Analyses of transcriptomic responses to heavy metal exposure in Manila clam collected in Venice lagoon areas characterized by different anthropogenic impact

A Ruditapes philippinarum microarray platform was developed to assess variations on transcritpomic response to copper exposures in Manila clam colelctted in Venice lagoon areas subjected to different anthropogenic impact A comparative analysis of gene expression was conducted in Manila clam R.philippinarum collected in different areas of Venice lagoon and exposed to copper. After sampling, clams were transferred to the laboratory in refrigerated boxes. Thirty clams per site were immediately used (WILD) for tissue collection. In particular, to perform gene expression analyses digestive gland has been dissected and transferred in RNAlater® solution. To increase homogeneity across samples the average size of all collected animals was similar. A total of 100 clams have been considered for chemical analyses to reveal concentration of dioxins and furans (PCDDs/PCDFs), polychlorinated biphenyls (PCBs), dioxin-like PCBs (PCBs-DL), hexachlorobenzene (HCB), Cu, Cd, Pb, Hg), in clam whole-body. The remaining clams were kept in the laboratory for 7 days in two different tanks with a sandy bottom and aerated seawater (31 ± 1 psu salinity, 8 ± 0.5 °C, 8.1 pH) and were fed microalgae (Isochrysis galbana). The seawater was renewed every other day. After the 7-day acclimation period (ACCL), 30 clams per tank (=site) were used for digestive gland collection, whereas the remaining clams were exposed to 50 µg Cu l-1 (as CuSO4) for 7 more days, which is an environmentally realistic concentration. In this case, the clams were maintained in glass aquaria (without sediment) containing aerated seawater (1 l per animal). The seawater was changed daily, and copper and microalgae (I. galbana at an initial concentration of approximately 150,000 cells l-1) were added. Digestive gland were collected after 1 (T1), 3 (T2) and 7 (T3) days of exposure. Gene expression profiling was performed using an Manila clam-specific oligo-DNA microarray of 15,037 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.