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Key metabolic pathways involved in xenobiotic biotransformation and stress responses revealed by transcriptomics of the mangrove oyster Crassostrea brasiliana.Aquatic Toxicology http://dx.doi.org/10. 1016/j.aquatox.2015.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Highlights-Thetranscriptome of the mangrove oyster Crassostrea brasilianawas sequenced.-The sequencing effort dramatically expanded the existing cDNA sequences available for the species.-Global analysis for transcription in the oyster treated with phenanthrene, diesel and domestic sewage was performed.-The pollutants altered mRNAs for genes in biotransformation, antioxidant and stress response pathways. Abstract 2The Brazilian oyster Crassostrea brasiliana was challenged to three common environmental contaminants: phenanthrene, diesel fuel water-accommodated fraction (WAF) and domestic sewage. Total RNA was extracted from the gill and digestive gland, and cDNA libraries were sequenced using the 454 FLX pla orm. The assembled transcriptome resulted in ˜20,000contigs, which were annotated to produce the first de novo transcriptome for C. brasiliana.Sequences were screened to identify genes potentially involved in the biotransformation of xenobiotics and associated antioxidant defence mechanisms. These gene families included those of the cytochrome P450 (CYP450), 70kDa heat shock, antioxidants, such as glutathione Stransferase, superoxide dismutase, catalase and also multi-drug resistance proteins. Analysis showed that the massive expansion of the CYP450 and HSP70 family due to gene duplication identified in the Crassostrea gigas genome also occurred in C. brasiliana, suggesting these processes form the base of the Crassostrea lineage. Preliminary expression analyses revealed several candidates biomarker genes that were up-regulated during each of the three treatments, suggesting the potential for environmental monitoring.

Diesel fuel can cause adverse effects in marine invertebrates by mechanisms that are not clearly understood. The authors used suppressive subtractive hybridization to identify genes up- and downregulated in Crassostrea brasiliana exposed to diesel fuel. Genes putatively involved in protein regulation, innate immune, and stress response, were altered by diesel challenge. Three genes regulated by diesel were validated by quantitative real-time polymerase chain reaction. This study sheds light on transcriptomic responses of oysters to diesel pollution.

Oysters Crassostrea rhizophorae and Crassostrea gigas were kept for fourteen days at four sites in São José, SC, Brazil, chosen according to a sewage discharge contamination gradient. Enzymatic activities of CAT, GST, G6PDH and GR were evaluated in gills and digestive glands. Higher CAT activity was observed in tissues of C. rhizophorae in response to sewage contamination possibly indicating peroxisome proliferation induction. C. gigas showed elevated G6PDH activity in digestive gland, and GR in gills, after domestic sewage exposure. In conclusion, C. rhizophorae and C. gigas showed different biochemical responses after in situ exposure to domestic sewage. However, C. rhizophorae showed more significant changes in CAT suggesting that this organism could be a better monitor to this kind of effluent.

In order to investigate the influence of domestic sewage on the gene expression of Pacific oysters Crassostrea gigas, suppression subtractive hybridization (SSH) method was employed. Oysters were sampled at a farming area and, after 10 days of acclimation in the laboratory, were exposed to untreated domestic sewage diluted 33% for 48 h. Gills of male oysters were excised for total RNA extraction. mRNA was purified and the differential gene expression was analyzed by SSH. We obtained 61 cDNA sequences but only 15 were identified, which includes fatty acid binding protein, multidrug resistance protein, omega glutathione S-transferase, cytochrome P450 isoform CYP356A1, among others. The identified genes are associated with different metabolic functions like biotransformation, membrane transport, aerobic metabolism and translational machinery, evidencing the potential toxic effects elicited by these effluents.