Data from: Comparative analysis of DNA extraction methods to study the body surface microbiota of insects: a case study with ant cuticular bacteria

High-throughput sequencing of the 16S rRNA gene has considerably helped revealing the essential role of bacteria living on insect cuticles in the ecophysiology and behavior of their hosts. However, our understanding of host-cuticular microbiota feedbacks remains hampered by the difficulties to working with low bacterial DNA quantities as in individual insect cuticle samples, which are more prone to molecular biases and contaminations. Herein, we conducted a methodological benchmark on the cuticular bacterial loads retrieved from two Neotropical ant species of different body size and ecology: Atta cephalotes (~15 mm) and Pseudomyrmex penetrator (~5 mm). We evaluated the richness and composition of the cuticular microbiota, as well as the amount of biases and contamination produced by four DNA extraction protocols. We also addressed how bacterial communities’ characteristics would be affected by the number of individuals or individual body size used for DNA extraction. Most extraction methods yielded similar results in term of bacterial diversity and composition for A. cephalotes (~15 mm). In contrast, greater amounts of artifactual sequences and contaminations, as well as noticeable differences in bacterial communities’ characteristics were observed between the extraction methods for P. penetrator (~5 mm). We also found that large (~15 mm) and small (~5 mm) A. cephalotes individuals harbor different bacterial communities. Our benchmark hence suggests that cuticular microbiota of single insect individuals can be reliably retrieved provided that blank controls, appropriate data cleaning, and standardization of individual body size are considered in the experiment.

16S rRNA基因（16S rRNA gene）高通量测序（high-throughput sequencing）已为我们揭示昆虫体壁定殖细菌在宿主生态生理学与行为中的核心作用提供了极大助力。然而，由于单个昆虫体壁样本的细菌DNA含量极低，极易引发分子偏差（molecular biases）与污染（contaminations），这极大限制了我们对宿主-体壁微生物群（cuticular microbiota）反馈互作机制的认知。为此，本研究针对两种体型与生态特征存在差异的新热带区蚂蚁物种——切叶蚁（Atta cephalotes，体长约15 mm）和伪切叶蚁（Pseudomyrmex penetrator，体长约5 mm）——的体壁细菌载量开展了方法学基准测试。我们评估了体壁微生物群的物种丰富度与群落组成，以及四种DNA提取方案（DNA extraction protocols）所引入的偏差与污染程度；同时探究了用于DNA提取的昆虫个体数量及个体体型对细菌群落特征的影响。对于体长约15 mm的切叶蚁，多数DNA提取方法在细菌多样性与群落组成层面的检测结果较为一致。与之形成鲜明对比的是，针对体长约5 mm的伪切叶蚁，不同DNA提取方法间的人工序列（artifactual sequences）与污染占比更高，且细菌群落特征亦存在显著差异。我们还发现，体长约15 mm与体长约5 mm的切叶蚁个体，其体壁细菌群落组成存在显著差异。综上，本方法学基准测试结果表明：若实验中设置空白对照（blank controls）、开展合理的数据清洗（data cleaning），并统一个体体型标准，则可可靠地获取单个昆虫个体的体壁微生物群信息。