian species, Stichopus japonicus; this provides evidence for structural homology between these proteins.