Morphological Structure Of Venom Apparatus From Anastatus Japonicus(Hymenoptera: Eupelmidae) And Analysis Of Venom Protein Components

Parasitoid wasp is an important natural enemy insect.Generally,the successful development of the offspring from the parasitoid wasps will lead to the death of the host,and thus,parasitoid wasps can be used as a biological controlling agent against pests.Its parasitic factors,such as venom,can be used to develop new biocontrol agents with a broad application prospect;however,the genes coding the venom proteins of egg parasitoids are largely unexplored.Therefore,an in-depth understanding of the interaction mechanism between the parasitic wasp and its host is needed to identify the key proteins that play a significant role in the parasitic process and to develop new biological control strategies.In the present study,the egg parasitoid-Anastatus japonicus was studied as a model research object.The morphology and structure of the female wasp venom apparatus were determined by the high ploidy anatomic microscope.The venom protein components of the A.japonicus were studied using the female transcriptome and venom proteome analysis.The differences and specificity of the venom proteins before（Be）and seven days after（Af）parasitization were analyzed and compared with other reported venom proteins of the parasitoid wasps.The results from this study help will help us to better understand the interaction mechanism between parasitoid wasps and hosts and provide a theoretical basis for the development of new biological control strategies.The results are as follows:1.Under the standard conditions（26±1℃,75±5%RH,L/D=14/10h）,the developmental duration of the A.japonicus from parasitism to emergence was 20-22 days,of which the egg stage is three days,Larval stage（including 1st instar,2nd instar,3rd instar,and mature larvae）6 days,pre-pupal stage 6-7 days,pupal stage（including early pupae,middle pupae,and late pupae）5-6 days.However,the male generally emerges two days earlier than the female.2.Morphological characterization and anatomical observation revealed that the venom apparatus of A.japonicus was bee-type.Interestingly,the present study found that,after the emergence of the female wasp,the venom reservoir gradually increased and reached saturation on the 7^thday.3.Transcriptomic library for at each developmental period of Anastatus sp before and after parasitization was constructed using a high-throughput Illumina sequencing platform,which yields 89,698,356 clean reads.Using sequence assembly tool Trinity,a total of 58160unigenes were obtained,with a total length of 51,712,053 nucleotides（nt）,a GC content of32.3169%,N50,and average lengths of 2113 nt and 889 nt,the longest unigene was 28184nt,and the shortest 201 nt.Using blastx alignment,a total of 21698 amino acid sequences were obtained by annotating 22044 unigenes,and the homology analysis of the annotated unigenes showed the highest homology with Nasonia vitripennis.For unannotated unigenes,we used ESTScan software to predict its coding region and obtained 3175 amino acid sequences.4.In this study,the transcriptome of the A.japonicus was used as the background,and label-free proteomics techniques were used to perform proteomic analysis of female venom before and after parasitism.A total of 2084 proteins were identifified,including 81 venom proteins,most of which were identifified as Hexamerin,Chitinase 2,Calreticulin,Heat shock protein 83-like,Serine protease,Arginine kinase,Phosphoserine aminotransferase,Actin.Together the before and after parasitization venom contains 1628 proteins,including212 DEPs with 181 and 31 signifificantly up-regulated and down-regulated respectively.In addition,nine proteins were specififically present in the pre-parasitic venom,whereas 26proteins were specifific to the post-parasitic treatments.The results of differential analysis of venom protein before and after parasitization were verified by RT-q PCR technology.5.In this study,through dissecting the eggs of Tessaratoma papillosa,it was found that female wasps of A.japonicus injected eggs into the gap between the host eggs shell and the embryos,rather than in host embryo.Based on the results obtained in this study,we proposed a hypothetical model of the parasitic process of A.japonicus.（1）When A.japonicus lays eggs,the eggs and venom are injected into the gap between the host’s embryo and the egg shell.（2）The arginine kinase in the venom may rapidly paralyze the host,causing it to lose resistance.（3）A certain amount of hexamerin may cause the host to stagnate development,into the diapause.（4）While under the action of chitinase,it accelerates the diffusion of immune-related proteins,such as calreticulin into the host body and inhibits the host’s immune response and ensures that the offspring of parasitoids feed on the host embryo,leading to the death of the host and achieving the purpose of pest control.In conclusion,The present study identified that venom apparatus of A.japonicus was bee-type;The transcriptome of A.japonicus and the venom proteomics data before and after the parasitism of female wasps were detected,and the differentially expressed protein genes were preliminarily analyzed by RT-q PCR.Microscopic observation that female wasp of A.japonicus injected eggs into the gap between the host egg shell and the embryos,rather than in host embryo.Taken together,results obtained from this study can provide the basis for further research on the function of these venoms proteins and the accurate localization of the key proteins in the parasitic process.It also opened the way to a better understanding of the evolution of venom in the interaction between egg parasitoids and hosts,leading to the development of novel biological control strategies.