| The oriental fruit fly,Bactrocera dorsalis(Diptera;Tephritidae)is a serious menace to agricultural production worldwide.Chemical control remains the predominant method for the control of the target pests.Due to pest resistance,environmental and human health concerns,there is an urgent demand for development of environmental friendly control strategies.Chief among these are the Sterile Insect Technique(SIT)and related biological control applications.However,the disadvantage of the sterile fruit flies subjected to SIT is competitive than wild males.Besides,the mass-reared sterile males after irradiation are more fragile than the natural counterparts.As such,in the present study,we used Bactrocera dorsalis as model insects to identify the bacterial community and evaluate the effects of bacteria enriched diets on the fitness of B.dorsalis.Further,probiotics were supplemented to the diets to improve sterile male performance and mating competitiveness.We also detected the presence of endosymbionts bacteria,Wolbachia,Cardinium and Spiroplasma in Bactrocera dorsalis,B.tau,B.papayae,and B.carambolae.The purpose is to expand our knowledge in insect-bacteria interactions and lends support to develop novel management programs in the long run.The main results are as follows:1 Identification of symbiotic bacteria by 16 s r RNA in the gut and reproductive organ of B.dorsalisThe culture dependent method of the bacteria with the reference to 16 s r RNA gene was used to isolate and identify bacteria in adult oriental fruit flies,B.dorsalis GSS laboratoryreared colonies.We identified the cultivable bacteria inhabiting the intestinal and reproductive organs of B.dorsalis.The results revealed highly diverse bacterial communities from the flies composing of Firmicutes,Proteobacteria,Actinobacteria and Bacteroidetes.Firmicutes and Proteobacteria were mostly abundant in all tested samples.However,the bacterial diversity varied with sex and tissue.Lactococcus was strongly presented with 38% in female gut,while Enterococcus was strongly presented in reproductive organ with 45% in male testis and 40% in female ovary.These results show the symbionts associated with B.dorsalis,in which the symbionts may play vital biological functions in their insect hosts.2 Effects of symbiotic bacteria on the fitness of B.dorsalisThe effects of different gut bacteria isolates on the fitness of B.dorsalis were investigated by supplementing 13 different bacteria to the larval diets.The response of the fly to different gut isolates varied greatly.It showed that diets enriched with Enterococcus phoeniculicola had shorter larval developmental duration,higher pupal weight,and increased percentage of host survival.In contrast,larvae reared on Microbacterium sp.,K.pneumoniae,V.fluvialis and M.morganii treatments had a significantly longer developmental time.The inoculation of L.lactis was the most detrimental to B.dorsalis by extending the development time with an average developmental time of 19.73 days and reduced host survival(P < 0.001).This study provides clues for beneficial and harmful symbionts that might be useful in mass rearing of fruit fly pests.3 Enriched bacteria diets to boost the SIT application in B.dorsalisTo evaluate the effects of the probiotics on the mating competitiveness of the sterile males,the probiotic bacteria were inoculated to adult diet(probiotic group)and the diet without bacteria referred to control group.The sterile insects were fed with each group of diet prior to the test.The results showed that enriching the sterile male diet with E.phoeniculicola significantly improved sterile male mating success,66.2% of probiotic males successfully mating with the females whereas 33.8% of males from control group(P< 0.0001).Moreover,re-mating of females first mated to E.phoeniculicola or C.freundii enriched sterile male diet were significantly lower.What is more,E.phoeniculicola or C.freundii group showed positive effects in terms of the enhancing glucose content of sterile flies.The data demonstrate that beneficial bacterial species supplement to the diets can improve sterile male dietary and mating performance.4 Investigation of bacteria community of sterile flies fed with probiotic dietThe bacteria community of sterile B.dorsalis flies fed with probiotic enriched diet was evaluated by the Illumina sequencing technology.We performed the comparison of bacteria diversity between probiotic group(enriched E.phoeniculicola)and the control group.The results showed Firmicutes was the predominant phylum in both groups.Bacteroidetes occurred significantly different between probiotic and control sample with 2% and 0.01%,respectively.Streptococcaceae was the most dominant family in both probiotic and control samples with the abundance 52.19% to 65.17%,respectively.The Agrobacterium,Fructobacillus,Brevundimonas and Dysgonomonas bacteria were only presence in sterile males fed with probiotics.Thus,the sterile male fed with probiotic bacteria has more diverse of bacteria community than that in control group.5 Detection of endosymbiotic bacteria in B.dorsalis and other tephritidsThe 420 individuals of B.dorsalis,B.tau,B.papayae,and B.carambolae from China and Thailand were sampled to monitor the presence of endosymbionts bacteria,Wolbachia,Cardinium and Spiroplasma through 16 S r RNA gene-based PCR.According to BLAST at the Genbank database,the sequence of bacteria were close to Wolbachia,Cardinium and Spiroplasma genera with 94%,98% and 97% similarity,respectively.Among the native populations of fruit fly species,B.dorsalis in Guangxi was the only population to be infected with Wolbachia(0.24%).The Cardiniun infection was detected only in B.tau(0.24%).Among these four detected Bactrocera species,B.dorsalis,B.papaya and B.carambolae were infected with the Spiroplasama bacteria,whereas no infection was observed in B.tau.The infection rate of Spiroplasama was varied with the populations,ranging from 0.24% to 2.88%.In accordance to these data,we revealed that reproductive bacteria also presented in tephritids Bactrocera species.In conclusion,the intimate relationship between B.dorsalis and symbiotic bacteria were well-addressed in this study.Enriched bacteria diets improved the fitness of B.dorsalis GSS significantly.Further,the probiotic bacteria,E.phoeniculicola can be manipulated to promote mating performance of laboratory reared flies towards the SIT implementation.In addition,the occurrence of Wolbachia,Cardinium and Spiroplasma were detected in Bactrocera natural populations which will help to develop IIT.Overall,the results provide novel insight into the insect-bacteria interaction and empower the future successful application of environmental friendly managements against Tephritids fruit fly pests including SIT and IIT. |
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