Suarez-Ulloa Victoria, Fernandez-Tajes Juan, Aguiar-Pulido Vanessa, Prego-Faraldo M Veronica, Florez-Barros Fernanda, Sexto-Iglesias Alexia, Mendez Josefina, Eirin-Lopez Jose M
Chromatin Structure and Evolution Group (Chromevol), Department of Biological Sciences, Florida International University , Miami, FL , United States of America.
McCarthy Group, Wellcome Trust Center for Human Genetics, University of Oxford , Oxford , United Kingdom.
PeerJ. 2015 Nov 19;3:e1429. doi: 10.7717/peerj.1429. eCollection 2015.
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.
背景。导致腹泻性贝类中毒（DSP）的有害藻华（HABs）对食用贝类的人类构成重大威胁。生物毒素冈田酸（OA）是一种著名的磷酸酶抑制剂和肿瘤促进剂，是急性DSP中毒的主要原因。尽管多项研究描述了高浓度OA对哨兵生物（如双壳贝类）的分子影响，但长期暴露于低（亚致死）浓度OA的影响仍不清楚。为了填补这一空白，本研究结合下一代测序和定制微阵列技术，对DSP藻华早期贻贝的转录组反应进行无偏表征。方法。将贻贝样本暴露于模拟DSP藻华早期的有害藻华事件中（200个细胞/L的利马原甲藻，持续24小时）。使用两种互补的cDNA文库制备方法对OA引发的转录组反应进行无偏表征：标准化和抑制性消减杂交（SSH）。对文库进行测序，并将读取的数据集映射到基因本体论和KEGG数据库。基于这些数据开发了定制的寡核苷酸微阵列，完成了消化腺和鳃组织的表达分析。结果。我们的研究结果表明，暴露于亚致死浓度的OA足以诱导贻贝Mytilus的基因表达改变。转录组分析显示，贻贝的蛋白酶体活性、分子转运、细胞周期调控、能量产生和免疫活性增加。相反，一些假设对OA有反应的转录本（特别是丝氨酸/苏氨酸磷酸酶PP1和PP2A）并未显示出实质性改变。消化腺和鳃组织对OA的反应相似，尽管前者的表达改变更为显著，这支持了选择该组织进行未来生物监测研究的选择。讨论。暴露于贝类安全食用法定限量内的OA浓度足以扰乱贻贝中的重要细胞过程，从而引发急剧的转录变化。通过结合cDNA文库研究和定制的OA特异性微阵列，我们的工作提供了OA特异性转录组的全面表征，与单重复RNA-seq方法相比，提高了表达谱分析的准确性。我们的数据与相关研究相结合，有助于理解海洋生物对DSP事件分子反应的潜在分子机制，为开发新一代OA污染监测工具提供有用信息。
