Rhinella arenarum transcriptomics: assembly, annotation and gene prediction after organophosphate exposure

PIRES, NATALIA SUSANA; LASCANO, CECILIA INÉS; MARDIROSIAN, MARIANA NOELIA; VENTURINO, ANDRÉS; CESCHIN, DANILO GUILLERMO

Águas de Lindóia, SP

Congreso; GENÉTICA 2017 - Brazilian-International Congress of Genetics; 2017

Sociedade Brasileira de Genética

Introduction: Northern Patagonia is an irrigated area holding 95% of the exportable production of apple and pear of Argentina. Assessing the impact of pesticides and other environmental toxicants released during anthropogenic activities requires the inclusion of studies on native species. It is known that there are biochemical and molecular responses which are manifested in toxicant concentration ranges below those that cause physiological alterations and lethal effects. The determination of the mechanisms of action and response is needed to develop sensitive and early biomarkers of toxic exposure. Biomonitoring of environmental impact of pesticides using native species is a preferred resource for its ecological significance. However, it proves to be an arduous task in the absence of knowledge, for instance, of their genomes. To obtain the genome sequence is still very expensive, but a transcriptomic approach using Next Generation Sequencing (RNA-Seq) is affordable and provides a huge set of information on gene expression. Rhinella arenarum is a toad widely distributed in Argentina and less abundant in Brazil, Bolivia and Uruguay. This amphibian can be used in ecotoxicological research and environmental toxicology.Objective: To obtain, assemble, annotate and gene predict using the transcriptome of Rhinella arenarum, acquired after exposure to organophosphate pesticides. Materials and Methods: R. arenarum embryos were obtained by in vitro fertilization. Larvae (complete operculum (CO) + 11 days) were exposed to sublethal concentrations of the organophosphates Azinphos Methyl and Chlorpyrifos. Samples were collected for RNA purification and massive sequencing. Results and Conclusions: After verification of the quality of the sequences, the R. arenarum transcriptome was assembled using the Trinity software (https://github.com/trinityrnaseq/trinityrnaseq/wiki) with the default parameters. A total of 204,302,781 PE sequences were used for de novo assembly. The set generated a total of 250,650 transcripts, with a Median of 372 bp and a Mean of 815 bp in length. The transcripts were compared using the BlastX tool with the non-redundant SwissProt protein database for annotation and gene prediction. Thus, 19,357 annotations were obtained, of which 4,635 were aligned with more than one species, and finally a total of 14,722 unique annotated transcripts were obtained. Moreover, primers were designed for several transcripts and were positively validated by RT-PCR. The candidates belonging to different pathways were selected: Glutathione S-Transferase (GST) and Glutathione Reductase (GSHR) for Detoxification Pathway; Catalase (CAT) and Superoxide Dismutase (SOD) for Oxidative Stress Pathway and Spermine Oxidase (SMOX), Polyamine Oxidase (PAO), Diamine Oxidase (DAO), Adenosylmethionine Decarboxylase 1(AMD1) and Ornithine Decarboxylase (ODC) for Polyamines Pathway. Thus, the availability of R. arenarum's transcriptome opens new possibilities for research in environmental toxicology at the molecular level.