During long-term coevolution, the gut microbiota is an essential part of the host insect's growth and development, digestion and detoxification processes. The rice stem borer (RSB), Chilo suppressalis, is one of the most destructive rice pests and has caused serious economic losses to major rice-producing areas of China. However, little is known about the gut microbiota of this moth linked to adaptability to different geographical environments. We investigated the gut microbiota of RSB populations collected from five sites in the typical areas of northern China using 16S rDNA gene sequencing on an Illumina MiSeq platform. In total, 453,853,707 reads and 1650 operational taxonomic units (OTUs) were obtained in 25 RSB samples. A total of 697 bacterial genera belonging to 30 phyla were detected, and Proteobacteria and Firmicutes were the most dominant phyla. Richness and alpha-diversity metrics revealed variation in the RSB gut bacteria among RSB populations. The highest bacterial diversity was found in Beizhen, western Liaoning Province. Principal coordinates analysis (PCoA) and clustering analysis (UPGMA) showed that the gut microbial community structure in western Liaoning differs greatly from that in other regions. LDA effect size (LEfSe) analysis indicated that the microbial groups had significant effects among the middle, northern and western regions. PICRUSt analysis demonstrated that microbial functions closely associated with the gut microbiome were membrane transport, amino acid metabolism and carbohydrate metabolism. In addition, redundancy analysis (RDA) of microbial community composition revealed the average annual temperature, average annual relative humidity and cumulative annual precipitation to be a significant source of variation in patterns of gut microbial abundance. Accordingly, our study provided important insights into the investigation of insect-bacteria symbioses and evaluated gut microorganisms as biocontrol agents for this pest and other agricultural lepidopterans.

Bacterial 16S rRNA gene amplification and high-throughput sequencing

The universal primer pair 341F/805R (341 F 5'-CCTACGGGNGGCWGCAG-3', 805 R 5'-GACTACHVGGGTATCTAATCC-3') was used to amplify the V3 and V4 hypervariable regions of the 16S rRNA gene (Claesson et al., 2010), where the barcodes are patterns based on eight-base sequences unique to each sample. Polymerase chain reaction (PCR) amplification was performed in triplicate 20 μL mixture containing1 μL template DNA (about 50 ng), 2 μL dNTPs (2.5 mM), 0.5 μL FastPfu polymerase (5 μM/μL), 2.5 μL 5 × FastPfu Buffer, 0.5 μL each primer (5 μM) and 13 μL ddH2O. The reaction steps of the mixture were 98°C for 30 s, 98°C for 30 s, 98°C for 10 s, 54°C for 30 s, 72°C for 45 s, 30 annealing cycles, and 72°C for 10 min. The concentration and purity of the DNA samples were detected by agarose gel electrophoresis and a Nanodrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). The products of PCR were purified and analysed using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and Qubit (Invitrogen, USA). According to the Illumina genomic DNA library preparation procedure, the pooled DNA products were used to construct an Illumina peer library. Then, the library was paired-end sequenced (2 × 250) on an Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd) in accordance with the standard protocols. 

Gut microbiome of Chilo suppressalis data.

Gut bacterial diversity and communities of 5 Chilo suppressalis populations sampled in the typical bivoltine areas of northern China.