Evaluation of Microbial Contamination in Bivalve Mollusks: Epidemiology and Diagnosis

Shellfish are filter-feeding organisms that can accumulate many bacteria and viruses. Considering that depuration procedures are not effective in removal of certain microorganisms, shellfish-borne diseases are frequent in many parts of the world, and their control must rely primarily on investigation of prevalence of human pathogens in shellfish and water environment. However, the diffusion of enteric viruses and Vibrio bacteria is not known in many geographical areas, for example in Sardinia, Italy. A survey aimed at investigating the prevalence of Norovirus (NoV), hepatitis A virus (HAV), V. parahaemolyticus, V. cholerae and V. vulnificus was carried out, analyzing both local and imported purified, non-purified and retail shellfish from North Italy and Sardinia. Shellfish from both areas were found contaminated by NoVs, HAV and Vibrio, including retail and purified animals. Molecular analysis evidenced different NoV genogroups and genotypes, including bovine NoVs, as well as pathogenic Vibrio strains, underlining the risk for shellfish consumers. However, also other approaches are needed to control the diffusion of shellfish-borne diseases. It was originally thought that enteric viruses are passively accumulated by shellfish. Recently, it was proven that NoVs bind to specific carbohydrate ligands in oysters, and various NoV strains are characterized by a different bioaccumulation pattern. To deepen the knowledge on this argument, a study was carried out, analyzing bioaccumulation of up to 8 different NoV strains in four different species of shellfish. Different bioaccumulation patterns were observed for each shellfish species and NoV strain used, potentially important in setting up effective shellfish purification protocols. Finally, a novel study of evaluation of viral contamination in shellfish from the French Atlantic coast was carried out following the passage of Xynthia tempest over Western Europe which caused massive destruction. Different enteric viruses were found over a one month period, evidencing the potential of these events of contaminating shellfish.

Characterization of mitochondrial genomes in bivalve species with doubly uniparental inheritance of mitochondria

Many bivalve species possess two distinct mtDNA lineages, called F and M, respectively inherited maternally and paternally: this system is called doubly uniparental inheritance (DUI). The main experimental project of my PhD was the quantification of the two mtDNAs during the development of the DUI species Ruditapes philippinarum, from early embryos to sub-adults, using Real-Time qPCR. I identified the time interval in which M mtDNA is lost from female individuals, while it is retained in males (which are heteroplasmic through all of their life cycle). The results also suggested absence of mtDNA replication during early embryogenesis, a process constituting a bottleneck that highly reduces the copy number of mtDNA molecules in cells of developing larvae. In males this bottleneck may produce cells homoplasmic for M mtDNA, and could be considered as a first step of the segregation of M in the male germ line. Another finding was the characterization, in young clams approaching the first reproductive season, of a significant boost in copy number of F mtDNA in females and of M in males. Given the age of animals in which this mtDNA-specific growth was observed, the finding could probably be the outcome of the first round of gonads and gametes production. Other lines of research included the characterization of the unassigned regions in mt genomes of DUI bivalves. These regions can harbor signals involved in the control of replication and/or transcription of the mtDNA molecule, as well as additional open reading frames (ORFs) not related to oxidative phosphorylation. These features in DUI species could be associated to the maintenance of separate inheritance routes for the two mtDNAs. Additional ORFs are also found in other animal mt genomes: I summarized the presence of gene duplications as a co-author in a review focusing on animal mt genomes with unusual gene content.

An Aquaculture-Based Method for Calibrated Bivalve Isotope Paleothermometry

To quantify species- specific relationships between bivalve carbonate isotope geochemistry ( delta O-18(c)) and water conditions ( temperature and salinity, related to water isotopic composition [delta O-18(w)]), an aquaculture-based methodology was developed and applied to Mytilus edulis ( blue mussel). The four- by- three factorial design consisted of four circulating temperature baths ( 7, 11, 15, and 19 degrees C) and three salinity ranges ( 23, 28, and 32 parts per thousand ( ppt); monitored for delta O-18(w) weekly). In mid- July of 2003, 4800 juvenile mussels were collected in Salt Bay, Damariscotta, Maine, and were placed in each configuration. The size distribution of harvested mussels, based on 105 specimens, ranged from 10.9 mm to 29.5 mm with a mean size of 19.8 mm. The mussels were grown in controlled conditions for up to 8.5 months, and a paleotemperature relationship based on juvenile M. edulis from Maine was developed from animals harvested at months 4, 5, and 8.5. This relationship [ T degrees C = 16.19 (+/- 0.14) - 4.69 (+/- 0.21) {delta O-18(c) VPBD - delta O-18(w) VSMOW} + 0.17 (+/- 0.13) {delta O-18(c) VPBD - delta O-18(w) VSMOW}(2); r(2) = 0.99; N = 105; P < 0.0001] is nearly identical to the Kim and O'Neil ( 1997) abiogenic calcite equation over the entire temperature range ( 7 - 19 degrees C), and it closely resembles the commonly used paleotemperature equations of Epstein et al. ( 1953) and Horibe and Oba ( 1972). Further, the comparison of the M. edulis paleotemperature equation with the Kim and O'Neil ( 1997) equilibrium- based equation indicates that M. edulis specimens used in this study precipitated their shell in isotopic equilibrium with ambient water within the experimental uncertainties of both studies. The aquaculture- based methodology described here allows similar species- specific isotope paleothermometer calibrations to be performed with other bivalve species and thus provides improved quantitative paleoenvironmental reconstructions.

Stable isotopic ratios, shell lengths and organochlorine compounds in four bivalve species collected in July 2009 around Svalbard

Organochlorine compounds (OC) were determined in Arctic bivalves (Mya truncata, Serripes groenlan-dicus, Hiatella arctica and Chlamys islandica) from Svalbard with regard to differences in geographic location, species and variations related to their size and age. Higher chlorinated polychlorinated biphenyls (PCB 101-PCB 194), chlordanes and alpha-hexachlorocyclohexane (alpha-HCH) were consistently detected in the bivalves and PCBs dominated the OC load in the organisms. OC concentrations were highest in Mya truncata and the lowest in Serripes groenlandicus. Species-specific OC levels were likely related to differences in the species' food source, as indicated by the d13C results, rather than size and age. Higher OC concentrations were observed in bivalves from Kongsfjorden compared to the northern sampling locations Liefdefjorden and Sjuoyane. The spatial differences might be related to different water masses influencing Kongsfjorden (Atlantic) and the northern locations (Arctic), with differing phytoplankton bloom situations.

Surface sediment analyses, bivalve and gastropod taxa of marine sediments from the Banc d'Arguin, Mauritania

High-nutrient tropical carbonate systems are known to produce sediments that, in terms of skeletal composition, are reminiscent of their extra-tropical counterparts. Such carbonate systems and associated carbonate grain assemblages in the tropics are rare in the present-day world. Nonetheless, it is crucial to gain a better understanding of those ecosystems, including their drivers and players because such settings potentially represent models for ancient depositional systems as well as for predicted future environmental conditions. One of the modern occurrences of eutrophic tropical carbonate systems is the northern Mauritanian Shelf. The marine environment is characterized by an eastern boundary upwelling system that pushes cool and nutrient-rich intermediate waters onto a wide epicontinental platform (Golfe d'Arguin) where the waters warm up to tropical temperatures. The resulting facies is mixed carbonate-siliciclastic with a dominant foramol association grading into bimol and barnamol grain assemblages in the shallowest areas forming the Banc d'Arguin. Besides this cool water-related heterozoan association, the carbonate sediment is characterized by tropical molluskan species, while chlorozoan biota (e.g., corals and algal symbiont-bearing foraminifers) are entirely absent. We here present a first comprehensive facies analysis of this model example of eutrophic tropical carbonates. Furthermore, we reconstruct the loci of carbonate production and provide a conclusive depositional model of the Banc d'Arguin that received little attention to date due to its poorly accessible nature.

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

Phenotypic evolution in a venerid bivalve species lineage from the late Middle Miocene Central Paratethys Sea: a multi-approach morphometric analysis

A morphometric analysis was performed for the late Middle Miocene bivalve species lineage of Polititapes tricuspis (Eichwald, 1829) (Veneridae: Tapetini). Specimens from various localities grouped into two stratigraphically successive biozones, i.e. the upper Ervilia Zone and the Sarmatimactra Zone, were investigated using a multi-method approach. A Generalized Procrustes Analysis was computed for fifteen landmarks, covering characteristics of the hinge, muscle scars, and pallial line. The shell outline was separately quantified by applying the Fast Fourier Transform, which redraws the outline by fitting in a combination of trigonometric curves. Shell size was calculated as centroid size from the landmark configuration. Shell thickness, as not covered by either analysis, was additionally measured at the centroid. The analyses showed significant phenotypic differentiation between specimens from the two biozones. The bivalves become distinctly larger and thicker over geological time and develop circular shells with stronger cardinal teeth and a deeper pallial sinus. Data on the paleoenvironmental changes in the late Middle Miocene Central Paratethys Sea suggest the phenotypic shifts to be functional adaptations. The typical habitats for Polititapes changed to extensive, very shallow shores exposed to high wave action and tidal activity. Caused by the growing need for higher mechanical stability, the bivalves produced larger and thicker shells with stronger cardinal teeth. The latter are additionally shifted towards the hinge center to compensate for the lacking lateral teeth and improve stability. The deepening pallial sinus is related to a deeper burrowing habit, which is considered to impede being washed out in the new high-energy settings.

Bivalve mollusks in the sediments of the Anapa Bay Bar (Black Sea) in October 2010

Sandy beaches of the Anapa Bay Bar are a unique natural resource, but they are gradually being degrade under both natural and anthropogenic factors. Emissions of sand and shelly ground from the adjacent sea bottom partly compensate for this process. Concentration of carbonates may reach up to 50% in beach sands, and most of these carbonates are of mollusk origin. The major deposit formation role belongs to the key bivalve species: Chamelea gallina (Linnaeus, 1758). Average biomass of this mollusk species reaches up to 450 g/m**2 at depths 5-10 m. The other two subdominating mollusk species, bivalve Donax trunculus (Linnaeus, 1758) and gastropod Rapana venosa (Valenciennes, 1846), may impact as 16 g/m**2 and 6 g/m**2, respectively. Annually, 350 kg of shelly ground per running meter are newly deposited on the Anapa beach.