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Abstract: The autometallographic approach was applied to three filter-feeder bivalves from differently polluted sites of the Lagoon of Venice. The area density (AD) values of black silver deposits (BSDs) were therefore seasonally quantified by image analysis in digestive cell lysosomes. Sediments were characterised in terms of grain size, organic content and metal concentrations (Cd, Cu, Ni, Pb, Zn). The physiological status of the bivalves was also evaluated by applying the “survival in air” test as a generic stress index. Chemical results showed that Zn contributed to the total metal load for more than 60% in sediments and for 85%–94% in mussel and clam digestive glands, respectively (data processed from published results obtained on the same samples). Regression analyses between extent of BSDs and both total metal and Zn concentrations in digestive gland followed a linear pattern in mussels and clams, but not in arks. Similarly, in mussels and clams only, BSD amount was significantly correlated with total metal concentrations in sediments according to a logarithmic model. Consequently, results obtained from autometallography can discriminate sites on the basis of their metal pollution levels, like chemical analyses on both sediments and digestive glands, evaluations being supported by the results of the physiological stress index. As a conclusion, it is suggested that autometallography in bivalves may provide overall estimates of metal fractions moving from sediments towards organisms through the ingestion of metal-enriched particles. Future work is needed to define the bioaccumulation model in arks and to clarify their possible use as metal biomonitors. [Copyright &y& Elsevier]

Gross biochemical composition, energy value and condition index were investigated in two populations of Tapes philippinarum differently exploited by fishing in the Lagoon of Venice during the period July 2000–July 2001. Proteins, lipids, total carbohydrates and glycogen showed similar patterns in clams from all areas, highlighting the importance of the reproductive cycle and physiological features of the species in determining biochemical composition. The influence of environmental conditions and the effects of different fishing impact could also be observed. Carbohydrates exhibit minima in winter and maxima in spring–summer, while proteins have an irregular trend. Lipid seasonal variations are inversely related to glycogen contents; furthermore, lipids are higher in females than in males. Condition index and total energy content were influenced by both endogenous stress and environmental factors. In particular, the condition index, which gives an indication of the general physiological status of the animals, shows highest values in clams from the control area, not affected by fishing. [Copyright &y& Elsevier]

The impact of experimental hydraulic dredging was assessed on Chamelea gallina populations in two sites along the north-western Adriatic coast (Lido and Jesolo) by detecting and quantifying shell damage caused by fishing operations on both captured and discarded clams. Various levels of stress were applied, the highest being that used by commercial fishing vessels, which employ high water pressure and mechanised sorting and the lowest manual sampling of clams by scuba divers. Water pressure and sorting significantly increased shell damage, the highest levels always being observed in commercially dredged clams. At Lido, damage was mostly due to the action of the mechanised sorter; at Jesolo, the effect of high water pressure was more clearcut. Moreover, clams collected at Jesolo had both higher mean damage level and higher numbers of damaged individuals compared to the Lido samples. These differences seem to be mostly related to differing bottom features in the two sites. A positive relationship was observed between damage level and clam size: small-sized samples (length <17 mm) were less damaged than medium-sized ones (25 mm > length >17 mm) and commercial size clams (>25 mm) showed the highest damage level. The severe and harmful physical impact of hydraulic dredging was apparent in captured and then discarded animals, a small fraction of which appears able to recover, as shown by the presence of clams with repaired shells. [Copyright &y& Elsevier]

Anthropogenic emissions of carbon dioxide are leading to decreases in pH and changes in the carbonate chemistry of seawater. Ocean acidification may negatively affect the ability of marine organisms to produce calcareous structures while also influencing their physiological responses and growth. The aim of this study was to evaluate the effects of reduced pH on the survival, growth and shell integrity of juveniles of two marine bivalves from the Northern Adriatic sea: the Mediterranean mussel Mytilus galloprovincialis and the striped venus clam Chamelea gallina . An outdoor flow-through plant was set up and two pH levels (natural seawater pH as a control, pH 7.4 as the treatment) were tested in long-term experiments. Mortality was low throughout the first experiment for both mussels and clams, but a significant increase, which was sensibly higher in clams, was observed at the end of the experiment (6 months). Significant decreases in the live weight (−26%) and, surprisingly, in the shell length (−5%) were observed in treated clams, but not in mussels. In the controls of both species, no shell damage was ever recorded; in the treated mussels and clams, damage proceeded via different modes and to different extents. The severity of shell injuries was maximal in the mussels after just 3 months of exposure to a reduced pH, whereas it progressively increased in clams until the end of the experiment. In shells of both species, the damaged area increased throughout the experiment, peaking at 35% in mussels and 11% in clams. The shell thickness of the treated and control animals significantly decreased after 3 months in clams and after 6 months in mussels. In the second experiment (3 months), only juvenile mussels were exposed to a reduced pH. After 3 months, the mussels at a natural pH level or pH 7.4 did not differ in their survival, shell length or live weight. Conversely, shell damage was clearly visible in the treated mussels from the 1st month onward. Monitoring the chemistry of seawater carbonates always showed aragonite undersaturation at 7.4 pH, whereas calcite undersaturation occurred in only 37% of the measurements. The present study highlighted the contrasting effects of acidification in two bivalve species living in the same region, although not exactly in the same habitat. [ABSTRACT FROM AUTHOR]

Abstract: Intertidal area is characterized by several fluctuations in natural agents and anthropogenic factors (oxygen levels, temperature, salinity, B[a]P presence) that cause a noticeable increase in the expression rate of heat shock protein 70 (HSP70). HSPs acting as molecular chaperones and their induction represent a specific cellular defence mechanism in response to several stress. Chamelea gallina specimens from the North Adriatic coast were exposed to different experimental conditions: varying oxygen levels (48h of anoxia followed by 24h of normoxic recovery), temperatures (20, 25, 30°C for 7days), salinity (28, 34, 40‰ for 7days) and B[a]P concentrations (0.5mg/L for 24h, 7 and 12days). Following the extraction of the digestive gland and gills, HSP70 levels were identified in the cytosolic fraction by immunoblotting using primary monoclonal antibodies. An increase in the rate of HSP70 expression under anoxic conditions in the digestive gland was observed at high temperatures, at low salinity and in the presence of B[a]P. The protein was overexpressed in the absence of oxygen and after 12days of B[a]P exposure, while it was underexpressed in hyposaline conditions in the gills. HSP70 induction can be considered an adaptation mechanism associated with changes in environmental parameters, but also with xenobiotic presence. The overexpression of HSP70 is therefore induced by protein damage due to stressogenic factors. HSP recruitment renders them available for the processes of folding and refolding of denatured proteins or for their transport to a degradation system. The evident sensitivity of HSP70 to natural and anthropogenic stressogenic agents was examined in the present research. The results of this research revealed an interesting response of heat shock protein 70 in C. gallina, underlining the sensitivity of this important commercial species to natural and anthropogenic stress agents. [ABSTRACT FROM AUTHOR]

Abstract: CYP1A sub-family represents the main form of cytochrome P450 involved in benzo[a]pyrene (B[a]P) detoxification, but there are no clear evidences about its presence in invertebrates. 7-Ethoxy resorufin O-deethylase (EROD) activity is strictly related to CYP1A presence, at the same time P450-dependent oxidative metabolism leads to reactive oxygen species (ROS) production, thought to be an important mechanism of pollutant-mediated toxicity in aquatic organisms. Superoxide dismutases (SODs), EROD and CYP1A activities and/or expressions were detected in haemocytes of pooled clams (Chamelea gallina) and cell-free haemolymph after 24 h, 7 and 12 days of exposure to 0.5 mg/L of B[a]P. After 24 h, B[a]P content was maximum in whole tissues. A 61 kDa band was recognized in haemocytes and cell-free haemolymph by polyclonal anti-fish CYP1A, while 53.5 and 63.8 kDa CYP1A immunopositive proteins were discriminate without differences of expression. Differently, EROD, MnSOD activity/expression and ECSOD expression decreased in haemocytes and haemolymph. C. gallina immune system presents an interesting response dose/time exposure of B[a]P and the 7 days condition highlights the major effects of xenobiotic action. The identification of basal EROD levels supports the possible presence of the CYP1A, never identified in C. gallina and more specifically never isolated in immune cells, as confirmed by CYP1A-immunopositive proteins identification. [Copyright &y& Elsevier]

This study is focused on evaluating biological effects on the target species Chamelea gallina as a result of repeated exploitation of clam beds by hydraulic dredging. A biomarker approach was adopted and two physiological indices were applied: adenylate energy charge (AEC) (biochemical level) and reburrowing behaviour (organism level). The aim was to investigate the biological response to various types of fishing, i.e., commercial systems using water at high pressures (HP), and experimental systems at lower pressures (LP) in the field, and to evaluate the response of clams to mechanical stress in the laboratory. Field results showed that HP-treated clams exhibited significantly lower levels of AEC compared with LP treatments. A similar trend was shown by reburrowing behaviour; HP-treated clams reburrowed less. Laboratory results were less clear: a very low level of AEC was measured in both control and treated clams. These poor conditions in foot muscle did not indicate worsening at organism level, as no dead or dying clams were recorded throughout the experiment. However, repeated mechanical stress reduced the percentage of reburrowing clams, which suggested that harvesting may affect reburrowing behaviour. [Copyright &y& Elsevier]

Glyphosate, the most widely used herbicide worldwide, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. While the potential for glyphosate to induce a broad range of biological effects in exposed organisms has been demonstrated, the global molecular mechanisms of toxicity and potential effects in bacterial symbionts remain unclear, in particular for ecologically important marine species such as bivalve molluscs. Here, the effects of glyphosate (GLY), its degradation product aminomethylphosphonic acid (AMPA), and a mixture of both (MIX) on the mussel M. galloprovincialis were assessed in a controlled experiment. For the first time, next generation sequencing (RNA-seq and 16S rRNA amplicon sequencing) was used to evaluate such effects at the molecular level in both the host and its respective microbiota. The results suggest that the variable capacity of bacterial species to proliferate in the presence of these compounds and the impairment of host physiological homeostasis due to AMPA and GLY toxicity may cause significant perturbations to the digestive gland microbiota, as well as elicit the spread of potential opportunistic pathogens such as Vibrio spp.. The consequent host-immune system activation identified at the molecular and cellular level could be aimed at controlling changes occurring in the composition of symbiotic microbial communities. Overall, our data raise further concerns about the potential adverse effects of glyphosate and AMPA in marine species, suggesting that both the effects of direct toxicity and the ensuing changes occurring in the host-microbial community must be taken into consideration to determine the overall ecotoxicological hazard of these compounds. • Ecotoxicological effects of glyphosate and AMPA were investigated in mussels. • GLY and AMPA exposures suggest alterations of mussel physiological homeostasis. • AMPA and GLY toxicity may cause perturbations to the mussels microbiota. • Microbiota perturbations lead to host-immune system activation. • Host-microbiota changes following GLY exposure must be taken into consideration. [ABSTRACT FROM AUTHOR]