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.

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).

Gut microbiome of Spodoptera exigua data.

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