Development of a microarray for Enchytraeus albidus (Oligochaeta): Preliminary tool with diverse applications. Enchytraeus albidus

Standard bioassays allow hazard assessment at the population level, but much remains to be learned about the molecular level response of organisms to stressors. The main aim of this study was the development of a DNA microarray for Enchytraeus albidus, a common soil worm species. Further, this microarray was tested using worms exposed to Cu, phenmedipham, and different soil types. Hybridization onto the developed microarray revealed several genes with homology to known sequences. Genes of interest were confirmed through real-time polymerase chain reaction. It was possible to discriminate between natural and chemical stressors and chemical concentrations. Gene responses were detected under conditions known to have effects in the reproduction of individuals. It was confirmed that the integration of different endpoints improves the assessment process and enhances the understanding of the modes of action of stressors. The chemical stress–induced genes were related to factors such as immune response, stress response, metabolic processes, and/or signal transduction. The present study represents the first step of a gene-level study in the ecologically relevant and standard test species E. albidus. It demonstrates the usefulness of cDNA normalization in the production of cDNA libraries of ecotoxicological standard organisms that are not genome models like E. albidus. Overall design: Fluorescently labelled cDNA, from enchytraeids exposed during 2 days to control LUFA 2.2 (Cy3) soil and to the different exposure conditions (Cy5), was synthesized for microarray analysis and hybridizations were performed. After scanning, spots were identified and ratios quantified using the Genepix software 5.0 (Axon Instruments). Statistical analysis of the microarrays was performed using limmaGUI package (1.18.0) (Smyth, 2005) in the R (2.8.0) software environment (http://www.R-project.org/). After being submitted to local background subtraction, microarrays were normalized using global loess method. To statistically evaluate the differential gene expression between the different conditions, a gene-per-gene linear model (limma – linear model for microarray analysis) and empirical Bayes methods were applied. The results were then corrected for multiple testing using the Benjamini-Hochberg’s method (adjusted p<0.05 was considered significant) (Benjamini and Hochberg, 1995).

传统生物测定法可开展种群水平的危险度评估，但关于生物体对胁迫因子的分子水平响应，仍有诸多问题有待阐明。本研究的核心目标是构建针对白色仙女虫（Enchytraeus albidus）——一种常见土壤环节动物——的DNA微阵列（DNA microarray）。随后，本研究利用暴露于铜（Cu）、甜菜安（phenmedipham）以及不同类型土壤的仙女虫对该微阵列进行测试。将样本与构建完成的微阵列进行分子杂交后，鉴定出多个与已知序列具有同源性的基因。目标基因通过实时聚合酶链式反应（real-time polymerase chain reaction）得以验证。本研究可区分自然胁迫因子与化学胁迫因子，同时也能识别不同的化学胁迫浓度。在已知会对个体繁殖产生影响的实验条件下，成功检测到了基因表达响应。研究证实，整合多维度生物学终点指标可优化评估流程，并加深对胁迫因子作用模式的理解。化学胁迫诱导的基因与免疫应答、应激反应、代谢过程以及信号转导等生物学过程密切相关。本研究针对生态相关的标准试验物种白色仙女虫（E. albidus）开展，是该物种基因水平研究的第一步，同时证实了cDNA归一化（cDNA normalization）在构建非基因组模式生物（如白色仙女虫）的生态毒理学标准物种cDNA文库中的应用价值。

整体实验设计：从暴露2天的仙女虫中合成荧光标记的cDNA：对照组采用LUFA 2.2标准土壤样本并以Cy3标记，不同胁迫处理组样本以Cy5标记，将上述标记产物用于微阵列分析并完成分子杂交。扫描完成后，使用Genepix 5.0软件（Axon仪器公司）对芯片点样信号进行识别并计算荧光比值。微阵列数据的统计分析基于R（2.8.0）软件环境（http://www.R-project.org/）中的limmaGUI包（版本1.18.0，Smyth, 2005）完成。在进行局部背景扣除后，采用全局loess法对微阵列数据进行归一化处理。为统计评估不同处理组间的基因差异表达情况，本研究采用了逐基因线性模型（limma——微阵列分析线性模型）以及经验贝叶斯方法。随后采用Benjamini-Hochberg法对多重检验结果进行校正（校正后p值<0.05视为具有统计学显著性，Benjamini和Hochberg, 1995）。