RESUMEN
Background. Harmful Algal Blooms (HABs) responsible for Diarrhetic Shellfish Poisoning (DSP) represent a major threat for human consumers of shellfish. The biotoxin Okadaic Acid (OA), a well-known phosphatase inhibitor and tumor promoter, is the primary cause of acute DSP intoxications. Although several studies have described the molecular effects of high OA concentrations on sentinel organisms (e.g., bivalve molluscs), the effect of prolonged exposures to low (sublethal) OA concentrations is still unknown. In order to fill this gap, this work combines Next-Generation sequencing and custom-made microarray technologies to develop an unbiased characterization of the transcriptomic response of mussels during early stages of a DSP bloom. Methods. Mussel specimens were exposed to a HAB episode simulating an early stage DSP bloom (200 cells/L of the dinoflagellate Prorocentrum lima for 24 h). The unbiased characterization of the transcriptomic responses triggered by OA was carried out using two complementary methods of cDNA library preparation: normalized and Suppression Subtractive Hybridization (SSH). Libraries were sequenced and read datasets were mapped to Gene Ontology and KEGG databases. A custom-made oligonucleotide microarray was developed based on these data, completing the expression analysis of digestive gland and gill tissues. Results. Our findings show that exposure to sublethal concentrations of OA is enough to induce gene expression modifications in the mussel Mytilus. Transcriptomic analyses revealed an increase in proteasomal activity, molecular transport, cell cycle regulation, energy production and immune activity in mussels. Oppositely, a number of transcripts hypothesized to be responsive to OA (notably the Serine/Threonine phosphatases PP1 and PP2A) failed to show substantial modifications. Both digestive gland and gill tissues responded similarly to OA, although expression modifications were more dramatic in the former, supporting the choice of this tissue for future biomonitoring studies. Discussion. Exposure to OA concentrations within legal limits for safe consumption of shellfish is enough to disrupt important cellular processes in mussels, eliciting sharp transcriptional changes as a result. By combining the study of cDNA libraries and a custom-made OA-specific microarray, our work provides a comprehensive characterization of the OA-specific transcriptome, improving the accuracy of the analysis of expresion profiles compared to single-replicated RNA-seq methods. The combination of our data with related studies helps understanding the molecular mechanisms underlying molecular responses to DSP episodes in marine organisms, providing useful information to develop a new generation of tools for the monitoring of OA pollution.
RESUMEN
Individuals of Mytilus galloprovincialis, contaminated with Diarrheic Shellfish Poisoning (DSP) toxins, were studied with the aim to correlate the okadaic acid (OA) body burden and the percentage of damaged haemocytes by quantifying annexin V positive cells by flow cytometry. Results showed less percentage of damaged haemocytes in high OA contaminated samples. These data were compared with results of in vitro assays of mussel haemocytes exposed to increased concentrations of OA. Similarly, haemocytes exposed to the most concentrated OA solution were less damaged.