Seasonal environmental changes may affect the physiology of Mytilus galloprovincialis (Lam.), an intertidal filter-feeder bivalve occurring commonly in Mediterranean and Atlantic coastal areas. We investigated seasonal variations in relative transcript abundance of the digestive gland and the mantle (gonads) of males and females. To identify gene expression trends – in terms of relative mRNA abundance- we used a medium-density cDNA microarray (1.7 K probes) in dual-color competitive hybridization analyses. Hierarchical clustering of digestive gland microarray data showed two main branches, distinguishing profiles associated with the “hot” months (May–August) from the other months. Genes involved in chitin metabolism, associated with mussel nutrition and digestion showed higher mRNA levels during summer. Moreover, we found different gene transcriptomic patterns in the digestive glands of males when compared to females, during the four stages of mussel gonadal development. Microarray data from gonadal transcripts also displayed clear patterns during the different developmental phases respect to the resting period (stage I) with peak relative mRNA abundance at the ripe phase (stage III) for both sexes. These data showed a clear temporal pattern in transcriptomic profiles of mussels sampled over an annual cycle. Physiological response to thermal variation, food availability, and reproductive status across months may contribute to variation in relative mRNA abundance.

BackgroundMixtures of chemicals present in aquatic environments may elicit toxicity due to additive or synergistic effects among the constituents or, vice versa, the adverse outcome may be reduced by antagonistic interactions. Deviations from additivity should be explained either by the perturbations of toxicokinetic parameters and/or chemical toxicodynamics. We addressed this important question in marine mussels exposed subchronically to a binary mixture made of two wide-spread pollutants: the heavy metal nickel and the organic phosphorus pesticide Chlorpyrifos. To this aim, we carried out in tissues of Mytius galloprovincialis (Lam) a systems approach based on the evaluation and integration of different disciplines, i.e. high throughput gene expression profiling, functional genomics, stress biomakers and toxicokinetics.ResultsCellular and tissue biomarkers, viz. digestive gland lysosomal membrane stability, lysosomal/cytosol volume ratio, neutral lipid content and gill acetylcholinesterase activity were, in general, altered by either the exposure to nickel and Chlorpyrifos. However, their joint action rendered (i) an overall decrease of the stress syndrome level, as evaluated through an expert system integrating biomarkers and (ii) statistically significant antagonistic deviations from the reference model systems to predict mixture toxicity. While toxicokinetic modeling did not explain mixture interactions, gene expression profiling and further Gene Ontology-based functional genomics analysis provided clues that the decrement of toxicity may arise from the development of specific toxicodynamics. Multivariate statistics of microarray data (238 genes in total, representing about 14% of the whole microarray catalogue) showed two separate patterns for the single chemicals: the one belonging to the heavy metal -135 differentially expressed genes (DEGs) was characterized by the modulation of transcript levels involved in nucleic acid metabolism, cell proliferation and lipid metabolic processes. Chlorpyrifos exposure (43 DEGs) yielded a molecular signature which was biased towards carbohydrate catabolism (indeed, chitin metabolism) and developmental processes. The exposure to the mixture (103 DEGs) elicited a composite complex profile which encompassed the core properties of the pesticide but also a relevant set of unique features. Finally, the relative mRNA abundance of twelve genes was followed by Q-PCR to either confirm or complement microarray data. These results, in general, were compatible with those from arrays and indeed confirmed the association of the relative abundance of two GM-2 ganglioside activator genes in the development of the hyperlipidosis syndrome observed in digestive gland lysosomes of single chemical exposed mussels.ConclusionThe transcriptomic assessment fitted with biological data to indicate the occurrence of different toxicodynamic events and, in general, a decrease of toxicity, driven by the mitigation or even abolition of lysosomal responses. Furthermore, our results emphasized the import...

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Global warming is a major factor that may affect biological organization, especially in marine ecosystems and in coastal areas that are particularly subject to anthropogenic pollution. We evaluated the effects of simultaneous changes in temperature and copper concentrations on lysosomal membrane stability (N-acetyl-hexosaminidase activity) and malondialdehyde accumulation (MDA) in the gill of the blue mussel Mytilus galloprovincialis (Lam.). Temperature and copper exerted additive effects on lysosomal membrane stability, exacerbating the toxic effects of metal cations present in non-physiological concentrations. Mussel lysosomal membrane stability is known to be positively related to scope for growth, indicating possible effects of increasing temperature on mussel populations in metal-polluted areas. To clarify the molecular response to environmental stressors, we used a cDNA microarray with 1,673 sequences to measure the relative transcript abundances in the gills of mussels exposed to copper (40 µg/L) and a temperature gradient (16°C, 20°C, and 24°C). In animals exposed only to heat stress, hierarchical clustering of the microarray data revealed three main clusters, which were largely dominated by down-regulation of translation-related differentially expressed genes, drastic up-regulation of protein folding related genes, and genes involved in chitin metabolism. The response of mussels exposed to copper at 24°C was characterized by an opposite pattern of the genes involved in translation, most of which were up-regulated, as well as the down-regulation of genes encoding heat shock proteins and “microtubule-based movement” proteins. Our data provide novel information on the transcriptomic modulations in mussels facing temperature increases and high copper concentrations; these data highlight the risk of marine life exposed to toxic chemicals in the presence of temperature increases due to climate change.

The endospores of Bacillus subtilis can serve as a tool for surface presentation of heterologous proteins. The unique properties of the spore protective layers make them perfect vehicles for orally administered vaccines. In this study, we successfully displayed a fragment of Clostridium difficile FliD protein on the surface of B. subtilis spores using the CotB, CotC, CotG and CotZ spore coat proteins. The presence of the fusion proteins in the spore coat was verified by Western blotting and immunofluorescence microscopy. The amount of recombinant proteins was assessed by a dot-blot technique. C. difficile is one of the most common infectious agents in nosocomial infections and is especially associated with antibiotic therapies. FliD is a flagellar cap protein of C. difficile and is known to be one of the immunogenic surface antigens of this bacterium. Therefore, its use in vaccine formulations gives a good perspective for successful immunization with a FliDbased vaccine. The recombinant spores presented here may be good candidates for C. difficile oral vaccines.

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