Functional categorization of transcriptome in the species Symphysodon aequifasciatus Pellegrin 1904 (Perciformes: Cichlidae) exposed to benzo[a]pyrene and phenanthrene. Symphysodon aequifasciata

This study aims to evaluate the transcriptome alterations, through cDNA libraries, associated with the effects of the combination of two PAHs, benzo[a]pyrene (0.5μg/L) and phenanthrene (50μg/L), present in crude oil, on specimens of Symphysodon aequifasciatus (discus fish) after 48h of exposure. The cDNA libraries were constructed according to the SOLiDTM SAGETM protocol for sequencing in the SOLiD v.3 Plus sequencer. The results were analyzed by bioinformatics and differentially expressed genes were categorized using the gene ontology program (AmiGO v.1.8). The functional categories (terms) found in the gene ontology and the gene network generated using STRING software were used to predict the adverse effects in the liver. In the present study, 27,127 genes (compared to Danio rerio database) were identified. Considering only those genes with a p-value less than or equal to 0.05 and a greater than or equal to two-fold change in expression across libraries, we found 804 genes, 438 down-regulated (54%) and 366 up-regulated genes (46%), in the experimental group compared to the control group. Out of this total, 327 genes were successfully categorized, 174 down-regulated and 153 up-regulated genes, using gene ontology tool. The final confidence gene network, analyzed in STRING, was composed by 199 nodes of proteins, 124 of them resulted in 274 interactions. The results showed that even an acute exposure of 48 h caused metabolic change in response to environmental contaminants, resulting in changes of cell integrity, changes in oxidation-reduction processes, disturbances of intracellular signaling and changes in the immune response of discus fish. Also the gene network interactions have showed no central interplay cluster, exhibiting interconnected clusters interactions and sparsely connected sub-networks. These findings highlight that even an acute and sublethal exposure of PAHs can cause metabolism changes that may affect this fish species survival rates. Our findings using the SAGE-method and the SOLiD sequencer showed that this is a powerful tool for gene expression analysis in discus fish, a non-model organism. Overall design: Transcriptome analysis of differentially expressed genes in liver in benzo[a]pyrene and phenanthrene-exposed and non-exposed fish (N=6 per treatment)

本研究旨在通过cDNA文库，探究原油中存在的两种多环芳烃（Polycyclic Aromatic Hydrocarbons, PAHs）——苯并[a]芘（0.5μg/L）与菲（50μg/L）联合暴露48小时后，对七彩神仙鱼（Symphysodon aequifasciatus）标本产生的相关转录组变化。本研究构建的cDNA文库遵循SOLiDTM SAGETM实验方案，使用SOLiD v.3 Plus测序仪进行测序。研究结果通过生物信息学手段进行分析，差异表达基因采用基因本体（Gene Ontology, GO）分析工具AmiGO v.1.8完成分类。基于基因本体注释得到的功能类别（术语）与使用STRING软件构建的基因互作网络，可预测肝脏组织的不良效应。本研究共鉴定出27127个基因（比对至斑马鱼Danio rerio参考数据库）。仅考虑p值≤0.05且表达量变化倍数≥2的基因，本研究在实验组与对照组间共筛选得到804个差异表达基因，其中438个基因表达下调（占比54%），366个基因表达上调（占比46%）。其中，327个基因可通过基因本体工具完成功能分类，包括174个下调基因与153个上调基因。经STRING分析得到的最终置信基因互作网络包含199个蛋白质节点，其中124个节点存在274条互作关系。研究结果显示，即便仅为48小时的急性暴露，也可诱导七彩神仙鱼产生针对环境污染物的代谢响应，进而引发细胞完整性改变、氧化还原过程异常、细胞内信号通路紊乱以及免疫应答失调。此外，该基因互作网络未呈现核心互作模块，仅存在模块间的相互连接与连接稀疏的子网结构。上述结果表明，即便为亚致死剂量的多环芳烃急性暴露，也可引发代谢改变，进而威胁该鱼类的生存存活率。本研究采用SAGE（Serial Analysis of Gene Expression, 基因表达系列分析）方法与SOLiD测序平台开展实验，结果证实该方案可有效用于非模式生物七彩神仙鱼的基因表达分析。实验整体设计：对苯并[a]芘与菲暴露组及非暴露组七彩神仙鱼肝脏组织的差异表达基因进行转录组分析（每组设置6个生物学重复）。