Diversity Analysis Of Gut Microbiota In Ceroplastes Japonicus Green And Screening Of Tannin-degrading Bacteria

Ceroplastes japonicus Green is an aggregative and phytophagous insect which lives on the succus of the host plant phloem with a piercing-sucking mouthpart.The gut microorganism is important for nutritional supplementation of C.japonica.In this study,the intestinal microbial diversity of C.japonica was analyzed,and the regulation of intestinal microorganisms on nutrient metabolism was explored.Moreover,we screened the tannin-degrading bacteria in the species.The main results as follows:1.Intestinal microbial diversity of C.japonica feeding on jujube and persimmomThe genomic DNA of the adult C.japonica from jujube and persimmon were extracted from guts for sequencing the variable region(V3 + V4)of 16 S r DNA by high-throughput sequencing technology.The diversity and difference of intestinal flora in the two groups of samples were analyzed.respectively.According to the OTU analysis,there were about 1,290 bacteria species in C.japonica on jujube,and about 1,306 species on persimmon.There was no significant difference between the two samples.Firmicutes,Proteobacteria and Bacteroidetes are the dominant intestinal flora in C.japonica gut.The three classes Clostridia in Firmicutes,Bacteroidia in Bacteroidetes and Alphaproteobacteria in Proteobacteria were dominant in the two groups.At the level of order,Clostridiales and Bacteroidales have an absolute high proportion,among which Bacteroidaceae,and Veillonellaceae have the highest richness.At the genus level,the dominant intestinal microflora of C.japonica were Bacteroides,Veillonella,Lactobacillus,Methylophilus,Brevundimonas and Lachnospira,but the proportions of different genera from different hosts were distinguished.The proportions of the three genera Bacteroides,Veillonella and Lactobacillus in C.japonica living on jujube were 13.87%,7.54% and 5.81% respectively,which slightly lower than that in C.japonica living on persimmon(15.71%,10.32% and 6.24%).While,Methylophilus(5.21%)and Brevundimonas(5.05%)in C.japonica on jujube is significantly higher than that in C.japonica on persimmon(1.12%,1.38%).2.The intestinal microbes of C.japonica and D.corpulenta feeding on persimmonThe intestinal microbial diversity of C.japonica and D.corpulenta from the persimmon were analyzed by high-throughput sequencing.C.japonica and D.corpulenta collectd on the same persimmon individual and foraged by piercing-sucking mouthpart,but the number and species of their intestinal microorganisms were different.By OTU analysis,there were about 53 species of microbes in the intestinal tract of D.corpulenta,while there are about 1,137 species in the intestinal tract of C.japonica.In C.japonica,the dominant bacteria mainly distributed in Bacteroides,Veillonella,Lactobacillus,Lachnospira and Faecalibacterium,accounting for 15.71%,10.32%,6.24%,3.88% and 3.69%,respectively.The dominant bacteria in D.corpulenta were Wolbachia,Sodalis and Candidatus_Uzinura,accounting for 35.51%,29.08% and 4.22% respectively.3.Effects of intestinal flora changes of C.japonica on host nutrient metabolismThe intestinal flora of C.japonica was examined by the treatment of the compound antibiotics,and analyzed by 16 S functional prediction.The nutritional components in C.japonica hemolymph were tested and compared with the control treatment.The Comparative results showed that the diversity of intestinal flora and the predicted metabolic pathways of C.japonica including carbohydrate,nucleotides and amino acids metabolism were significantly changed after antibiotics treatment.Simultaneously,the nutritional indexes of glucose,albumin and triglyceride in the host insect haemolymph were measured by assay.It conformities with the results of 16 S functional prediction,the concentrations of glucose,albumin and triglyceride in the control group were significantly higher than the treatment group.As a result,the number of bacteria involved in nutrient metabolism is reduced with antibiotics treatment which resulted in metabolic disorders.4.Screening of tannin-degrading bacteria in the intestinal tract of C.japonicaThree strains of tannin degrading bacteria in the intestinal tract of C.japonica were isolated and identified as Bacillus cereus,Acinetobacter johnsonii XBB1 and Bacillus oceanisediminis 2691 by 16 S RNA as molecular marker.The tannase activity of the three strains gradually increased and reached the peak on the third day with the detection at the culture condition of 45 ? and p H5.0.The peak tannase activity of Acinetobacter Johnson XBB1 was the highest with 22.33 U /m L,whereas Bacillus cereus and Bacillus oceanisediminis 2691 have similar value with 14.33 U /m L and 15 U /m L,respectively.In this study,the intestinal microorganisms of C.japonica was analyzed,and the dominant flora and functional prediction was obtained.The nutritional function of intestinal flora was realized,and three tannin degrading bacteria were isolated.These results provide more data for a better understanding of intestinal microbial function of C.japonica and may contribute to a new idea for pest control.