Permian non-marine bivalves of the Falkland Islands and their palaeoenvironmental significance

We describe the occurrence of non-marine bivalves in exposures of the Middle Permian (Capitanian) Brenton Loch Formation on the southern shore of Choiseul Sound, East Falklands. The bivalves are associated with ichnofossils and were collected from a bed in the upper part of the formation, within a 25 cm thick interval of dark siltstones and mudstones with planar lamination, overlain by massive sandstones. The shells are articulated, with the valves either splayed open or closed. At the top of the succession, mudstone beds nearly 1.5 m above the bivalve-bearing layers yielded well-preserved Glossopteris sp. cf. G. communis leaf fossils. The closed articulated condition of some shells indicates preservation under high sedimentation rates with low residence time of bioclasts at the sediment/water interface. However, the presence of specimens with splayed shells is usually correlated to the slow decay of the shell ligament in oxygen-deficient bottom waters. The presence of complete carbonized leaves of Glossopteris associated with the bivalve-bearing levels also suggests a possibly dysoxic-anoxic bottom environment. Overall, our data suggest that the bivalves were preserved by abrupt burial, possibly by distal sediment flows into a Brenton Loch lake, and may represent autochthonous to parautochthonous fossil accumulations. The shells resemble those of anthracosiids and are herein assigned to Palaeanodonta sp. aff. P. dubia, a species also found in the Permian succession of the Karoo Basin, South Africa. Our results confirm that (a) the true distributions in space and time of all Permian non-marine (freshwater) bivalves are not yet well known, and (b) there is no evidence for marine conditions in the upper part of the Brenton Loch Formation.

POPULATION BIOLOGY AND DIET OF THE SOUTHERN KINGCROAKER Menticirrhus americanus (LINNAEUS, 1758) (PERCIFORMES: SCIAENIDAE) IN CARAGUATATUBA BAY, SOUTHEASTERN BRAZIL

This study evaluated the spatio-temporal distribution, population biology and diet of Menticirrhus americanus in Caraguatatuba Bay. Samples were taken monthly between August 2003 and October 2004, by trawling in two previously selected areas. The northern area is more exposed to wave activity and is influenced by a river, functioning as a small estuary. In contrast, the southern area is relatively sheltered from wave energy and influenced to a lesser degree by smaller rivers. The fishes' length was measured, and the sex and gonadal stage macroscopically identified. The abundance of this species was compared between areas and among months. The diet was identified and quantified. M. americanus occurred in equal proportions in the two study areas, being most abundant in April 2004, followed by December 2003 and January 2004. The population was dominated by small immature individuals. The few individuals in maturation or mature that were captured showed no seasonal pattern of distribution. This species had a varied diet, feeding on worms (nemerteans, sipunculans and echiurans), mollusks (bivalves and cephalopods), polychaetes, crustaceans and fish. The presence of intact nematodes in the intestine suggests that these are parasites. The results demonstrated that M. americanus has a homogeneous spatial and temporal distribution in Caraguatatuba Bay, being uniformly distributed between the south and north areas as well as across the months. This species can be considered a carnivorous predator, showing a preference for consuming benthic sandy-beach species such as glycerids and other polychaetes, crustaceans, and bivalve siphons.

Evaluation of tropical water sources and mollusks in southern Brazil using microbiological, biochemical, and chemical parameters

Florianopolis, a city located in the Santa Catarina State in southern Brazil, is the national leading producer of bivalve mollusks. The quality of bivalve mollusks is closely related to the sanitary conditions of surrounding waters where they are cultivated. Presently, cultivation areas receive large amounts of effluents derived mainly from treated and non-treated domestic, rural, and urban sewage. This contributes to the contamination of mollusks with trace metals, pesticides, other organic compounds, and human pathogens such as viruses, bacteria, and protozoan. The aim of this study was to perform a thorough diagnosis of the shellfish growing areas in Florianopolis, on the coast of Santa Catarina. The contamination levels of seawater, sediments, and oysters were evaluated for their microbiological, biochemical, and chemical parameters at five sea sites in Florianopolis, namely three regular oyster cultivation areas (Sites 1, 2, and oyster supplier), a polluted site (Site 3), and a heavily polluted site (Site 4). Samples were evaluated at day zero and after 14 days. Seawater and sediment samples were collected just once, at the end of the experiment. Antioxidant defenses, which may occur in contaminated environments in response to the increased production of reactive oxygen species (ROS) by organisms, were analyzed in oysters, as well as organic compounds (in oysters and sediment samples) and microbiological contamination (in oysters and seawater samples). The results showed the presence of the following contaminants: fecal coliforms in seawater samples (four sites), human adenovirus (all sites), human noroviruses GI and GII (two sites), Hepatitis A viruses (one site), JC Polyomavirus in an oyster sample from the oyster supplier, Giardia duodenalis cysts, and Cryptosporidium sp oocysts (one site). Among organochlorine pesticides, only DDT (dichlorodiphenyltrichloroethane) and HCH (hexachlorocyclohexane) were detected in some sediment and oysters samples in very low levels; site 4 had the highest concentrations of total aliphatic hydrocarbons. PAHs, and linear alkylbenzenes (LABs) found either in oysters or in sediment samples. The major concentration of fecal sterol coprostanol was found at site 4, followed by site 3. After 14 days of allocation in the four selected sites, there was a significant difference in the enzymes analyzed at the monitored spots. The detection of different contaminants in oysters, seawater, and sediment samples in the present study shows the impact untreated or inadequately treated effluents have on coastal areas. These results highlight the need for public investment in adequate wastewater treatment and adequate treatment of oysters, ensuring safe areas for shellfish production as well as healthier bivalve mollusks for consumption.

NEW FOSSIL RECORDS OF PLEISTOCENE MARINE MOLLUSKS IN SOUTHERN BRAZIL

Fossils of the gastropods Diodora patagonica, Zidona dufresnei, Olivancillaria carcellesi, Lamniconus lemniscatus carcellesi and the bivalve Arcinella brasiliana are registered for the first time from the outcrops of Chui Creek, on the coastal plain of Rio Grande do Sul State, southernmost Brazil, together with other taxa previously known elsewhere. The specimens were collected in a shallow Pleistocene marine facies exposed at the base of the banks of the creek, in a fossil concentration possibly formed by storm events. The taxa described here live in shallow environments (with the exception of A. brasiliana and Z. dufresnei) with sandy bottoms (except for D. patagonica, T patagonica, B. odites, C. rhizophorae and A. brasiliana). The presence of L. lemniscatus carcellesi, found living today only in Uruguay and Argentina, indicates a wider distribution for this taxon during the late Pleistocene.

A new Pennsylvanian Oriocrassatellinae from Brazil and the distribution of the genus Oriocrassatella in space and time

Oriocrassatella Etheridge Jr., 1907 is a long range crassatellid bivalve genus well recognized in shallow waters of epeiric seas throughout the upper part of Paleozoic. The first occurrences of this genus are recorded in the sedimentary successions of the Gondwana, both in Australia and South America. However, the geographic and age distribution of Oriocrassatella in Late Mississippian deposits of Australia and Argentina may indicate an earliest Visean or even a pre-Visean origin for the genus. Following its origin in Early Carboniferous a complex paleobiogeographic history from Southern to Northern Hemisphere took place in the Permian. During its initial dispersal phase from Late Carboniferous to the Early Permian the genus thrived in cold water environments associated to the Late Paleozoic Gondwana glaciation. Shallow-water bottoms of the warm waters of the central Gondwana fringe and Laurussia were colonized by Oriocrassatella only during Early Permian times when the genus became cosmopolitan. A new species of this genus is described herein, Oriocrassatella piauiensis n. sp., recorded from the Piaui Formation, Pennsylvanian of the Parnaiba Basin. This new species may represent an early adaptation to warm waters. However, based on available data, species of this genus seem to have adapted definitely to warm water environments probably related the Late Pennsylvanian interglacial phases. In these phases, climatic barrier were interrupted allowing the faunal interchange and larval dispersion following a South to North migration route through the eastern margins of Gondwana and the eastern Paleotethys.

Interannual variation in commercial oyster (Crassostrea gigas) farming in the sea (Florianopolis, Brazil, 27 degrees 44 ' S; 48 degrees 33 ' W) in relation to temperature, chlorophyll a and associated oceanographic conditions

Aquaculture of filter-feeding bivalve mollusks involves the fruitful conversion of marine particulate organic matter into premium protein of high nutritive value. Culture performance of bivalves is largely dependent on hydrological conditions and directly affected by e. g. temperature and chlorophyll levels. Accordingly, these parameters may be related with seasonality but also with oceanographic features combined with climate events. Yields of Pacific cupped oyster (Crassostrea gigas) reared at commercial procedures in suspended structures (long-lines) in a sheltered bay in Southern Brazil (Santa Catarina State, 27S 43'; 48 W 30') were evaluated in relation to local environmental conditions: sea surface temperature, chlorophyll a concentration, and associate effects of cold fronts events and El Nino and La Nina periods. Outputs from four consecutive commercial crop years were analyzed (2005/06, 2006/07, 2007/08, 2008/09) in terms of oyster survival and development time during the following grow-out phases of the culture cycle: seed to juvenile, juvenile to adult, adult to marketable. Since culture management and genetics were standardized significant differences verified among crop performance could be mostly related to environmental effects. Time series of temperature and chlorophyll a (remote sensing data) from crop periods displayed significant seasonal and interannual variation. As expected, performance during initial grow-out stages (seed to juvenile) was critical for final crop yield. Temperature was the main factor affecting survival in these initial stages with a trend of negative correlation, though not statistically significant. On the other hand, oyster development rate was significantly and positively affected by chlorophyll a concentration. Chlorophyll a values could be increased by upwelled cold nutrient-rich South Atlantic Central Water (SACW, related to predominant Northern winds) though further dependent on occurrence of Southern winds (cold fronts) to assist seawater penetration into the sheltered farming area. Lower salinity nutrient-rich northward drifted waters from La Plata River discharge may also result in chlorophyll a rise in the farming area. The El Nino period (July 2006 to February 2007) coincided with lower chlorophyll a levels in the farming site that may be related to both decreased number of cold fronts as well as predominance of Northern winds that retain northward spreading of La Plata River discharge waters. In contrast, the La Nina period (August 2007 to June 2008) corresponded to higher chlorophyll a values in the farming area by both upwelling of SACW and penetration of La Plata River discharge water assisted by increased occurrence of Southern winds and cold fronts. The recognition of the potentially changing climate and effects upon the environment will be an important step in planning future development of bivalve aquaculture.

Life history of three catfish species (Siluriformes: Ariidae) from southeastern Brazil

This study evaluated the spatio-temporal distribution, reproduction and diet of the catfishes Genidens genidens, G. barbus and Aspistor luniscutis in Caraguatatuba Bay. Their sizes were recorded and the sex and reproductive stage identified. The abundance was compared between areas (South and North) and among months (August 2003 - October 2004). The species had different spatial distributions, allowing them to coexist. The temporal distribution reflected their tendencies to migrate in the reproductive period, as evidenced by the dominance of small immature individuals in the bay. G. genidens tended to reproduce in winter, and A. luniscutis in spring. The diet of G. genidens consisted of crustaceans, mollusks (shells), fish scales, ostracods, and bivalve siphons. G. barbus consumed a high proportion of mysids, followed by fish (bones and scales). For A. luniscutis, the diet was based on fish scales and crustaceans. High quantities of particulate organic matter were observed in the diet of all three species, as previously known for estuarine catfishes. The consumption of fish scales may reflect a lepidophagic habit. A small overlap was observed among the diets, reflecting differences in their environments as well as in the proportions of each item ingested.

Population biology and diet of the southern kingcroaker Menticirrhus americanus (Linnaeus, 1758) (Perciformes: Sciaenidae) in Caraguatatuba Bay, southeastern Brazil

This study evaluated the spatio-temporal distribution, population biology and diet of Menticirrhus americanus in Caraguatatuba Bay. Samples were taken monthly between August 2003 and October 2004, by trawling in two previously selected areas. The northern area is more exposed to wave activity and is influenced by a river, functioning as a small estuary. In contrast, the southern area is relatively sheltered from wave energy and influenced to a lesser degree by smaller rivers. The fishes' length was measured, and the sex and gonadal stage macroscopically identified. The abundance of this species was compared between areas and among months. The diet was identified and quantified. M. americanus occurred in equal proportions in the two study areas, being most abundant in April 2004, followed by December 2003 and January 2004. The population was dominated by small immature individuals. The few individuals in maturation or mature that were captured showed no seasonal pattern of distribution. This species had a varied diet, feeding on worms (nemerteans, sipunculans and echiurans), mollusks (bivalves and cephalopods), polychaetes, crustaceans and fish. The presence of intact nematodes in the intestine suggests that these are parasites. The results demonstrated that M. americanus has a homogeneous spatial and temporal distribution in Caraguatatuba Bay, being uniformly distributed between the south and north areas as well as across the months. This species can be considered a carnivorous predator, showing a preference for consuming benthic sandy-beach species such as glycerids and other polychaetes, crustaceans, and bivalve siphons.

Currents controlling sedimentation, deposits recording palaeo-hydrodynamics – lessons from the continental shelf-slope system off SE South America

The continental margin off SE South America hosts one of the world’s most energetic hydrodynamic regimes but also the second largest drainage system of the continent. Both, the ocean current system as well as the fluvial runoff are strongly controlled by the atmospheric circulation modes over the region. The distribution pattern of particular types of sediments on shelf and slope and the long-term built-up of depositional elements within the overall margin architecture are, thus, the product of both, seasonal to millennial variability as well as long-term environmental trends. This talk presents how the combination of different methodological approaches can be used to obtain a comprehensive picture of the variability of a shelf and upper-slope hydrodynamic system during Holocene times. The particular methods applied are: (a) Margin-wide stratigraphic information to elucidate the role of sea level for the oceanographic and sedimentary systems since the last glacial maximum; (b) Palaeoceanographic sediment proxies combined with palaeo-temperature indicating isotopes of bivalve shells to trace lateral shifts in the coastal oceanography (particularly of the shelf front) during the Holocene; (c) Neodymium isotopes to identify the shelf sediment transport routes resulting from the current regime; (d) Sedimentological/geochemical data to show the efficient mechanism of sand export from the shelf to the open ocean; (e) Diatom assemblages and sediment element distributions indicating palaeo-salinity and the changing marine influence to illustrate the Plata runoff history. Sea level has not only controlled the overall configuration of the shelf but also the position of the main sediment routes from the continent towards the ocean. The shelf front has shifted frequently since the last glacial times probably resulting from both, changes in the Westerly Winds intensity and in the shelf width itself. Remarkable is a southward shift of this front during the past two centuries possibly related to anthropogenic influences on the atmosphere. The oceanographic regime with its prominent hydrographic boundaries led to a clear separation of sedimentary provinces since shelf drowning. It is especially the shelf front which enhances shelf sediment export through a continuous high sand supply to the uppermost slope. Finally, the Plata River does not continuously provide sediment to the shelf but shows significant climate-related changes in discharge during the past centuries. Starting from these findings, three major fields of research should, in general, be further developed in future: (i) The immediate interaction of the hydrodynamic and sedimentary systems to close the gaps between deposit information and modern oceanographic dynamics; (ii) Material budget calculations for the marginal ocean system in terms of material fluxes, storage/retention capacities, and critical thresholds; (iii) The role of human activity on the atmospheric, oceanographic and solid material systems to unravel natural vs. anthropogenic effects and feedback mechanisms

Deposit-feeding in benthic macrofauna : Tracer studies from the Baltic Sea

A low content of organic matter, which is largely refractory in nature, is characteristic of most sediments, meaning that aquatic deposit-feeders live on a very poor food source. The food is derived mainly from sedimenting phytodetritus, and in temperate waters like the Baltic Sea, from seasonal phytoplankton blooms. Deposit-feeders are either bulk-feeders, or selective feeders, which preferentially ingest the more organic-rich particles in the sediment, including phytodetritus, microbes and meiofauna. The soft-bottom benthos of the Baltic Sea has low species biodiversity and is dominated by a few macrobenthic species, among which the most numerous are the two deposit-feeding amphipods Monoporeia affinis and Pontoporeia femorata, and the bivalve Macoma balthica. This thesis is based on laboratory experiments on the feeding of these three species, and on the priapulid Halicryptus spinulosus. Feeding by benthic animals is often difficult to observe, but can be effectively studied by the use of tracers. Here we used the radioactive isotope 14C to label food items and to trace the organic matter uptake in the animals, while the stable isotopes 13C and 15N were used to follow feeding on aged organic matter in the sediment. The abundance of M. balthica and the amphipods tends to be negatively correlated, i.e., fewer bivalves are found at sites with dense populations of amphipods, with the known explanation that newly settled M. balthica spat are killed by the amphipods. Whether the postlarvae are just accidentally killed, or also ingested after being killed was tested by labelling the postlarvae with 14C and Rhodamine B. Both tracer techniques gave similar evidence for predation on and ingestion of postlarval bivalves. We calculated that this predation was likely to supply less than one percent of the daily carbon requirement for M. affinis, but might nevertheless be an important factor limiting recruitment of M. balthica. The two amphipods M. affinis and P. femorata are partly vertically segregated in the sediment, but whether they also feed at different depths was unknown. By adding fresh 14C-labelled algae either on the sediment surface or mixed into the sediment, we were able to distinguish surface from subsurface feeding. We found M. affinis and P. femorata to be surface and subsurface deposit-feeders, respectively. Whether the amphipods also feed on old organic matter, was studied by adding fresh 14C-labelled algae on the sediment surface, and using aged, one-year-old 13C- and 15N-labelled sediment as deep sediment. Ingestion of old organic matter, traced by the stable isotopes, differed between the two species, with a higher uptake for P. femorata, suggesting that P. femorata utilises the older, deeper-buried organic matter to a greater extent. Feeding studies with juveniles of both M. affinis and P. femorata had not been done previously. In an experiment with the same procedure and treatments as for the adults, juveniles of both amphipod species were found to have similar feeding strategies. They fed on both fresh and old sediment, with no partitioning of food resources, making them likely to be competitors for the same food resource. Oxygen deficiency has become more wide-spread in the Baltic Sea proper in the last half-century, and upwards of 70 000km2 are now devoid of macrofauna, even though part of that area does not have oxygen concentrations low enough to directly kill the macrofauna. We made week-long experiments on the rate of feeding on 14C-labelled diatoms spread on the sediment surface in different oxygen concentrations for both the amphipod species, M. balthica and H. spinulosus. The amphipods were the most sensitive to oxygen deficiency and showed reduced feeding and lower survival at low oxygen concentrations. M. balthica showed reduced feeding at the lowest oxygen concentration, but no mortality increase. The survival of H. spinulosus was unaffected, but it did not feed, showing that it is not a surface deposit-feeder. We conclude that low oxygen concentrations that are not directly lethal, but reduce food intake, may lead to starvation and death in the longer term.

Climatic and stratigraphic paleoindicators from the Canary Island

[EN] The last 5 Myr  are characterized by cliamatic variations globally and are reflected in ancient fossiliferous marine deposits visible in the Canary Islands. The fossils contained are identificated as paleoecological and paleoclimatic indicators. The Mio-Pliocene Transit is represented by the coral Siderastrea micoenica Osasco, 1897; the gastropods Rothpletzia rudista Simonelli, 1890; Ancilla glandiformis (Lamarck, 1822); Strombus coronatus Defrance, 1827 and Nerita emiliana Mayer, 1872 and the bivalve Gryphaea virleti Deshayes, 1832 as most characteristic fossils  and typical of a very warm climate and littoral zone. Associated  lava flows  have been dated radiometrically  and provides a range between  8.9 and about 4.2 Kyr. In the mid-Pleistocene, about 400,000 years ago, the called Marine Isotope Stage 11, a strong global warming that caused a sea level rise happens. Remains of the MIS 11  are preserved on the coast of Arucas (Gran Canaria), and associated with a tsunami in Piedra Alta (Lanzarote). These fossilifeorus  deposits contains the bivalve Saccostrea cucullata (Born, 1780), the gastropod Purpurellus gambiensis (Reeve, 1845) and the corals Madracis pharensis (Heller, 1868) and Dendrophyllia cornigera (Lamarck, 1816). Both sites have been dated by K-Ar on pillow lavas (approximately 420,000 years) and by Uranium Series on corals (about 481,000 years) respectively. The upper Pleistocene starts with another strong global warming known as the last interglacial or marine isotope  stage (MIS) 5.5, about 125,000 years ago, which also left marine  fossil deposits exposed in parallel to current in Igueste of San Andrés (Tenerife),  El Altillo, the  city of Las Palmas de Gran Canaria  and Maspalomas (Gran Canaria),  Matas Blancas, the Playitas and Morrojable (Fuerteventura ) and in Playa Blanca and Punta Penedo (Lanzarote ). The fossil coral Siderastrea radians (Pallas , 1766 ) currently living in the Cape Verde Islands , the Gulf of Guinea and the Caribbean has allowed Uranium series dating. The gastropods Strombus bubonius Lamarck, 1822 and Harpa doris (Röding , 1798 ) currently living in the Gulf of Guinea. Current biogeography using synoptic data obtained through satellites provided by the ISS Canary Seas provides data of Ocean Surface Temperature (SST) and Chlorophyll a (Chlor a) . This has allowed the estimation of these sea conditions during interglacials compared to today .

Organismi acquatici e ambiente: meccanismi biochimici di interazione, risposta e adattamento

The research is focused on the relationship between some Mg2+-dependent ATPase activities of plasma- and mitochondrial membranes from tissues of cultured marine bivalve molluscs and potentially stressful environmental conditions, such as the exposure to contaminants both of natural origin (ammonia nitrogen, the main contaminant of aquaculture plants) and of anthropic source (alkyltins). The two filter-feeding bivalve species selected colonize different habitats: the common mussel Mytilus galloprovincialis binds to hard substrates and the Philippine clam Tapes philippinarum burrows into sea bottom sandy beds. The choice of typical species of coastal waters, extremely suitable for environmental studies due to their features of poor motility, resistance to transport and great filtering efficiency, may constitute a model to evaluate responses to contaminants of membrane-bound enzyme activities involved in key biochemical mechanisms, namely cell ionic regulation and mitochondrial energy production. In vitro and in vitro approaches have been pursued. In vitro assays were carried out by adding the contaminants (NH4Cl and alkyltins) directly to the ATPase reaction media. In vivo experiments were carried out by exposing mussels to various tributyl tin (TBT) concentrations under controlled conditions in aquaria. ATPase activities were determined spectrophotometrically according to the principles of the method of Fiske and Subbarow (1925). The main results obtained are detailed below. In Tapes philippinarum the interaction of NH4 +, the main form of ammonia nitrogen at physiological and seawater pHs, with the Na,K-ATPase and the ouabaininsensitive Na-ATPase was investigated in vitro on gill and mantle microsomal membranes. The proven replacement by NH4 +of K+ in the activation of the Na,KATPase and of Na+ in the activation of the ouabain-insensitive ATPase displayed similar enzyme affinity for the substituted cation. on the one hand this finding may represent one of the possible mechanisms of ammonia toxicity and, on the other, it supports the hypothesis that NH4 + can be transported across the plasma membrane through the two ATPases. In this case both microsomal ATPases may be involved and co-operate, at least under peculiar circumstances, to nitrogen excretion and ammonia detoxification mechanisms in bivalve molluscs. The two ATPase activities stimulated by NH4 + maintained their typical response to the glycoside ouabain, specific inhibitor of the Na,K-ATPase, being the Na++ NH4 +-activated ATPase even more susceptive to the inhibitor and the ouabain-insensitive ATPase activity activated indifferently by Na+ or NH4 + unaffected by up to 10-2 M ouabain. In vitro assays were carried out to evaluate the response of the two Na-dependent ATPases to organotins in clams and mussels and to investigate the interaction of TBT with mussel mitochondrial oligomycin-sensitive Mg-ATPase. Since no literature data were available, the optimal assay conditions and oligomycin sensitivity of mussel mitochondrial MgATPase were determined. In T. philippinarum the ouabain-insensitive Na-ATPase was found to be refractory to TBT both in the gills and in the mantle, whereas the Na,K-ATPase was progressively inhibited by increasing TBT doses; the enzyme inhibition was more pronounced in the gills than in the mantle. In both tissues of M. galloprovincialis the Na,K-ATPase inhibition by alkyltins decreased in the order TBT>DBT(dibutyltin)>>MBT(monobutyltin)=TeET(tetraethyltin) (no effect). Mussel Na-ATPase confirmed its refractorimess to TBT and derivatives both in the gills and in the mantle. These results indicate that the Na,K-ATPase inhibition decreases as the number of alkyl chains bound to tin decreases; however a certain polarity of the organotin molecule is required to yield Na,K-ATPase inhibition, since no enzyme inhibition occurred in the presence of tetraalkyl-substituted derivatives such as TeET . Assays carried out in the presence of the dithioerythritol (DTE) pointed out that the sulphhydrylic agent is capable to prevent the Na,K-ATPase inhibition by TBT, thus suggesting that the inhibitor may link to -SH groups of the enzyme complex.. Finally, the different effect of alkyltins on the two Na-dependent ATPases may constitute a further tool to differentiate between the two enzyme activities. These results add to the wealth of literature data describing different responses of the two enzyme activities to endogenous and exogenous modulators . Mussel mitochondrial Mg-ATPase was also found to be in vitro inhibited by TBT both in the gills and in the mantle: the enzyme inhibition followed non competitive kinetics. The failed effect of DTE pointed out that in this case the interaction of TBT with the enzyme complex is probably different from that with the Na,K-ATPase. The results are consistent with literature data showing that alkyltin may interact with enzyme structures with different mechanisms. Mussel exposure to different TBT sublethal doses in aquaria was carried out for 120 hours. Two samplings (after 24 and 120 hrs) were performed in order to evaluate a short-term response of gill and mantle Na,K-ATPase, ouabain-insensitive Na-ATPase and Mg-ATPase activities. The in vivo response to the contaminants of the enzyme activities under study was shown to be partially different from that pointed out in the in vitro assays. Mitochondrial Mg-ATPase activity appeared to be activated in TBTexposed mussels with respect to control ones, thus confirming the complexity of evaluating in vivo responses of the enzyme activities to contaminants, due to possible interactions of toxicants with molluscan metabolism. Concluding, the whole of data point out that microsomal and mitochondrial ATPase activities of bivalve molluscs are generally responsive to environmental contaminants and suggest that in some cases membrane-bound enzyme activities may represent the molecular target of their toxicity. Since the Na,K-ATPase, the Na-ATPase and the Mg-ATPase activities are poorly studied in marine bivalves, this research may contribute to enlarge knowledge in this quite unexplored field.

Ricerca, mediante tecniche di proteomica, di biomarcatori proteici utili nella valutazione della qualità degli alimenti di origine animale

In the last decades, the increase of industrial activities and of the request for the world food requirement, the intensification of natural resources exploitation, directly connected to pollution, have aroused an increasing interest of the public opinion towards initiatives linked to the regulation of food production, as well to the institution of a modern legislation for the consumer guardianship. This work was planned taking into account some important thematics related to marine environment, collecting and showing the data obtained from the studies made on different marine species of commercial interest (Chamelea gallina, Mytilus edulis, Ostrea edulis, Crassostrea gigas, Salmo salar, Gadus morhua). These studies have evaluated the effects of important physic and chemical parameters variations (temperature, xenobiotics like drugs, hydrocarbons and pesticides) on cells involved in the immune defence (haemocytes) and on some important enzymatic systems involved in xenobiotic biotransformation processes (cytochrome P450 complex) and in the related antioxidant defence processes (Superoxide dismutase, Catalase, Heat Shock Protein), from a biochemical and bimolecular point of view. Oxygen is essential in the biological answer of a living organism. Its consume in the normal cellular breathing physiological processes and foreign substances biotransformation, leads to reactive oxygen species (ROS) formation, potentially toxic and responsible of biological macromolecules damages with consequent pathologies worsening. Such processes can bring to a qualitative alteration of the derived products, but also to a general state of suffering that in the most serious cases can provoke the death of the organism, with important repercussions in economic field, in the output of the breedings, of fishing and of aquaculture. In this study it seemed interesting to apply also alternative methodologies currently in use in the medical field (cytofluorimetry) and in proteomic studies (bidimensional electrophoresis, mass spectrometry) with the aim of identify new biomarkers to place beside the traditional methods for the control of the animal origin food quality. From the results it’s possible to point out some relevant aspects from each experiment: 1. The cytofluorimetric techniques applied to O. edulis and C. gigas could bring to important developments in the search of alternative methods that quickly allows to identify with precision the origin of a specific sample, contributing to oppose possible alimentary frauds, in this case for example related to presence of a different species, also under a qualitative profile, but morpholgically similar. A concrete perspective for the application in the inspective field of this method has to be confirmed by further laboratory tests that take also in account in vivo experiments to evaluate the effect in the whole organism of the factors evaluated only on haemocytes in vitro. These elements suggest therefore the possibility to suit the cytofluorimetric methods for the study of animal organisms of food interest, still before these enter the phase of industrial working processes, giving useful information about the possible presence of contaminants sources that can induce an increase of the immune defence and an alteration of normal cellular parameter values. 2. C. gallina immune system has shown an interesting answer to benzo[a]pyrene (B[a]P) exposure, dose and time dependent, with a significant decrease of the expression and of the activity of one of the most important enzymes involved in the antioxidant defence in haemocytes and haemolymph. The data obtained are confirmed by several measurements of physiological parameters, that together with the decrease of the activity of 7-etossi-resourifine-O-deetilase (EROD linked to xenobiotic biotransformation processes) during exposure, underline the major effects of B[a]P action. The identification of basal levels of EROD supports the possible presence of CYP1A subfamily in the invertebrates, still today controversial, never identified previously in C. gallina and never isolated in the immune cells, as confirmed instead in this study with the identification of CYP1A-immunopositive protein (CYP1A-IPP). This protein could reveal a good biomarker at the base of a simple and quick method that could give clear information about specific pollutants presence, even at low concentrations in the environment where usually these organisms are fished before being commercialized. 3. In this experiment it has been evaluated the effect of the antibiotic chloramphenicol (CA) in an important species of commercial interest, Chamelea gallina. Chloramphenicol is a drug still used in some developing countries, also in veterinary field. Controls to evaluate its presence in the alimentary products of animal origin, can reveal ineffective whereas the concentration results to be below the limit of sensitivity of the instruments usually used in this type of analysis. Negative effects of CA towards the CYP1A- IPP proteins, underlined in this work, seem to be due to the attack of free radicals resultant from the action of the antibiotic. This brings to a meaningful alteration of the biotransformation mechanisms through the free radicals. It seems particularly interesting to pay attention to the narrow relationships in C. gallina, between SOD/CAT and CYP450 system, actively involved in detoxification mechanism, especially if compared with the few similar works today present about mollusc, a group that is composed by numerous species that enter in the food field and on which constant controls are necessary to evaluate in a rapid and effective way the presence of possible contaminations. 4. The investigations on fishes (Gadus morhua, and Salmo salar) and on a bivalve mollusc (Mytilus edulis) have allowed to evaluate different aspects related to the possibility to identify a biomarker for the evaluation of the health of organisms of food interest and consequently for the quality of the final product through 2DE methodologies. In the seafood field these techniques are currently used with a discreet success only for vertebrates (fishes), while in the study of the invertebrates (molluscs) there are a lot of difficulties. The results obtained in this work have underline several problems in the correct identification of the isolated proteins in animal organisms of which doesn’t currently exist a complete genomic sequence. This brings to attribute some identities on the base of the comparison with similar proteins in other animal groups, incurring in the possibility to obtain inaccurate data and above all discordant with those obtained on the same animals by other authors. Nevertheless the data obtained in this work after MALDI-ToF analysis, result however objective and the spectra collected could be again analyzed in the future after the update of genomic database related to the species studied. 4-A. The investigation about the presence of HSP70 isoforms directly induced by different phenomena of stress like B[a]P presence, has used bidimensional electrophoresis methods in C. gallina, that have allowed to isolate numerous protein on 2DE gels, allowing the collection of several spots currently in phase of analysis with MALDI-ToF-MS. The present preliminary work has allowed therefore to acquire and to improve important methodologies in the study of cellular parameters and in the proteomic field, that is not only revealed of great potentiality in the application in medical and veterinary field, but also in the field of the inspection of the foods with connections to the toxicology and the environmental pollution. Such study contributes therefore to the search of rapid and new methodologies, that can increase the inspective strategies, integrating themselves with those existing, but improving at the same time the general background of information related to the state of health of the considered animal organism, with the possibility, still hypothetical, to replace in particular cases the employment of the traditional techniques in the alimentary field.

Mitochondrial Genomics: structure, inheritance and phylogenetic utility of the Mitochondrial genome in Bivalvia and Insecta

This PhD Thesis is the result of my research activity in the last three years. My main research interest was centered on the evolution of mitochondrial genome (mtDNA), and on its usefulness as a phylogeographic and phylogenetic marker at different taxonomic levels in different taxa of Metazoa. From a methodological standpoint, my main effort was dedicated to the sequencing of complete mitochondrial genomes, and the approach to whole-genome sequencing was based on the application of Long-PCR and shotgun sequences. Moreover, this research project is a part of a bigger sequencing project of mtDNAs in many different Metazoans’ taxa, and I mostly dedicated myself to sequence and analyze mtDNAs in selected taxa of bivalves and hexapods (Insecta). Sequences of bivalve mtDNAs are particularly limited, and my study contributed to extend the sampling. Moreover, I used the bivalve Musculista senhousia as model taxon to investigate the molecular mechanisms and the evolutionary significance of their aberrant mode of mitochondrial inheritance (Doubly Uniparental Inheritance, see below). In Insects, I focused my attention on the Genus Bacillus (Insecta Phasmida). A detailed phylogenetic analysis was performed in order to assess phylogenetic relationships within the genus, and to investigate the placement of Phasmida in the phylogenetic tree of Insecta. The main goal of this part of my study was to add to the taxonomic coverage of sequenced mtDNAs in basal insects, which were only partially analyzed.

Alterazione di risposte fisiologiche in invertebrati marini esposti nella Baia di Cadice (Spagna) come indici della qualità dell'ambiente

La forte crescita nella pescicoltura ha portato ad una significativa pressione ambientale di origine antropica nei sistemi costieri. Il bivalve locale Scrobicularia plana è stato usato come bioindicatore per valutare la qualità ambientale di un ecosistema affetto da scarichi di acque residuali di una piscifattoria nel braccio di mare Rio San Pedro (Spagna sud-occidentale). I bivalvi sono stati raccolti nei sedimenti intertidali nell'ottobre del 2010 da cinque siti del braccio di mare, seguendo un gradiente di inquinamento decrescente dall'effluente al sito di controllo. Per valutare l'esposizione e l'effetto di contaminanti legati alle acque residuali delle piscifattorie è stata selezionata una batteria di biomarker. Sono state misurate nei tessuti delle ghiandole digestive dei bivalvi: l'attività di enzimi del sistema di detossificazione della Fase I (etossiresorufina-O-deetilasi, EROD e dibenzilfluoresceina, DBF) l'attività di un enzima del sistema di detossificazione di Fase II (glutatione S-transferasi, GST), l'attività di enzimi antiossidanti (glutatione perossidasi, GPX e glutatione reduttasi, GR) e parametri di stress ossidativo (perossidazione lipidica, LPO, e danno al DNA). In parallelo sono state misurate in situ, nelle aree di studio, temperatura, pH, salinità e ossigeno disciolto nelle acque superficiali; nelle acque interstiziali sono stati misurati gli stessi parametri con l'aggiunta del potenziale redox. Sono state trovate differenze significative (p<0,05) tra siti di impatto e sito di controllo per quanto riguarda l'attività di EROD e GR, LPO e danno al DNA; è stato osservato un chiaro gradiente di stress riconducibile alla contaminazione, con alte attività di questi biomarker nell'area di scarico delle acque residuali della pescicoltura e livelli più bassi nel sito di controllo. È stata trovata inoltre una correlazione negativa significativa (p<0,01) tra la distanza alla fonte di inquinamento e l’induzione dei biomarker. Sono state analizzate le componenti abiotiche, inserendole inoltre in una mappa georeferenziata a supporto. Ossigeno disciolto, pH, salinità e potenziale redox mostrano valori bassi vicino alla fonte di inquinamento, aumentando man mano che ci si allontana da esso. I dati ottenuti indicano nel loro insieme che lo scarico di acque residuali dalle attività di pescicoltura nel braccio di mare del Rio San Pedro può indurre stress ossidativo negli organismi esposti che può portare ad un'alterazione dello stato di salute degli organismi.