The Venom Components Of Leptopilina Drosophilae And Their Functions

Parasitoid wasps are highly abundant and essential in natural ecosystem.They have venom and other parasitic factors to regulate host behavior,overcome host immunity and affect host metabolism for their survival.The parasitic and host-killing abilities make them become valuable biological control agents.Drosophila suzukii is a highly invasive pests for soft fruits.At present,spraying chemical insecticide is main method to control the populations of D.suzukii.However,overuse of chemical insecticide causes environmental pollution and seriously threatens people’s public health.Moreover,the emergence of insecticide resistance also weakens the control effect of those chemicals.In this study,we obtained two species of Drosophila parasitoids in Zhejiang province.We did morphology and DNA barcode analysis of this parasitoid to verify the identification.Furthermore,the biological characteristics of the parasitoids were detected,one of them could successfully parasitize D.suzukii.Then its venom compositions and functions were also investigated.Finally,we discussed the potential capability of this parasitoid wasp for biological control of D.suzukii.The results are listed below:1.Identification of Leptopilina drosophilae and its biological characteristicsThrough morphology and DNA barcode analysis,the parasitoid species was identified as Leptopilina drosophilae（Ld）.The developmental duration of Ld was 18days for male and 20 days for female,which includes egg,1^st-5^th instar larvae,pupae and adult stages.We found Ld had strong resistance to extreme low or high temperatures,and even under the starvation stress.This parasitoid presented a high parasitism rate on D.suzukii,indicating that Ld is a very good biological control agent.2.Genome sequence of Ld and the RNAi technique platformThe Ld genome was sequenced at a chromosome level,and a reference genome with high continuity and completeness was obtained,including 10 chromosome with a mount rate of 95.7%.We then annotated 14,793 protein-coding genes based on the Ld genome.Next,we successfully set up the RNAi technique platform for Ld by using the Orco as a maeker gene.3.Ld paralyzes host for successful egg-laying relaying on some small molecules in the venom glandDuring parasitism,the Drosophila larvae were immediately paralyzed by Ld,which benefits the wasp egg-laying.From the results of injection the venom components of different molecular weight or the protease-treated venom,we found that the small molecules could paralyze Drosophila larvae.Next,we used non-targeted metabolomics and HPLC to analyze the small molecules in the Ld venom,and found four neurotransmitters with high content in Ld venom,includingγ-aminobutyric acid,taurine,dopamine and adenosine.We then injected the four small molecules into the larvae of D.melanogaster and D.suzukii,and found all host larvae showed paralysis behavior,indicating that the four small molecules are important for Ld to paralyze their host,leading to the successful egg-laying.4.Ld suppresses host immune system via GTPase venom proteinsCombining venom gland transcriptome and venom protein mass spectrometry analysis,192 venom genes were annotated.We screened the functions of the top 40venom protein genes through RNA interference,and found that when knocking down expression of Ld3919,Ld2901 and Ld7079 venom genes,Ld larvae were encapsulated in the body of D.melanogaster and D.suzukii,but no obvious encapsulation was observed after knocking down the other 37 genes.The results indicate that Ld ulilizes Ld3919,Ld2901 and Ld7079 venom proteins to suppress host immunity.The following gene sequence analysis of Ld3919,Ld2901 and Ld7079 revealed that they all belong to GTPase family.In conclusion,a parasitoid species of D.suzukii was trapped and identified as Ld.Ld has short developmental duration,long lifespan,strong resistance to low or high temperatures and starvation stress.Most importantly,Ld has high parasitism rates on D.suzukii.Our findings also show Ld has a sensitive olfactory system,which helps them accurately locate the host larvae.After targeting the host,Ld will oviposit efficiently using small molecules in the venom to paralyze host larvae.To ensure the survival of his offspring within the host larvae,Ld has recruited specific GTPase venom proteins to suppress host immunity.Our findings are not only helpful to strengthen the basic theoretical knowledge of parasitiod venom regulating host behavior and immunity,but also will improve the efficiency of pest control of D.zuzukii.