Gut Symbiont CF-BD Enhances Trichlorphon Resistance Of Oriental Fruit Fly Bactrocera Dorsalis (Hendel) And Mechanism Research

The oriental fruit fly Bactrocera dorsalis?Hendel??Diptera:Tephritidae?is a significant dangerous pest species thatdamages a wide range of fruit and other horticultural products,causing major financial losses to horticulture.Chemical pesticides are the main method to control the B.dorsalis so far.As one of them,trichlorphon is a moderately toxic organophosphateinsecticide that has been widely used to control this pestbecause of its low toxicity to humans and its high efficacy.However,pesticide resistance ofB.dorsalis to trichlorphon has beenincreasing in recently years.The gut of B.dorsalisharbored a large number of symbiont,previous research found that trichlorphon resistance strain of B.dorsalis had more Citrobacter bacteria in gut.After isolation and identification,this symbiont was confirmed as Citrobacter freundii-BD?CF-BD?.To clarify the relationship between gut symbiont CF-BD and trichlorphonresistance of B.dorsalis,the effect of CF-BD on resistance of B.dorsaliswas studied in this research.The mechanism of CF-BD enhance the insecticide resistance of B.dorsalis was clarified,and we elucidate the transmission route of CF-BD in B.dorsalis population.This research provided significant theoretical reference for the control of insect resistance.The main results are as follows:1 Effect of CF-BD on trichlorphon resistance ofB.dorsalisWe manipulated insecticide resistance byadding or removing CF-BD and provided evidencefor a close relationship between this bacterium and thepesticide resistance in B.dorsalis.After feeding susceptible?SS?flies by CF-BD,the number of gutCF-BD was significantly higher than in the control fliesthat were given water.Moreover,the SS flies thatwere fed the CF-BD inoculum were show more resistant to trichlorphon than the control flies(LC₅₀=2.35 mg/L for SS+CF-BDinocula,LC₅₀=1.28 mg/L for SS).After feeding with streptomycin,the numberof CF-BD colonies obtained from resistant?RS?flies gut samples wassignificantly reduced compared with the RS gut samplesfrom flies that were fed sterile water.Moreover,the RS flies fed the streptomycinsolution were more sensitive to trichlorphon than the RSflies(LC₅₀=89.78 mg/L for RS+streptomycin,LC₅₀=133.31 mg/Lfor RS).These results indicated that CF-BD was required forenhanced resistance to trichlorphon.The result of fluorescence in situ hybridization?FISH?showed that CF-BD signals were consistently localized in the midgut and hindgut crypts of the whole gut ofB.dorsalis,and the fluorescence signal intensity in the regionnear the rectal pad of female adult was distinctly higher than that of the other sections of the gut.2 Mechanism of trichlorphon degradation by CF-BDTo clarify the mechanism of trichlorphon degradation by CF-BD,we further examined the filtrate by GasChromatograph-MassSpectrometer-computer?GC-MS?to identify the degradation products.GC-MS analysis revealed a greater amount of trichlorphon inthe filtrate purified from the trichlorphon-enriched mediumwithout CF-BD,and trichlorphon was degraded into chloralhydrate and dimethyl phosphite,which are significantly lesstoxic than trichlorphon.To predict the CF-BD trichlorphon degradation ability,the CF-BD genome was further sequenced and analyzed.Among the function-annotated genes,55phosphatase genes were identified in CF-BD.In particular,there are five phosphatase genes?gene ID 0086,1012,1747,2752and 4498?were organophosphate-hydrolyzing?OPH?like genes.In order to verify the function of the five identifiedOPH-like genes,the expression of these genes wasmeasured with the CF-BD exposed to trichlorphon atdifferent concentrations.The results showed the expression of genes 0086,1012,1747,and 4498weresignificantly higher when exposed to trichlorphon.3Detection of CF-BD symbionts from diverse populations and effects of CF-BD on B.dorsalis fitnessFor understanding CF-BD infections in flies from wild populations,CF-BD was detected in several individuals from 21 populations.As a result,CF-BD was detected in all these populations,with 100%infection.To confirm the resistance levels of a field population flies,a stomach toxicity assay of trichlorphon was performed.The results showed that the correlation coefficient between the LC₅₀ and mean Ct values is 0.7147.To further evaluate the effect of CF-BD on B.dorsalis,we established populations of B.dorsalis with and without the CF-BD symbiont.Strikingly,the CF-BD-fed flies as well as the control insects exhibited significantly higher larval and pupal dry weights,and more female fecunditythan the CF-BD-absent flies.4 Transmission route of CF-BD in B.dorsalisTo clarify the transmission route of CF-BD in B.dorsalis,using agar plate and Nested-PCR conducted a series of mixed rearing and mating experiments.The result showed that the symbiont CF-BD is mainly transmitted from maternal flies to their offspring via egg surfaces contaminated with CF-BD cells.In addition,the oral intake of CF-BD ensure that a high rate of horizontal transmission of CF-BD in B.dorsalis,but not by mating.The 16S rDNA sequence analysis of bacterial diversity in P.guajava revealed that Citrobacterbacteria can rapidly reproduce and presence stable in the environment around larvae of B.dorsalis.