Approved Abstracts

Analysis of Crassostrea gasar transcriptome reveals candidate genes involved in metal metabolism



Author(s): Ferreira CP; Moreira RS; Toledo-Silva G; Bainy ACD; Lüchmann KH;
Presenter: Karim Hahn Lüchmann

The oyster Crassostrea gasar shows a wide geographic distribution along the Brazilian coast and is susceptible to contamination caused by anthropic origin, particularly by metals. Molecular biology techniques have been largely applied in aquatic ecotoxicological studies, but most studies performed with C. gasar are related to the effects of organic contaminants and very few with metals. When metals enter the cells, they induce the synthesis of cysteine-rich molecules, such as metallothionein (MT) and glutathione (GSH), resulting in decreased intracellular levels of free metal ions. These thiol-containing peptides and proteins have an important role in metal metabolism by controlling the uptake, transport, accumulation, and cellular detoxification. Thus, this study focused on searching for genes involved in metal metabolism with ecotoxicological interest to support environmental biomonitoring programs using C. gasar as a bioindicator. To accomplish our goals, we have revisited a total of 399,291 454-reads which were deposited at the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA) [GenBank: SRX790900]. A clear consensus C. gasar transcriptome from a set of de novo assemblies (Trinity v2.11.0, SOAPdenovo-Trans v1.04, Velvet v1.2.10, Oases v0.2.09, Trans-ABySS v2.0.1, and rnaSPAdes v1) was generated by using the EvidentialGene program, which significantly reduced the presence of false transcripts within the final assembly of isogroups. We found a total number of assembled contigs of 7,911. The Trinotate pipeline was utilized to perform the functional annotation due to the number of different well-referenced methods, such as homology search against sequence databases (BLAST+/SwissProt), protein domain identification (HMMER/PFAM), and comparison to currently curated annotation databases (like eggNOG, and Gene Ontology terms). Among the identified unigenes, 46.06% matched the Pfam, 54.81% to the Swissprot, 46.33% to COG, 53.84% to the GO, and 48.815% to the KEGG databases. Our analyses revealed key genes involved in metal metabolism in C. gasar. Hence, the sequences of metallothionein (MT) and zinc transport (ZIP) genes were identified and chosen as potential biomarkers of metal exposure and effect due to their central role in metal metabolism. The glutathione gamma-glutamyl cyclotransferase (CHAC) gene was also selected since its product is specific on breaking the gamma bond of glutathione and does not act on other gamma-glutamyl peptides. Additionally, the glutamate-cysteine ligase (GCLC) gene was chosen because it encodes for the catalytic subunit of the glutamate-cysteine ligase enzyme and is part of glutathione biosynthesis. Interestingly, all selected genes matched in identity with the oyster Crassostrea virginica; within three genes corresponding to MT with 85.92% (e-value 3e-28), 83.54% (e-value 1e-15), and 77.19% (e-value 5e-19) identity; three corresponding to ZIP genes 74.40% (e-value 1e-79), 89.22% (e-value 4e-59), and 91.39% (e-value 1e-149); and one copy of both CHAC and GCLC genes with 92.37% (e-value 1e-75) and 97.37% (e-value 4e-70) identity with C. virginica, respectively. These results provide valuable information about key genes for use as biomarkers of metal exposure and effects in C. gasar and contribute to our understanding of the molecular mechanisms and regulative pathways regulating metal levels and toxicity in estuarine oysters.

Keywords: Oysters; Biomarkers; Estuarine pollution

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