Identification Of Asobara Japonica And The Preliminary Study On Its Regulatory Mechanisms Of Host Immune Responses

Parasitoids,free-living as adults but,as juveniles,develop parasitically on other host species,generally killing the host.As such,many parasitoids have been widely used in massive release programs against a variety of pest insects around the world,and they have played crucial roles in reducing global agricultural losses and in maintenance of natural ecosystems.Over millions of years of co-evolution,hosts have evolved strong immune responses to recognize and kill the infectious parasites,meanwhile,parasitoids have developed a range of regulatory factors that are able to manipulate the immunity of their hosts.The interaction between parasitoids and hosts is interesting and the underlying molecular mechanisms will help to provide potential targets for finding some novel insecticidal proteins and increase the biological control efficiency.Here,we find a new Drosophila parasitoid species in Zhejiang Provience,and confirm that it is Asobara japonica through morphology identification and DNA barcode analysis.Furthermore,we have studied its basic biological characteristics and the effects on host immunity.Finally,we also indetify its venom proteins and test the function of two venom genes.The results are listed as below:1 Identification of A.japonica and its biological characteristicsWith the help of a combination of morphology and DNA barcode analysis,the parasitoid species is identified as A.japonica.The developmental duration of egg stage,larval stage and pupal stage of A.japonica under the conditions of 25 ± 1 ℃,50 ± 1%relative humidity and photoperiod of 16 L : 8 D is 2.38 ± 0.01,5.36 ± 0.07 and 8.30 ±0.04 d,respectively.The A.japonica can successfully parasitize all the tested 6 species of Drosophila,including D.suzukii,a well known fruits pest.2 The effects of A.japonica parasitization on host immunityThe host lymph glands gradually disappear at 24 h,48 h,and 72 h after A.japonica parasitization.When detecting the apoptotic signal in the host lymph glands at 24 h post infection,we find a higher apoptotic ratio(28.7% ± 2.0%)than that of the nonparasitized group.Parasitization will also affect the number of host hemocytes.24 h later,the number of host hemocytes in parasitized hosts is significantly higher than that of the non-parasitized group.However,the number of hemocytes in parasitized hosts is significantly lower than that of the non-parasitized group at 48 h and 72 h post infection.In addition,the melanization ability of host hemolymph will be inhibited after being parasitized for 24 h,48 h,72 h and 96 h,respectively.However,the expression of host antimicrobial peptides will increase significantly at 6 h and 12 h after parasitization.3 The venom protein genes of A.japonica and preliminary study of two selected venom proteinsAfter RNA sequencing of A.japonica venom glands,a total of 7379 proteincoding genes are obtained,of which 165 protein-coding genes containing signal peptides and FPKM > 10 are indetified as candidate venom protein-coding genes.When using q RT-PCR to verify the tissue expression specificity of candidate venom proteincoding genes,9 of the 10 selected genes are specifically expressed in the venom gland.The above 165 candidate venom protein-coding genes have been divided into seven categories according to their molecular functions,including Hydrolases,Oxidordeuctases,Transferases,Protease inhibitors,Recognition/Binding proteins,Paralytic factors and Others,of which Hydrolases accounts for the most abudance.Two venom genes which have been reported to involve in melanization suppression are selected to perform the function analysis.The results showed that the RNAi efficiency of the two genes are very high,but the host melanization rate,parasitism rate,and wasp emergence rate have no significant difference with the control group.