Supplementary Information - Chapter 2. Metabarcoding and genomic analysis of two insecticide-degrading bacteria in E. heros (Hemiptera: Pentatomidae) led to the identification of a new insect-associated Serratia species

The microbiota plays a diverse role in their hosts life. Insect-associated microbes can play a role in host nutrition, immunity, stress response, and xenobiotic metabolization, including synthetic pesticides. Gut associated insecticide-degrading bacteria are also selected by the selection pressure of intensive insecticide exposure as their hosts. For this reason, this study aimed at investigating the influence of the evolution of E. heros resistance to the neonicotinoid thiamethoxam and the pyrethroid lambda-cyhalothrin on the diversity of the gut microbiota, as well as to identify and characterize insecticide-degrading bacteria using comparative genomics strategies. We compared the gut microbiota composition of lab-maintained susceptible and insecticide-resistant strains and of field-collected adults of E. heros. The diversity of the gut microbiota was assessed using metabarcoding analysis of the V3-V4 regions of the 16S ribosomal gene (16S rRNA). The gut microbiota analysis of Euschistus heros revealed a predominance of Pseudomonadota - Gammaproteobacteria, which plays a key role in the insect gut environment. The Enterobacteriaceae Yokenella and Serratia, and the Erwiniaceae Pantoea and Erwinia were the most represented in the gut microbiota of E. heros. The gut microbial diversity of laboratory reared strains was higher than that of field-collected adults. The abundance of Serratia, Siccibacter, and Pantoea was enriched in the pyrethroid-resistant strains (RESPYR), and Serratia and Siccibacter were identified as potential biomarkers for pyrethroid-resistance. Two insecticide-degrading bacteria were isolated from the gut of E. heros and identified as Delftia acidovorans and Serratia toxivorans n. sp. using genomic comparative analyses. Our data emphasizes the importance of microbiota in metabolizing toxic compounds, and that the associated microbiota as well as the host are under selection pressure.微生物群在宿主生命活动中发挥着多样的功能。与昆虫共生的微生物可参与宿主的营养代谢、免疫调控、应激响应以及外源物质（包括合成杀虫剂）的降解过程。随着宿主持续接触杀虫剂所带来的选择压力，肠道中具有杀虫剂降解能力的共生细菌也会被筛选富集。鉴于此，本研究旨在探究Euschistus heros（以下简称E. heros）对新烟碱类杀虫剂噻虫嗪（thiamethoxam）以及拟除虫菊酯类杀虫剂氯氟氰菊酯（lambda-cyhalothrin）的抗性演化对其肠道微生物群多样性的影响，并通过比较基因组学策略鉴定并表征具有杀虫剂降解能力的共生细菌。本研究对比了实验室饲养的敏感品系、杀虫剂抗性品系，以及野外采集的E. heros成虫的肠道微生物群组成。本研究通过对16S核糖体RNA基因（16S rRNA）的V3-V4可变区进行宏条形码（metabarcoding）分析，评估肠道微生物群的多样性。对Euschistus heros的肠道微生物群分析结果显示，假单胞菌门（Pseudomonadota）γ-变形菌纲（Gammaproteobacteria）为其肠道微生物群的优势类群，该类群在昆虫肠道微环境中发挥关键作用。肠杆菌科（Enterobacteriaceae）的Yokenella、沙雷氏菌属（Serratia），以及欧文菌科（Erwiniaceae）的泛菌属（Pantoea）和欧文菌属（Erwinia）为E. heros肠道微生物群中丰度最高的类群。实验室饲养品系的肠道微生物群多样性高于野外采集的成虫。在抗拟除虫菊酯品系（RESPYR）中，沙雷氏菌属、Siccibacter以及泛菌属的丰度显著上调，且沙雷氏菌属与Siccibacter被鉴定为抗拟除虫菊酯性的潜在生物标志物。本研究从E. heros的肠道中分离得到两株具有杀虫剂降解能力的细菌，通过比较基因组学分析鉴定其分别为食酸戴尔福特菌（Delftia acidovorans）以及有毒沙雷氏菌新种（Serratia toxivorans n. sp.）。本研究结果凸显了微生物群在有毒物质代谢中的重要作用，同时证实宿主与其共生微生物群均处于选择压力之下。