The Biodiversity Of Helicoverpa Armigera Symbiotic Bacteria And Its Regulatory Effects On The Defense Response In Cotton

The cotton bollworm,Helicoverpa armigera is one of the most important crop pests worldwide.In response to insect feeding,plants have evolved a variety of defense responses,and insect symbionts can manipulate plant signaling pathways and affect plant defense responses directly or indirectly.However,the mechanism of cotton defense mediated by symbiotic bacteria is still unclear.In this study,the communities and the effect on cotton of the symbiotic bacteria in H.armigera were studied through a targeted approach using,16S r RNA amplicon sequencing,metagenome,transcriptome,metabonomic,and bacterial isolation.The results were as follows:1. The symbiotic bacterial community structure in H.armigera was affected by the development stage.Here,using 16S r RNA and metagenome,we investigated the biodiversity and activity of symbiotic bacterial communities across the holometabolous life cycle of H.armigera.The valid sequences obtained from each sample were clustered and analyzed according to the sequence similarity.25 phyla,60 classes,140 orders,264 families,548 genera,and 898 species of symbiotic bacteria were obtained from the H.armigera during the whole life cycle.Proteobacteria and Firmicutes dominate in the phylum;the relative abundance was greater than 70%,Enterobacter,and Enterococcus in the genus but undergo a structural“metamorphosis”in tandem with its host.The species diversity and richness of the 3^rd,4^th instar larvae and pupae were significantly lower than that of other developmental stages.In contrast,the 1^st instar larvae and adults had higher richness and diversity.2. Symbiotic bacterial community structure in H.armigera varies from location,and symbiotic bacteria collected from the oral secretion from H.armigera contribute to its growth and development.The richness and diversity of symbiotic bacteria in the whole larvae of the 3^rd instar larvae were higher than that of the other tissues,while similar composition and structure of symbiotic bacteria were observed in the oral section and intestinal microflora,followed by the midgut and salivary glands.In a word,symbiotic bacteria in the oral section was influenced by both intestinal and salivary gland symbiosis.We first analyze the composition and structure of the symbiotic bacteria of oral secretion in H.armigera;Firmicutes was the most abundant bacterial phylum present and,the most abundant genus was Enterococcus,followed by Klebsiella,Acinetobacter,Stenotrophomonas,and Pseudomonas.The Asaia were collected from OS and then isolated,and fed H.armigera,which showed that Asaia could accelerate the growth and development of H.armigera larvae.3. Symbiotic bacterial community structure in H.armigera vary from food.16S r RNA was performed to understand the biodiversity and activity of the symbiotic bacterial community of H.armigera feeding on different food and the bacterial community in different tissue of H.armigera.No significant differences were found in the diversity and richness of symbiotic bacteria between eggs and eggshells.However,eggshells significantly affected the microbial richness and diversity of the 1^st instar larvae of H.armigera,in which larvae feeding on it had reduced species richness.After feeding on cotton leaves,the species diversity and richness of symbiotic bacteria of the 1^st instar larvae decreased but were significantly higher than those of eggs and 3^rd instar larvae.The diversity of symbiotic bacteria of the 3^rd instar larvae fed on the cotton plant was higher than that of the 3^rd instar larvae fed on an artificial diet.4. Symbiotic bacterial community structure in H.armigera vary within geographical populations.16S r RNA was investigated for biodiversity and activity of symbiotic bacterial of H.armigera in different geographic populations of Yellow River basin?Anyang,Binzhou,Cangzhou,Qiuxian,Yanggu,and laboratory population?.Proteobacteria,Firmicutes,Bacteroidetes,and Actinobacteria dominated the microenvironment at all populations.Laboratory polulation harbored lower species richness and diversity,but the higher relative abundace of Proteobacteria?92.04%?than field populations?56.14%-66.87%?.Enterobacter,Morganella,Lactococcus,Asaia,Apibacter,and Enterococcus formed the core symbiotic bacteria,the total relative abundance was 69.13%,54.8%,68.13%,84.66%,79.97%,and 95.13%,respectively.Moreover,comparative functional analysis with PICRUSt indicated that the microbiome of laboratory population was less enriched for genes involved in the metabolism of amino acids,nucleotide metabolism,glycan biosynthesis and metabolites,and other metabolism,also decreased in transcription,translation,and other genetic information processions.5. Symbiotic bacteria affect the defense response of cotton to H.armigera.Through16S r RNA high-throughput sequencing,the community compositions and species diversities of symbiotic bacteria were determined in the neonate?L1?,third instar larva?L3?,and antibiotic-treated third instar larva?L3N?of H.armigera.Alpha diversity indices indicated that L1 habored higher rich and diverse,followed by L3,while the lowest shown in L3N.The RNA-Seq technology and non-targeted metabolomic analysis were performed to analyze in cotton undergoing different H.armigera feeding?L1,L3,L3N?.In comparison to control,1,767?790 up-and 977 down-regulated?,2,967?1,192 up-and 1,775down-regulated?,3,805?1,980 up-and 1,825 down-regulated?genes and 62?22 up-and 40down-regulated?,42?29 up-and 13 down-regulated?,48?43 up-and 5 down-regulated?metabolites and were found to be differentially expressed in L1,L3,L3N groups.As the diversity of symbionts decreased,the number of differential genes and up-regulated metabolites increased.These differential genes and metabolites and/or participated in many biological processes related to plant resistance,such as the systemic acquired resistance,the regulation of defense response to insects,and jasmonic acid-mediated signal pathway.They were also involved in linolenic acid metabolism,biosynthesis of secondary metabolites,diterpenoids synthesis,linoleic acid metabolism,MAPK plant signaling pathway,alanine metabolism,flavonoid,and flavonol biosynthesis.Moreover,the fold change of indicator genes?i.e.,lipoxygenase,allene oxide synthase,allene oxide cyclase,peroxidase,Polyphenol oxidase?were increased with the decrease of the diversity and richness of bacterial species in H.armigera.Indeed,flavonoid and tannin were significantly decreased in L1,whereas increased in L3 and L3N.The results indicated that H.armigera with low diversity of symbiotic bacteria would cause a stronger defensive response of cotton.In this study,the composition,structure,and effect of symbiotic bacteria on the cotton were studied,which greatly enriched the information of symbiotic bacteria in H.armigera,and confirmed that symbiotic bacteria could mediate the defense response of cotton.The results help to understand the regulation of symbiotic bacteria affecting cotton response to H.armigera,and to reveal the role of symbiotic bacteria in the prevention and control of H.armigera.