The first modern morphological description of Cercaria pennata and molecular evidence of its synonymy with Pronoprymna ventricosa in the Black Sea.

During the parasitological examination of molluscs Abra segmentum obtained from the Black Sea basin, parthenitae belonging to the family Faustulidae were found. The cercariae were obtained by natural emergence and were studied using differential interference contrast microscopy and scanning electron microscopy. Specimens resemble Cercaria pennata ex Tapes rugatus which was described from the Sevastopol area, in the shape and length of the body, tail length, location and shape of internal organs, suckers, pharynx, testicular rudiments, and the number and position of longitudinal lamellae on the tail finlets. To date, there are only limited descriptions of the parthenitae of C. pennata without detailed measurements, thus the taxonomic position of the individuals studied needs thorough revision and molecular verification. According to the molecular analyses, C. pennata was identical to that of published sequences of Pronoprymna ventricosa.

Adaptive potential of the Mediterranean mussel Mytilus galloprovincialis to short-term environmental hypoxia.

Environmental hypoxia naturally occurs in coastal ecosystems and bivalve mollusks have to frequently face fluctuations of dissolved oxygen concentrations. Exposure to hypoxia is often associated with the change of the antioxidant and functional status in bivalves, and restoration of the normal oxygen supply is considered to induce oxidative stress in tissues of mollusks. The study investigates changes in the activity of two antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD), as well as the expression level of SOD and CAT genes in gills of the Mediterranean mussel, Mytilus galloprovincialis, under exposure to low dissolved oxygen concentration (2.2 mg L-1) for 24 h and 72 h, and 24 h reoxygenation period. We also evaluated the intracellular level of reactive oxygen species (ROS), mortality and changes in mitochondrial membrane potential in hemocytes following hypoxia-reoxygenation cycle. 24 h exposure to hypoxia significantly decreased activity of both enzymes, which then recovered up to control levels at the end of 72 h experimental period for SOD and after reoxygenation for CAT. Expression of antioxidant enzyme genes was up-regulated following the 72 h hypoxic exposure period and returned to the basal normoxic level after 24 h reoxygenation. Hypoxia demonstrated a time-dependent effect on the functional state of hemocytes. The 24 h exposure period did not influence aerobic respiration of hemocytes, but prolonged hypoxia (72 h) was associated with a substantial decrease in mitochondrial membrane potential of hemocytes. The intracellular ROS level and mortality of hemocytes did not change under hypoxia. Reoxygenation period was accompanied with a significant decrease of intracellular ROS level. This study indicated that hypoxia did not induce the pronounced oxidative stress in gills and the changes in the antioxidant status were reversible within 24 h of reoxygenation. Hemolymph demonstrated a stable functional state indicating the tolerance of mussels to short-time hypoxia.

First morphological and phylogenetic data on Ligophorus kaohsianghsieni (Platyhelminthes: Monogenea) from the Black Sea and the Sea of Japan and molecular evidence of deep divergence of sympatric Ligophorus species parasitizing Planiliza haematocheilus.

Ligophorus kaohsianghsieni (Gusev, ) Gusev, was collected from the so-iuy mullet Planiliza haematocheilus (Temminck & Schlegel, 1845) from the Black Sea and the Sea of Japan. DNA sequences data for L. kaohsianghsieni, as well as its morphological characters from the Sea of Japan were obtained for the first time. Significant morphometric and genetic diversity between specimens of L. kaohsianghsieni from the Black-Azov Sea region and the Sea of Japan were not found. For the first time, the molecular phylogeny of L. kaohsianghsieni based on three fragments of the nuclear DNA ribosomal cluster (18S, internal transcribed spacer 1 and 28S) was reconstructed. Molecular analysis of Ligophorus species from the Atlantic and Pacific Oceans revealed a significant phylogenetic distance between L. kaohsianghsieni and two others species (Ligophorus pilengas and Ligophorus llewellyni) from the same host (P. haematocheilus) and region. This result indicates the lack of correspondence between the phylogenetic and geographical closeness of the hosts and the relation of their parasites from the genus Ligophorus.

Molecular-genetic approaches to species identif ication of platyhelminthes of the genus Ligophorus (Monogenea) parasitising f lathead mullet.

Mugil cephalus L., 1758 (flathead mullet) is a valuable commercial fish and a promising object of artificial breeding in the Black Sea and the Sea of Azov, and the study of its parasite fauna is important for fishery and mariculture. Monogeneans of the genus Ligophorus are common ectoparasites dwelling on the gills of mullets. Two representatives of this genus parasitise flathead mullet in the Azov-Black Sea region, namely Ligophorus mediterraneus Sarabeev, Balbuena et Euzet, 2005 and Ligophorus cephali Rubtsova, Balbuena, Sarabeev, Blasco- Costa et Euzet, 2006. Morphological identification of these species requires spending much time and a high level of experience in monogenean taxonomy. For quick and correct species identification of these parasites, we have developed a genotyping approach based on the polymerase chain reaction of allele-specific gene sites for various Monogenea species. A fragment of the 28S ribosomal gene, which includes conserved and variable sites, was chosen as a genetic marker. Three approaches were used as follows: amplified fragment length analysis, allelespecific PCR with endpoint detection and allele-specific real-time PCR using SYBR Green intercalating dye. The first approach was by obtaining PCR products of different lengths that were specific either to L. mediterraneus or to L. cephali. This approach was implemented due to the presence of several variable sites located at a distance from each other. The PCR mixture contained three primers: one forward and two reverse. The forward primer was complementary to the conserved site, which did not differ between species. Reverse primers were speciesspecific and, for each species, they were complementary to different DNA regions located 100 bp apart. As a result, L. mediterraneus was characterized by shorter amplicons than L. cephali. For the second and third approaches, a pair of primers was designed according to the following principle: the forward primer was complementary to both species, since it was selected for the conserved gene region. Reverse primers were species-specific and were designed for the 28S variable region. The two parasite species were distinguished by three-point mutations. Thus, one pair of primers was complementary to L. mediterraneus, the other, to L. cephali. The amplified fragment length analysis and the allele-specific real-time PCR demonstrated 100 % coincidence of genotyping results compared with Sanger sequencing. The developed genotyping protocols can be used not only to distinguish two species of Ligophorus from flathead mullet in ecological studies and veterinary practice but also for further development of similar approaches for other monogeneans, among which there are many pathogenic species.

Morphological and genetic variability of the cryptic Gyrodactylus sphinx and Gyrodactylus gerasevi n. sp. (Platyhelminthes: Monogenea) from the Mediterranean Sea and Black Sea: two new members of the cross-ocean distributed Gyrodactylus orecchiae species group.

Gyrodactylus sphinx Dmitrieva & Gerasev, 2000 is the only species of Gyrodactylus originally described from Aidablennius sphynx (Valenciennes) in the Black Sea. In the present study, monogeneans similar to G. sphinx are reported from the same host and from two other species of Blenniidae from the Black Sea, as well as from the Mediterranean Sea. This study aims to verify the taxonomic status of the specimens found in different hosts and localities, other than the type ones of G. sphinx. Twenty-two measurements of the haptoral structures of 169 gyrodactylids were used for the morphological study. Morphometric variability between different samples was analysed using analysis of variance, multivariate analysis of variance (MANOVA) and principal component analysis (PCA). Molecular studies were carried out using the nuclear internal transcribed spacer 2 and 5.8S ribosomal DNA regions. Network, Bayesian phylogenetic and species delimitation analyses were performed to infer the number of taxonomic units and the phylogeographic relationships occurring within and among them. MANOVA revealed a significant dependence of the morphometry of hamuli and marginal hooks on host species and regions, but a clear differentiation between samples was not confirmed by PCA. Moreover, the ranges of all dimensions overlapped between samples. However, molecular analyses suggested the occurrence of at least two taxonomic entities. The most common entity was present in individuals of the Black and Mediterranean seas, and is described here as Gyrodactylus gerasevi n. sp., whereas a second entity recognized as a G. sphinx was found only in individuals from two localities off Crimea. The monophyletic cluster grouping of these two species was placed within a large clade that also included a separate sister cluster with seven other species of the Gyrodactylus orecchiae cross-ocean species group.

Identification and characterization of the novel genes encoding glutathione S-transferases in Mytilus galloprovincialis.

The superfamily of glutathione S-transferases (GST) plays an essential role in the xenobiotic metabolism, binding compounds to the glutathione, and is like a cell protector during the influence of various negative external factors. Nevertheless, there are very few works devoted to the investigation of these genes in marine invertebrates. Up to this time, only three classes of cytosolic GSTs for one of the leading commercial molluscs Mytilus galloprovincialis were described. We sequenced the whole transcriptome from the gill tissues and, using bioinformatic analysis, detected ten classes of glutathione S-transferases, which are expressed in the mussel M. galloprovincialis. For the first time, two subfamilies were described: mitochondrial GST (kappa class) and microsomal (MAPEG), as well as five classes of the family of cytosolic GSTs (mu, omega, rho, tau, theta). Omega and sigma GST classes might be rapidly regulated genes due to the lack of introns and this assumption was confirmed by the investigation of short-term hypoxia on M. galloprovincialis. Seven new classes of GST revealed a greater gene variety of this detoxifying enzyme in mussels than expected. The obtained nucleotide sequences are necessary for future investigations of GSTs expression in response to various external factors (pollution, oxygen starvation, infection, etc.).

First rDNA sequence data for Haplosplanchnus pachysomus (Digenea: Haplosplanchnidae) ex Mugil cephalus from the Black Sea, and molecular evidence for cryptic species within Haplosplanchnus pachysomus (Digenea: Haplosplanchnidae) in Palaearctic and Indo-West Pacific regions.

Adult trematodes, morphologically similar to Haplosplanchnus pachysomus (Eysenhardt, 1829), were extracted from the intestine of Mugil cephalus Linnaeus, 1758, collected in the Black Sea basin. Morphological, morphometric and 28S ribosomal DNA (rDNA) partial sequence data were obtained for these trematodes following comparative analysis with previous data on this species. Worms from this study were morphologically identical to all previously reported H. pachysomus specimens from different locations. The results of the morphometric analysis indicated general similarity between H. pachysomus from the Black Sea and trematodes from Vietnam and Australia. Trematodes from the Black Sea and specimens from Spain were identical based on 28S rDNA partial sequences; however, these sequences differed from that of H. pachysomus from Vietnam and Australia by eight fixed substitutions. Overall, our results indicate that H. pachysomus from Spain and the Black Sea and from Vietnam and Australia can presently be considered as two cryptic species, one in Palaearctic and one in Indo-West Pacific regions. Our results provide a molecular base for including Haplosplanchnus purii in the genus Provitellotrema or to consider H. purii, P. crenimugilis and H. pachysomus within the same genus.

Acute hypoxic exposure: Effect on hemocyte functional parameters and antioxidant potential in gills of the pacific oyster, Crassostrea gigas.

Bivalve mollusks are frequently subjected to fluctuations of dissolved oxygen concentration in the environment which can represent a significant threat to bivalve antioxidant status. In this work the effects of hypoxia on hemocyte reactive oxygen species (ROS) production and level of mitochondrial potential as well as the activity and expression level of catalase (CAT) and superoxide dismutase (SOD) in gills of Crassostrea gigas were investigated after 24 h and 72 h exposure. 24 h hypoxia promoted an increase of mitochondrial membrane potential in agranulocytes and induced ROS accumulation in granulocytes. 72 h exposure substantially decreased hemocyte mitochondrial potential and intracellular ROS level in all hemocyte types. No significant changes in the activity of CAT in gills were observed following both 24 h and 72 h exposure periods compared to control. SOD activity in gills decreased after 72 h exposure to hypoxia but did not change under 24 h hypoxia. Significant up-regulation of SOD gene and no changes in expression level of CAT were observed in all experimental groups. The results indicate an overall shift in antioxidant status in gills and hemocytes of the Pacific oyster that may act as compensatory mechanisms to maintain redox homeostasis after a short-term (24 h) exposure and represent the occurrence of oxidative stress conditions at the end of 72 h hypoxia.