The Effects Of Gut Microbiota On The Growth And The Repair Of Irradiated Damage In Bactrocera Dorsalis

Sterile insect technique(SIT),a green pest control strategy,has made remarkable achievements in agriculture,forestry,and animal husbandry and the control of human insectborne diseases.It has been continuously improved and developed in recent decades.Compared with the traditional pest control strategies of chemical insecticides,SIT has the advantages of species specificity and environmentally friendly.Sterile insect technique based on irradiation has successfully controlled many fruit flies species,including prevention,suppression,and pests elimination.The successful large-scale implementation of insect sterility technique mainly depends on two crucial aspects:(1)rapid propagation under low-cost conditions to obtain a sufficient number of insects;(2)obtaining high-quality sterile males with good fitness and mating competitiveness in the wild.However,it has been reported that sterile males of fruit flies and mosquitoes are at a disadvantage in mating competitiveness when competing with wild males.Still,the reason and mechanism of the decline of sterile males' field ecological fitness are not clear.Insect symbiotic microorganisms can affect host growth and development,nutrient absorption,reproduction and speciation,central nervous system development,and behavior.It has been found that ionizing radiation can change the homeostasis of host gut microbiota,but whether gut microbiota is associated with the decline of the ecological adaptation of infertile males and the mechanism of gut microbiota in this is not clear.In this study,the structure and diversity of the gut microbiota community of Bactrocera dorsalis were studied by high-throughput 16 s rRNA gene amplicon sequencing,axenic larva feeding,bacterial genomics,?-Red homologous recombination system,transcriptome,and metabolic group and high-throughput sequencing techniques.The function and mechanism of gut microbiota of B.dorsalis on the growth and development of host larvae and the ecological adaptation of sterile males to radiation damage and repair.The main results are as follows: 1.Structure and diversity of gut microbiota of Bactrocera dorsalis larvae.The dynamic change law of gut microbiota of B.dorsalis larvae with the increase of instar was comprehensively explored by high-throughput 16 S rRNA gene amplicon sequencing and culturable bacteria technique.The results showed many low-abundance microbial communities in different instar periods,while the species and abundance of relatively high-abundance communities had little difference in different instar periods.With the larval growth,the richness and diversity of gut microbiota decreased gradually.Enterobacteriaceae and Leuconostocaceae are relatively abundant and stable gut microbiota in the gut of B.dorsalis larvae.The culturable bacteria that exist stably in the intestines of B.dorsalis larvae are mainly Providencia,Morganella,and Enterobacter,belonging to Enterobacteriaceae,and Lactobacillus,Leuconostoc and Fructobacillus belonging to lactic acid bacteria.2.Gut microbiota of Bactrocera dorsalis larvae promote the larvae growth and its mechanism.The effects of 27 strains on the growth of B.dorsalis larvae were studied by the gnotobiotic fly rearing technique,bacteria mono-association experiment,and bacterial genome association analysis.Through axenic biological techniques,it was found that the body length of axenic larvae plus egg-associated microbiota(AXA group)was significantly higher than axenic larvae(AX group)under the condition of low nutrition.The presence of gut microbiota was beneficial to the growth of larvae.The results showed that 7 bacteria strains of Enterobacteriaceae,including Enterobacter cloacae N29(9.56 ±0.16 mm),Morganella morganii V41(8.31 ±0.29 mm),Providencia alcalifaciens V29(7.44 ±0.66 mm),Providencia rettgeri V2(6.46 ±0.46 mm),Providencia stuartii N83(6.41 ±0.14 mm),Morganella morganii N94(6.07 ±0.25 mm)and Klebsiella quasipneumoniae N46(5.78 ±0.09 mm)isolated from the gut of B.dorsalis larvae,significantly promotes larva,compared with the axenic larvae.Five bacteria strains of Enterobacteriaceae isolated from the gut of adult fly,including Enterobacter hormaechei EH(7.92 ±0.34 mm,Enterobacter soli EA(8.02 ±0.12 mm),Klebsiella michiganensis BD177(5.96 ±0.10 mm),Citrobacter koseri BD195(6.52 ±0.09 mm)and Serratia nematodiphila SM(4.44 ±0.18 mm),three bacteria strains of Enterobacteriaceae from other environmental sources,including Klebsiella michiganensis KM700324(5.83 ±0.16 mm)? Citrobacter portucalensis CF2(5.24 ±0.19 mm)and Escherichia coli EC(4.76 ±0.08 mm)could significantly promote the growth of larvae.Among these strains,the larval length associated with single bacteria of N29,V41,V29,EH and EA was also significantly higher than that of AXA group.In particular,strain N29 has the most significant effect on the growth of larvae.Two lactic acid bacteria strains,including Lactobacillus plantarum M96(5.25 ±0.5 mm)and Leuconostoc citreum M58(4.50 ±0.27 mm)isolated from the gut of B.dorsalis larvae,and one bacteria strain Escherichia coli EC(4.76 ±0.08 mm)also significantly promote the growth of larvae.However,the other 8 bacteria strains have no significant effect on the growth of B.dorsalis larvae.The genomic information of the above 27 strains was obtained by high-throughput sequencing.Further correlation analysis between the whole genome and phenotype of bacteria showed that amino acid synthesis and metabolism,lipopolysaccharide biosynthesis,fatty acid degradation,vitamin synthesis,biofilm formation,oxidative phosphorylation,and sulfur metabolism related pathways in KEGG pathway were significantly positively correlated with larval growth.Through the bacterial gene knockout test,it was found that the body length of N29 wild type strain mono-association larvae(8.76 ± 0.21mm)was significantly increased by 58.70% compared with that of AX group(5.52 ±0.29).In strain N29,the deletion of gene pdx A in vitamin B6 biosynthesis pathway(6.55 ±0.3 mm)and tyrosine metabolism pathway gene p?hpc E(6.21 ± 0.28 mm)could lead to the loss of the promoting effect on larval growth.At the same time,the deletion of hpc E gene(6.82 ± 0.21 mm)in the tyrosine metabolism pathway of N29 led to a significant decrease in the promoting effect.Therefore,tyrosine metabolism and vitamin B6 pathway play an important role in promoting the growth of B.dorsalis larvae by strain N29.Thus,E.cloacae N29 strain can be used as probiotics in the sterile insect technique of B.dorsalis to improve the efficiency of large-scale propagation of insect populations.3.The renovation of ecological fitness irradiation damage of Bactrocera dorsalis sterile males by gut symbiotic bacteria and its potential mechanism.We found that ? irradiation significantly decreased the mating competitiveness,flight ability,and survival ability of B.dorsalis male flies.On the 14 th day after eclosion,compared with unirradiated males,the cumulative flight time,cumulative flight distance,average flight speed(p < 0.005),and maximum flight speed(p < 0.001)of irradiated males were significantly lower than those of unirradiated males.In the competitive mating experiment on the 14 th day of eclosion,only 31% of females successfully mated with irradiated males,which was significantly lower than(p < 0.0001)69% of females successfully mated with unirradiated males.Compared with the median survival time of 51 days of unirradiated males,irradiation treatment reduced males' median survival time to 30 days,about 46%.The sequencing of high-throughput 16 s rRNA gene amplicon and the analysis of culturable bacteria showed that the composition and structure of gut microbiota of B.dorsalis male fly changed significantly after irradiation.Compared with unirradiated males,the number of gut microbiota communities irradiated by males on the 7th day after eclosion,Observe,Chao1 index,ACE index,and Shannon index increased,while Simpson index decreased significantly.The gut microbiota community composition analysis showed that the relative abundance and bacterial load of Enterobacteriaceae bacteria in the main gut microbiota decreased significantly.In contrast,the relative abundance of(Bacillus aceae,Clostridiaceae,Xanthomonadaceae,Sphingobacteriaceae,Aeromonadaceae,Flavobacteriaceae)in the secondary gut microbiota increased significantly.In the analysis of culturable bacteria,it was found that the relative abundance of Citrobacter koseri BD195,Klebsiella michiganensis BD177,and Enterobacter soli BD400 in the gut of irradiated males decreased by 81.82%,54.17%,and 33.75%,respectively.Therefore,we speculate that the significant decrease of the ecological fitness of B.dorsalis male fly after irradiation may be closely related to the changes in the structure and composition of the host gut microbiota community.Through supplementary feeding of intestinal symbiotic bacteria C.koseri BD195 and K.michiganensis BD177,it was found that K.michiganensis BD177 could significantly repair the radiation damage to the ecological fitness of sterile males.The ecological fitness such as mating competitiveness,flight ability,survival rate,and life span of irradiated sterile males fed with K.michiganensis BD177 strain could be restored to the level of unirradiated males.On the 7th day after eclosion,the cumulative flight distance and the fastest flight speed of irradiated males fed with BD177 strain were 1-2.5 times higher than those of irradiated males without BD177 strain.The mating competitiveness experiment showed that the irradiated males fed BD177 successfully mated with 47% of the females.There was no significant difference in the number of females(53%)between the irradiated males and the unirradiated normal males.Compared with unirradiated males,the life span of irradiated males fed with BD177 was significantly longer than that of unirradiated males.Still,there was no significant difference between unirradiated males and unirradiated males.Further experiments showed that irradiation significantly decreased the feeding ability and the level of primary nutrients in the hemolymph of B.dorsalis male fly.The food intake and main nutrient level of irradiated males could be significantly restored by feeding K.michiganensis BD177 strain.On the first day after Eclosion,the food intake(p = 0.0141),total sugars(p = 0.0247),and total free amino acids(p = 0.0144)in the hemolymph of irradiated males were significantly lower than those of unirradiated males.On the 7th day after eclosion,the food intake and the levels of major nutrients(sugars and amino acids)in the hemolymph of irradiated males not fed with BD177 were significantly lower than those of unirradiated males in the control group.In contrast,the food intake and total amount of free amino acids of irradiated males fed with BD177 were significantly higher than those of irradiated males without BD177(p = 0.0475 and 0.0124,respectively).These results suggest that K.michiganensis BD177 may repair the radiation damage of ecological fitness in sterile males by changing the feeding behavior and nutrient absorption and metabolism of the host.Therefore,through the joint analysis of the transcriptome,metabolic group,and K.michiganensis BD177 genome,we found that phenylalanine,tyrosine,and tryptophan biosynthesis pathways were mainly found in the response of the host KEGG pathway to tissue transcription and hemolymph metabolism after supplementation of K.michiganensis BD177.Phenylalanine,tyrosine and tryptophan biosynthesis pathways were also found in the KEGG pathway mapped by specific genes of K.michiganensis BD177 genome.Therefore,we speculate that phenylalanine,tyrosine,and tryptophan biosynthesis pathway may play an essential role in K.michiganensis BD177,improving the ecological adaptation of sterile males.K.michiganensis BD177 strain can be used as probiotics to repair the ecological fitness radiation damage of sterile males and enhance the implementation efficiency of insect sterility technique in B.dorsalis.