Mediterranean Mussel Gene Expression Profile Induced by Okadaic Acid Exposure CHIARA MANFRIN, RENE’ DREOS, † SILVIA BATTISTELLA, ALFRED BERAN, ‡ MARCO GERDOL, LAURA VAROTTO, ‡ GEROLAMO LANFRANCHI, | PAOLA VENIER, § AND ALBERTO PALLAVICINI* Department of Life Sciences, Universit ` a di Trieste, P.le Valmaura, 9, Trieste, Italy 34148 Received November 19, 2009. Revised manuscript received September 15, 2010. Accepted September 15, 2010. Seasonal seawater temperature increases define optimal growth conditions for Dinoflagellate species which can reach high concentrations in water column and also in filter- feeding organisms like Mytilus galloprovincialis. Commonly produced by Dinophysis and Prorocentrum spp., okadaic acid (OA) and its analogues are responsible for the Diarrheic Shellfish Poisoning (DSP) syndrome in humans. Closure of shellfishing grounds is therefore recommended by the EU when DSP toxin levels in shellfish exceed 16 µg OA 100 g -1 flesh. Despite not being responsible for casualties either in humans or mussels, DSP outbreaks are considered natural events causing health and economic issues due to the frequency of their occurrence. Since gene expression studies offer a wide range of different solutions, we used a mussel cDNA microarray to evaluate gene expression changes in the digestive gland of mussels fed for five weeks with OA-contaminated nutrient. Among the differentially expressed genes we observed a general up-regulation of transcripts coding for stress proteins, proteins involved in cellular synthesis, and a few not annotated proteins. Overall, at the first time point analyzed we identified 58 candidate transcripts for OA-induced stress in mussels, half of which have unknown function. In this paper we present the first gene expression analysis performed on Mediterranean mussels exposed to okadaic acid. The characterization of these transcripts could be useful for the identification of an early physiological response to OA exposure. Introduction The Diarrheic Shellfish Poisoning (DSP) syndrome is a serious health issue caused in humans by the ingestion of molluscs contaminated with lipophilic toxins such as okadaic acid (OA) and the structurally related dinophysitoxins 1, 2, and 3 (DTX-1, -2, -3). OA production is commonly associated with Dinophysis and Prorocentrum spp. During Dinophysis spp. blooms, usually characterized by population densities of <10 4 cells L -1 , and abundance <10 3 cells L -1 , the typical filter-feeding behavior of mussels causes their toxification. The “toxic season” usually occurs in summer (June to September), and the breeders adapt their marketing strategies to face it. In 1994, in Scotland, toxicity caused by DTX-2 persisted until February even though toxic Dinophysis species had not been recorded since September. When DSP toxin (DST) levels in shellfish exceed 16 µg OA 100 g -1 mussel meat, closure of shellfishing grounds is recommended by the EU (1, 2). However, vectoring of DSTs through the food chain and their effect on marine organisms are poorly understood (3, 4). McMahon et al. (5) suggest the pairing of bioassays and chemical tests for the confirmation of the toxin presence in the monitoring of Dinophysis spp. Okadaic acid is known to inhibit the phosphatase activity of protein serine/threonine phosphatases in mammals, yeasts, and higher plants; hence, it blocks the dephospho- rylation of proteins that serve as substrates for several protein kinases with consequences on many basic processes ranging from cytoskeleton dynamics and cell adhesion to cell-cycle control, and the overall regulation of gene expression, even in M. edulis (4, 6, 7). On the other hand, the glycogen synthase is not affected by the OA levels that naturally occur in blue mussels in vivo. Although little is known about the direct effects of OA on mussel physiology, it is likely that some molecular pathways are affected by the presence of the toxin and involved in its metabolism. Suggestive of protective mechanisms against the harmful effects of OA, digestive cells of molluscan hepatopancreas are particularly rich in lyso- somes (8), which can accumulate and seize foreign com- pounds such as lipophilic xenobiotics by increasing autoph- agic activity (9). However, the DSP toxins are accumulated mainly in the digestive gland of bivalves where their transformation of the ingested DSP toxins, for example from DTX-1 to DTX-3, might also occur (3). For the reasons mentioned above the digestive gland represents the optimal target tissue to analyze possible changes at a molecular level induced by the presence of biotoxins, such as okadaic acid. The detection of biomarkers sensitive to stress at a molecular and a cellular level represents an integrated tool able to contribute in determining the toxicity of pollutants, assuming that their activation should give early warning signals of toxic chemical effects on the animals (10, 11). Microarray platforms and experimental functional genomics tools could greatly enhance the molecular understanding of toxicity mechanisms in sentinel organisms. In 1999, Nuwaysir et al. (12) described how microarray technologies could transform toxicology, leading to the creation of a new scientific field called toxicogenomics (13); subsequently, gene expression profiling has been used to identify and confirm mechanisms of action of different toxicants (14, 15), discern- ing the effects of chemical mixtures and identifying novel biomarkers of exposure. In the past few years, we have supported the development of mussel functional genomics through the production of ESTs and DNA microarrays (16, 17). Using MytArray 1.0, the first mussel cDNA microarray resulting from nonredundant ESTs, we analyzed the tran- scriptional signatures of Mediterranean mussels exposed to OA toxin in the laboratory. Following the preparation of new cDNA libraries, 7112 unique expressed sequences from about 25,000 ESTs have been organized in MytiBase, the first ESTs catalogue of M. galloprovincialis, annotated and publicly available (17). * Corresponding author phone: +39040812237; e-mail: pallavic@ units.it. † Current address: John Innes Centre Norwich Research Park, Colney, Norwich, UK NR47UH. ‡ Current address: Biological Oceanography, National Institute of Oceanography and Experimental Geophysics - OGS, Via A. Piccard 54, Trieste, IT 34010. § Current address: Department of Biology, University of Padova, Via U. Bassi 58/B, Padova, IT 35121. | Current address: CRIBI Biotechnology Centre, University of Padova, Via U. Bassi 58/B, Padova, IT 35121. Environ. Sci. Technol. XXXX, xxx, 000–000 10.1021/es102213f  XXXX American Chemical Society VOL. xxx, NO. xx, XXXX / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 A