Gut bacterial community structure shifts in successive generations of Spodoptera exigua under short-term thermal stress

Long-term studies that advance our mechanistic understanding of gut bacterial symbionts of insect hosts in response to the successive generations of short-term thermal stress are lacking. The beet armyworm, Spodoptera exigua is a notorious agricultural pest worldwide and has often experienced stressful temperature fluctuations in field environments. In this study, 1,795,224 reads and 2,565 operational taxonomic units (OTUs) were detected in 23 gut samples of S. exigua fed for five successive generations using 16S rRNA high-throughput sequencing technology. Overall, we identified 618 bacterial genera from 30 phyla, and Proteobacteria and Firmicutes were the most dominant phyla. Alpha-diversity of gut microbiome revealed significant differences among these generation treatment groups. We detected the highest bacterial richness and alpha diversity in the fifth generation and the lowest in the first generation under short-term thermal stress. Beta diversity indicated that the gut microbial community structure of S. exigua in the first generation was significantly different from that of other generations. Finally, PICRUSt analysis showed that most functional prediction categories were related to RNA processing and modification. Our findings represent the first investigation of the successive generations of short-term thermal stress that can affect the microbial communities associated with lepidopteran insects and broaden our understanding of the ecological adaptation of this species.

Methods

Amplification, library preparation and sequencing of the bacterial 16S rRNA gene

The highly variable V3-V4 region of the 16S rRNA gene was amplified from genomic DNA using the universal bacteria-specific primers 341 F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHVGGGTATCTAATCC-3') (Claesson et al. 2010). Polymerase chain reaction (PCR) was performed in triplicate using a 20 卩L mixture containing 1 卩L DNA template, 2 卩L dNTPs (2.5 mM), 0.5 皿 FastPfu polymerase (5 卩M/pL), 2.5 卩L 5 x FastPfu Buffer, 0.5 卩L each of forward and reverse primer (5 pM) and 13 pL ddH?O. PCR cycle conditions were initial denaturation at 98°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 45 s, and final extension at 72°C for 10 min. The concentration and purity of DNA samples were measured by Nanodrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). PCR products were purified with AMPure XT beads (Beckman Coulter, Brea, CA, USA) and Qubit (Invitrogen, USA). According to Illumina genomic DNA library preparation program, the pooled DNA products were used to construct an Illumina peer library. Amplified libraries were sequenced using 2 x 250 peer protocol (Hangzhou Lc-Bio Technologies Co., Ltd) on an Illumina MiSeq platform. Raw reads were deposited in the National Center for Biotechnology Information (NCBI) sequence read archive (SRA) database (BioProject accession no. PRJNA807462).

目前尚缺乏能够从机制层面阐明昆虫宿主肠道细菌共生体如何响应多代短期热胁迫的长期研究。甜菜夜蛾（Spodoptera exigua）是全球范围内极具危害性的农业害虫，在田间环境中经常会遭遇极端温度波动。本研究采用16S rRNA高通量测序技术，对连续5代饲养的甜菜夜蛾的23份肠道样本进行分析，共检测到1795224条读长（reads）与2565个操作分类单元（operational taxonomic units, OTUs）。本研究共从30个菌门中鉴定出618个细菌属，其中变形菌门（Proteobacteria）和厚壁菌门（Firmicutes）为优势菌门。肠道微生物组的α多样性分析显示，各代次处理组间存在显著差异：短期热胁迫下，第5代的细菌丰富度与α多样性最高，而第1代最低。β多样性分析表明，第1代甜菜夜蛾的肠道微生物群落结构与其余各代存在显著差异。最后，通过PICRUSt分析发现，绝大多数功能预测类别与RNA加工及修饰相关。本研究首次探究了短期热胁迫的多代暴露对鳞翅目昆虫相关微生物群落的影响，加深了我们对该物种生态适应性的认知。

方法

细菌16S rRNA基因的扩增、文库构建与测序
采用细菌通用引物341 F（5'-CCTACGGGNGGCWGCAG-3'）与805R（5'-GACTACHVGGGTATCTAATCC-3'），从基因组DNA中扩增16S rRNA基因的高变V3-V4区（Claesson等，2010）。聚合酶链式反应（PCR）设置3次重复，反应体系总体积为20 μL，包含1 μL DNA模板、2 μL dNTPs（2.5 mM）、0.5 μL FastPfu聚合酶（5 μM/μL）、2.5 μL 5×FastPfu缓冲液、上下游引物各0.5 μL（5 μM）以及13 μL 双蒸水（ddH₂O）。PCR循环程序为：98℃预变性5 min；随后30个循环：94℃变性30 s，54℃退火30 s，72℃延伸45 s；最终72℃延伸10 min。采用Nanodrop 2000C分光光度计（Thermo Scientific，美国马萨诸塞州沃尔瑟姆）检测DNA样本的浓度与纯度。PCR产物经AMPure XT磁珠（Beckman Coulter，美国加利福尼亚州布雷亚）与Qubit（Invitrogen，美国）纯化。参照Illumina基因组DNA文库构建流程，将混合后的DNA产物构建为Illumina双端文库。扩增后的文库采用2×250 bp双端测序策略，由杭州LC-Bio科技有限公司在Illumina MiSeq平台上完成测序。原始读长数据已提交至美国国家生物技术信息中心（NCBI）的序列读取归档（SRA）数据库，生物项目编号为PRJNA807462。