Diversity Of Intestinal Bacteria Communities From Atrijuglans Hetaohei(Lepidoptera: Heliodinidae) And Dichocrocis Punctiferalis(Lepidoptera: Pyralidae) Larvae Estimated By PCR-DGGE And T-RFLP Analysis

This study was aimed to exam the structure and diversity of intestinal bacteria community from Atrijuglans hetaohei Yang and Dichocrocis punctiferalis Guenee larvae consuming walnut’s green husk. 16 S r RNA PCR-denaturing gradient gel electrophoresis(PCR-DGGE) and terminal-restriction fragment length polymorphism(T-RFLP) and high-throughput sequencing were employed to understand the characteristics of intestinal bacteria community from the two main pests. The results were as follows:(1)PCR-DGGE showed Wolbachia, Enterococcus, Enterobacter, Rhizobium and Pseudomonas were in the intestinal tract of A. hetaohei larvae, with phylotypes of Wolbachia, 40% of the measured sequence, being the predominant group; Wolbachia, Enterococcus, Rhizobium and Pseudomonas were in the intestinal tract of D. punctiferalis larvae, with phylotypes of Enterococcus, 62.9% of the measured sequence, being the predominant group.(2)Phylogenetic analysis of the DGGE band revealed that in all the isolated strains, 72.5% of the strains belonged to Proteobacteria, 12.5% to Firmicutes, and 15% to Bacteroidetes in the intestinal tract of A. hetaohei larvae; 37% of the strains belonged to Proteobacteria and 63% belonged to Firmicutes in the intestinal tract of D. punctiferalis larvae.(3)T-RFLP data showed 76-bp and 122-bp T-RFs were the predominant group in the intestinal tract of A. hetaohei and D. punctiferalis larvae respectively. Principal component analysis(PCA) and cluster analysis indicated that there were two structure categories for the intestinal bacteria communities in A. hetaohei and D. punctiferalis.(4)The high-throughput sequencing showed there were 23 phylum 119 family and 107 genus in samples of A. hetaohei; there were 22 phylum 108 family and 108 genus in samples of D. punctiferalis. The abundance of Firmicutes, Proteobacteria, Cyanobacteria and Actinobacteria were more than 0.5% and average relative abundance respectively were 63.33%, 29.84%, 3.49% and 1.45% in samples of A. hetaohei; The abundance of Proteobacteria, Firmicutes, Cyanobacteria and Actinobacteria were more than 0.5% and average relative abundance respectively were 80.18%, 10.00%, 5.88% and 2.14% in samples of D. punctiferalis 。 Enterococcus, Rubrobacter, Wolbachia, Halomonas, Pseudomonas, Agrobacterium were the predominant group in samples of A. hetaohei, and average relative abundance respectively were 9.44%, 2.26%, 1.88%, 1.87%, 0.87%, 0.71%; Wolbachia, Pseudomonas, Agrobacterium, Enterococcus, Halomonas, Rubrobacter were the predominant group in samples of D. punctiferalis and average relative abundance respectively were 21.33%, 5.09%, 3.74%, 1.55%, 1.45%, 1.14%。Principal coordinate analysis(PCo A)and clustal analysis indicated that there were two structure categories for the intestinal bacteria communities in A. hetaohei and D. punctiferalis.(5)DGGE and T-RFLP and high-throughput sequencing analysis showed the diversity differences of intestinal bacteria from A. hetaohei larvae and D. punctiferalis larvae were not significant, and there was a difference in the proportion of bacteria composition. The study revealed the characteristics of the intestinal microbial composition in two different insects, which provided a basis for further discussion on the relationship between the diets and intestinal microbes of the two different diet larvae consuming walnut’s green husk.