Sviluppo e validazione di un cDNA microarray a scala genomica in Mytilus galloprovincialis

Marine bivalves of the genus Mytilus are intertidal filter-feeders commonly used as biosensors of coastal pollution. Indeed, mussels readily bioaccumulate both organic and metal pollutants, and react to changes with physiological and genetic mechanisms. However, one main problem in using mussels as bio-sensors is the poor characterization of their functional and defence reactions. The currently used biomarkers provide insufficient understanding of mussel physiology status or stressor-induced effects, and knowledge of mussel genome structure, function and evolution are still lacking. Therefore, genomic approaches are needed to increase our knowledge of physiological processes and to better understanding molecular and cellular mechanisms involved in the stress responses. Based on the production and massive sequencing of 3'ESTs library from main mussel tissues, we arrayed, in collaboration of C.R.I.B.I., University of Padova, the first collection of selected transcript 1714 tags on glass slides as MytArray 1.0. In order to use this molecular platform defined in 2003, it has been necessary to refresh the physical collection of the bacterial clones bearing cDNA inserts in recombinant plasmids. Then, microarrays have been spotted and hybridisation experiments realized. The potential use of this novel tool was evaluated by analyzing gene expression changes in mussels exposed to mixture of toxic and genotoxic metals in laboratory and in their natural environment (Venice lagoon, Italy). Among the potential toxic contaminants, heavy metals can damage cell components, disturb cell signalling and are expected to modulate the expression of many genes. After competitive ibridisation experiments on MytArry 1.0, we found gene expression changes in gill and digestive gland in mussels treated with increasing micromolar doses of a metal mixture (Cd, Cu, Hg). Results appear instructive and consistent with the enhance of chromosomal damage in gill cells of same mussels (evaluated as increases of micronuclei and other nuclear abnormalities). The transcriptional changes raised in dose-dependent manner and transcripts showing consistent expression trends revealed the complexity of the induced cellular response, with the most evident changes referring to: ion homeostasis (i.e metallothionein 10IV isoform, ferritin), protein turnover (sequestosome 1 and proteasome subunits) and chaperones (hsp70, hsp90, shsp24), regulation of apoptosis and DNA damage-inducible transcripts (gadd45, apoptosis inhibitor 2), cell motility and adhesion. The subsequent real-time PCR performed supports further these results. To assess the potential use of the mussel microarray in environment, I evaluated the transcriptional digestive gland profiles of mussels living in the Venice lagoon. Venice lagoon is a unique case and significant concentrations of cadmium, mercury, PAHs, PCBs and dioxin-like compounds are recurrently detected in theindustrial area near the town. Native mussels were sampled in the early summer in 2005, 2006, 2007, from zones affected differently by chemical pollution: from industrial district channels and from Lido lagoon inlet relatively uncontaminated. Offshore mussel farm, was chosen as a source of reference. I performed the competitive hybridisation experiments on DG samples and detected differentially expressed genes are grouped into different functional categories. In mussels of the industrial canals (Marghera, Venice) microarray analysis performed on individual mussels indicated a general profile similarity which make them distinguishable from mussels living in less polluted sites. Chemical data support this work hypothesis. The number of biological replicates influence the study size but, how much the individual variability can influence our studies? To try to answer to this question, I performed new hybridisation experiments by using the pool from the same samples. The overall clustering of transcriptional profiles can be compared with data already obtained from individual mussel tissues even though only transcripts with significant expression values are found. In all three years, the suggestive presence of gene markers, tracing organic contaminants and heavy metals in mussels from the industrial district is consistent with reported trends of chemical contamination. Finally, I contributed to the preparation of samples generating new primary cDNA libraries and a unique normalized library from mussels treated with with chemical and biological contaminants in order to enlarge the transcript collection and better understanding transcriptional mussel responses (work in collaboration with C.R.I.B.I and UniTrieste). Massive sequencing of the primary and normalized libraries yielded positive results and information obtained are going to be organized in the first intergrated Mytilus databatase.