Project description:BACKGROUND: Mytilus species are important in marine ecology and in environmental quality assessment, yet their molecular biology is poorly understood. Molecular aspects of their reproduction, hybridisation between species, mitochondrial inheritance, skewed sex ratios of offspring and adaptation to climatic and pollution factors are priority areas. METHODOLOGY/PRINCIPAL FINDINGS: To start to address this situation, expressed genetic transcripts from M. galloprovincialis were pyrosequenced. Transcripts were isolated from the digestive gland, foot, gill and mantle of both male and female mussels. In total, 175,547 sequences were obtained and for foot and mantle, 90% of the sequences could be assembled into contiguous fragments but this reduced to 75% for the digestive gland and gill. Transcripts relating to protein metabolism and respiration dominated including ribosomal proteins, cytochrome oxidases and NADH dehydrogenase subunits. Tissue specific variation was identified in transcripts associated with mitochondrial energy metabolism, with the digestive gland and gill having the greatest transcript abundance. Using fragment recruitment it was also possible to identify sites of potential small RNAs involved in mitochondrial transcriptional regulation. Sex ratios based on Vitelline Envelop Receptor for Lysin and Vitelline Coat Lysin transcript abundances, indicated that an equal sex distribution was maintained. Taxonomic profiling of the M. galloprovincialis tissues highlighted an abundant microbial flora associated with the digestive gland. Profiling of the tissues for genes involved in intermediary metabolism demonstrated that the gill and digestive gland were more similar to each other than to the other two tissues, and specifically the foot transcriptome was most dissimilar. CONCLUSIONS: Pyrosequencing has provided extensive genomic information for M. galloprovincialis and generated novel observations on expression of different tissues, mitochondria and associated microorganisms. It will also facilitate the much needed production of an oligonucleotide microarray for the organism.

Project description:Transcriptional profiling of mussel (Mytilus galloprovincialis) digestive gland tissue comparing control tissue with tissue obtained from animals exposed for four days to sublethal amounts of Nickel, Chlorpyrifos, or their mixture. Background: Mixtures 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 proposed the use of 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. Results: Stress biomarkers showed statistically significant antagonistic deviations from the reference model systems to predict mixture toxicity which are based either on simple additivity or non-interaction. While toxicokinetic modeling did not explain mixture interactions, gene expression profiling and further functional genomics analysis provided clues that the decrement of toxicity may arise from the development of specific toxicodynamics. Multivariate statistics of microarray and quantitative PCR data showed two separate patterns for the single chemicals and a composite complex profile for the mixture suggesting the occurrence of interactive molecular responses. The latter signature accounted for differentially expressed genes whose relative expression values were either in trend or in contrast with those found due to single substance treatments and a relevant set of sequences which were exclusive of the mixture gene list. Conclusion: The functional genomics assessment fits with biological data to indicate the occurrence of different toxicodynamic events and ‘in general’ a decrease of toxicity, driven by the mitigation or even abolishment of lysosomal responses such as the lipid metabolic disorder observed exclusively in single chemical-exposed samples. Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecological relevant organisms.

Project description:BACKGROUND: Sessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes. RESULTS: We have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways. CONCLUSIONS: The Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world.

Project description:Transcriptional profiling of mussel (Mytilus galloprovincialis) digestive gland tissue comparing control tissue with tissue obtained from animals exposed for four days to sublethal amounts of Nickel, Chlorpyrifos, or their mixture. Background: Mixtures 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 proposed the use of 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. Results: Stress biomarkers showed statistically significant antagonistic deviations from the reference model systems to predict mixture toxicity which are based either on simple additivity or non-interaction. While toxicokinetic modeling did not explain mixture interactions, gene expression profiling and further functional genomics analysis provided clues that the decrement of toxicity may arise from the development of specific toxicodynamics. Multivariate statistics of microarray and quantitative PCR data showed two separate patterns for the single chemicals and a composite complex profile for the mixture suggesting the occurrence of interactive molecular responses. The latter signature accounted for differentially expressed genes whose relative expression values were either in trend or in contrast with those found due to single substance treatments and a relevant set of sequences which were exclusive of the mixture gene list. Conclusion: The functional genomics assessment fits with biological data to indicate the occurrence of different toxicodynamic events and ‘in general’ a decrease of toxicity, driven by the mitigation or even abolishment of lysosomal responses such as the lipid metabolic disorder observed exclusively in single chemical-exposed samples. Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecological relevant organisms. Mussel MytArray 1.0 (Platform GPL1799)-based arrays: Three-condition experiment. Dual color competitive hybridizations. Common reference (vehicle treated animals, 0.02% Dimethyl sulfoxide); Pools of six animals. Biological replicates: 4 controls, 4 Nickel, 4 Chlorpyrifos. One replicate per array. Mussel MytArray 1.1 (Platform GPL10269)-based arrays: Four-condition experiment. Dual color competitive hybridizations. Common reference (vehicle treated animals, 0.02% Dimethyl sulfoxide); Pools of six animals. Biological replicates: 5 controls, 1 Nickel, 3 Chlorpyrifos, 5 mixture. One replicate per array.

Project description:Transcriptional profiling of natural population of mussels (Mytilus galloprovincialis) -digestive gland tissue- comparing female individuals sampled in the Bizerta Lagoon, Tunisia, across May 2007 - April 2008. Background: 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, we used a medium-density cDNA microarray (1.7 K probes) in dual-color competitive hybridization analyses. Results: Hierarchical clustering of digestive gland microarray data showed two main branches, distinguishing profiles associated with the M-bM-^@M-^\hotM-bM-^@M-^] months (MayM-bM-^@M-^SAugust) from the other months. Genes involved in chitin metabolism, associated with mussel nutrition and digestion, showed higher expression during summer. Moreover, we found different gene expression patterns in the digestive glands of males and females during the four stages of mussel gonadal development. Microarray data from gonadal transcripts also displayed clear patterns during the different developmental phases with peak relative mRNA abundance at the ripe phase (stage III) for both sexes. Conclusion: These data showed a clear temporal pattern in gene expression 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 gene expression. Loop design: Female individuals from 12 different months across one year (May 2007 to April 2008). Dual color competitive hybridizations (month n vs month n+1) including dye swap. Single individuals. Three (May 2007 to December 2007) or four biological replicates (January 2008 to April 2008 ). One replicate per array.

Dataset Information

The occurrence of tissue-specific twitchin isoforms in the mussel Mytilus galloprovincialis.

Publications

The occurrence of tissue-specific twitchin isoforms in the mussel Mytilus galloprovincialis.

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