Exploring the effects of seasonality and chemical pollution on the hepatopancreas transcriptome of the Manila clam MASSIMO MILAN,* † SERENA FERRARESSO,* CLAUDIO CIOFI, † GUIDO CHELAZZI, † CLAUDIO CARRER, ‡ GIORGIO FERRARI, § LINO PAVAN, ¶ TOMASO PATARNELLO* and LUCA BARGELLONI* *Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell’Universita ` 16, 35020 Legnaro (PD), Italy, †Department of Evolutionary Biology, University of Florence, Via Romana 17, 50125 Florence, Italy, ‡Thetis S.P.A, Laboratorio Centro Studi Microinquinanti Organici (C.S.M.O.). Magistrato alle Acque, Via Asconio Pediano, 9, 35127 Padova, §Ufficio Tecnico per l’Antinquinamento, Magistrato alle Acque di Venezia, San Polo 737, Riva del Vin, 30125 Venezia, ¶G3 Industriale, Via Milano 18, 30020 Marcon (VE) Abstract The assessment of marine environmental health is a complex but fundamental task both for ecosystem conservation and food safety related to the human consumption of marine products. Manila clams inhabiting the Venice Lagoon constitute an excellent case study for evaluating the effects of complex mixtures of industrial and urban efflu- ents on aquatic organisms. Clams were collected in different seasons at four locations within the Venice Lagoon. The sampling sites were characterized by a range of pollu- tant concentrations and included Porto Marghera, a highly polluted industrial area where clam harvesting for human consumption is strictly forbidden. Pooled soft tissues were subjected to mass spectroscopy analysis to measure the concentrations of PCDDs/PCDFs/PCBs-DL, PCBs, PBDEs, HCB and PAHs, and pooled digestive gland samples were used for gene expression profiling. While seasonal variation was found to be responsible for the largest proportion of transcriptional changes, significance analysis of microarrays quantitative correlation analysis identified 162 transcripts that were correlated with at least one class of chemicals measured in the samples from the four different sampling sites. Prediction Analysis of Microarrays (PAM) identified a minimal set of seven genes that correctly assigned samples collected in the restricted polluted area (Porto Marghera), independent of the season in which they were collected. An integrated approach combining transcriptomics and chemical analyses of the Manila clam provided a global picture of how Manila clams respond to complex mixtures of xenobiotics and their interplay with other biotic and abiotic factors. We were also able to identify gene expression signatures for different classes of chemicals and a set of robust biomarkers of exposure to these chemicals. Keywords: biotechnology, ecotoxicology, invertebrates, molluscs, population ecology, transcri- ptomics Received 25 July 2012; revision received 21 December 2012; accepted 12 January 2013 Introduction Monitoring the levels of pollutants and associated bio- logical responses in sentinel species could play an important role in environmental risk assessment and in evaluating the impact of human activities on natural habitats. Since the early 1970s, great efforts have been devoted to investigating the biological effects of con- taminants on aquatic organisms and to identifying bio- markers of chemical exposure that could indicate environmental quality and ensuing risks (e.g. Hagger Correspondence: Massimo Milan, Fax: +39 049 8272973; E-mail: massimo.milan@unipd.it © 2013 Blackwell Publishing Ltd Molecular Ecology (2013) doi: 10.1111/mec.12257