Microbes that live inside insects play various roles in host biology, ranging from nutrient supplementation to host defense. Although Lepidoptera (butterflies and moths) are one of the most diverse insect taxa and important in natural ecosystems, their microbiotas are little-studied, and to understand their structure and function, it is necessary to identify potential factors that affect microbiome analysis. Using a model organism, the silkworm Bombyx mori, we investigated the effects of different sample types (whole gut, gut content, gut tissue, starvation, or frass) and metagenomic DNA extraction methodologies (small-scale versus large-scale) on the composition and diversity of the caterpillar gut microbial communities. High-throughput 16S rRNA gene sequencing and computational analysis of the resulting data unraveled that DNA extraction has a large effect on the outcome of metagenomic analysis: significant biases were observed in estimates of community diversity and in the ratio between Gram-positive and Gram-negative bacteria. Furthermore, bacterial communities differed significantly among sample types. The gut content and whole gut samples differed least, both had a higher percentage of Enterococcus and Acinetobacter species; whereas the frass and starvation samples differed substantially from the whole gut and were poor representatives of the gut microbiome. Thus, we recommend a small-scale DNA extraction methodology for sampling the whole gut under normal insect rearing conditions whenever possible, as this approach provides the most accurate assessment of the gut microbiome. Our study highlights that evaluation of the optimal sample-processing approach should be the first step taken to confidently assess the contributions of microbiota to Lepidoptera.

生活在昆虫体内的微生物在宿主生物学中发挥着各种作用，从营养补充到宿主防御。尽管鳞翅目（蝴蝶和飞蛾）是最多样化的昆虫类群之一，并且在自然生态系统中很重要，但对其微生物群的研究很少，为了了解它们的结构和功能，有必要确定影响微生物组分析的潜在因素。使用模式生物，家蚕Bombyx mori，我们研究了不同样本类型（整个肠道、肠道内容物、肠道组织、饥饿或粗纤维）和宏基因组 DNA 提取方法（小规模与大规模）对毛虫肠道微生物群落组成和多样性的影响。高通量 16S rRNA 基因测序和所得数据的计算分析表明 DNA 提取对宏基因组分析的结果有很大影响：在群落多样性估计以及革兰氏阳性和革兰氏阴性之间的比率中观察到显着偏差细菌。此外，细菌群落在样本类型之间存在显着差异。肠道内容物和全肠道样本差异最小，均含有较高比例的肠球菌和不动杆菌物种; 而 frass 和 starvation 样本与整个肠道有很大不同，并且是肠道微生物组的不良代表。因此，我们建议尽可能采用小规模 DNA 提取方法在正常昆虫饲养条件下对整个肠道进行采样，因为这种方法可以最准确地评估肠道微生物群。我们的研究强调，评估最佳样品处理方法应该是自信地评估微生物群对鳞翅目昆虫的贡献的第一步。