| Insects live in close association with symbiotic bacteria across life history.Bacteria are increasingly being recognized as playing an important role in many aspects of insect host biology and physiology,for example,host development,reproduction,immunity,defense against natural enemies and nutrition.The location and biological function of the symbionts vary from colonization inside or outside of the host cells,and transmission may occur vertically or horizontally.Insect dependence on microbes can be integrated into novel control strategies,such as Incompatible Insect Technique(IIT)based on the mechanism of Wolbachia-induced CI.Further,bacterially enriched diets can improve the fitness of artificial rearing insects.In the present study,we used the Chinese citrus fly,Bactrocera minax,a detrimental tephritids fruit fly pest as a model to address the roles of symbionts.The oligophagous and univoltine traits of the target fly make it tough to domesticate thereby delimiting the basic and applied research.As such we firstly investigated the population genetics of the Chinese citrus fly.Then the bacteria communities of B.minax were identified using culture-dependent and culture-independent techniques.Bacteria enriched diets were utilized to estimate the effects of symbionts on the fitness of adult fly.In addition,the dynamics of GFP or RFP modified bacteria were monitored over time in the gut of B.minax.The goal is to improve the efficacy of rearing of B.minax and understand the intimate relationship between the Chinse citrus fly and symbiotic bacteria.The main results are as follows.1.Population genetics and structure of the Chinese citrus flyThe pest colonizing structures,phylogeography,and their pattern of distribution have key importance in the management and quarantine of the fruit flies.The Chinese citrus fly has a critical impact on Chinese citrus industry due to its destructive nature.Nevertheless,we know little about the population genetic structure of this pest.In this study,population genetic structure of B.minax was inferred by using mitochondrial gene cytochrome c oxidase I(COI).A range of nucleotide diversity and haplotype diversity from 0.0007 to 0.0030 and 0.275 to 0.909 respectively were observed.Twenty-nine haplotypes were obtained from 266 individuals belonging to twelve different localities of China.The populations were grouped into four regions that were geographically consistent and contiguous by SAMOVA.The result of AMOVA showed a significant difference and indicated molecular variance was higher within-population 49.72% as compared to among groups 43.70%.Mantel test revealed a significant relationship between genetic differentiation and geographical distance suggesting that B.minax occurred in China for a long period of time.There was a significant value of Tajima's D and Fu's Fs for the populations of Zigui and Xunyang and non-significant mismatch distribution,which showed a recent demographic expansion.Overall high levels of genetic diversity and a strong genetic structure suggest that the Chinese citrus fly,B.minax existed in China over a long period of time.2.Identification of bacteria from the Chinese citrus flyThe bacterial communities of the B.minax gut and reproductive organs were explored by using 454-Pyrosequencing technology.Some highly diverse bacterial communities from 17 bacterial phyla with almost 361 microbial operational taxonomic units(OTUs)were obtained from B.minax.However,whether and how these abundant symbiotic bacteria contribute to fitness in B.minax is largely unknown.In order to elucidate the effects of bacteria on the fitness of B.minax,gut resident bacteria were isolated from the adult flies using culture-dependent techniques with reference to 16 s r RNA gene.Ten different bacterial species were identified,belonging to the Enterobacteriaceae family including Klebsiella oxytoca,Klebsiella pneumoniae,Citrobacter braakii,Pantoea dispersa,Enterobacter intermedius,Enterobacter aerogenes,Enterobacter cloacae,Proteus penneri,Proteus mirabilis and Erwinia cacticida.3.Localization of GFP or RFP modified bacteria in the gut of B.minaxThe discovery of fluorescent protein provided the potential to monitor the cellular processes in living systems using optical microscopy.To further explore the impact of gut isolated bacteria and figure out their spatiotemporal dynamics,we carried out the experiments regarding the localization of GFP/RFP modified bacteria in the gut of B.minax.Some of the pre-identified bacteria such as Klebsiella oxytoca,Klebsiella pneumoniae,and Citrobacter braakii were genetically modified with GFP or RFP.The results showed that after 24 hours RFP modified Klebsiella pneumoniae,Klebsiella oxytoca and GFP modified Citrobacter braakii were present in small quantity in the gut and they started to form biofilms after 48 hours.Over 72 hours a thick biofilm of RFP-labelled K.pneumoniae and GFP-labelled C.braakii were observed at the wall of midgut,while RFPlabelled K.oxytoca was seen in lower quantity.4.Impact of bacteria on the fitness of the Chinese citrus flyIn this chapter,we supplemented with four bacterial isolates(Klebsiella pneumonia,Klebsiella oxytoca,Citrobacter braakii and Pantoea dispersa)into the adult diets to estimate the effects on the longevity and the fecundity of B.minax fly.The bacteria were removed from flies by antibiotic treatments to generate aposymbiotic fly.Adults fitness parameters such as male and female longevity,female fecundity,male copulation number were measured for the two treatments and one control group.Results were complex depending on the fitness parameters measured and the bacterial species.Compared to the controls,antibiotic-treated B.minax had significantly decreased fecundity,but male and female longevity were increased.When flies were fed diets supplemented with any of the bacterial isolates,female fecundity was significantly enhanced.However,only Citrobacter braakii and Klebsiella pneumoniae significantly increased male mating frequency than control males.The results show that gut bacteria directly influence the fitness of B.minax,but impacts are dependent on the bacterial species and the fitness parameters measured.In conclusion,high levels of genetic diversity and a strong genetic structure give the hint that the Chinese citrus fly,B.minax occurred in China over a long period of time.The identified bacteria from the gut of B.minax belong to family Enterobacteriaceae.Supplementation of probiotics affected the fitness and contributed to the reproductive activity in the Chinese citrus fly.However,the impact that different bacteria have on this fly is different.Additionally,genetically modified RFP and GFP bacteria fully established in the midgut of B.minax after supplementation over time.The above results suggest that symbiotic bacteria have intimate ties with B.minax and make a significant contribution to their fitness,which provides better understanding in terms of fly-bacteria interactions and shed light on developing novel control strategies against the target fly. |
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