| Diaphorina citri as the main known vector for the transmission of Candidatus Liberibacter asiaticus,which causes citrus huanglongbing(HLB),poses a serious threat to the citrus industry.At present,chemical control methods are mainly adopted for D.citri,and long-term,large-scale application of chemical pesticides,not only does it make D.citri resistant to the drug,but also brings many negative effects on agricultural products and the environment,so the search for biological control is particularly urgent.In the study of parasitic natural enemies,parasitic wasps inject venom,polydnavirus,teratocyte and other parasitic factors into the parasitic process to regulate various physiological processes in the host and thus control the pest population.Tamarixia radiata Waterston,an ectoparasitic wasp of D.citri,has a good control effect on D.citri,but the mechanism of interaction between T.radiata and D.citri and whether its venom plays a key role in the parasitic process have not been reported.In this study,D.citri and its natural enemy T.radiata were used as the experimental objects,then the nymph of D.citri was treated with natural parasitism and in vitro injection of venom respectively,to investigate the effects of venom on host immunity,development and behavior,and analyzed them in conjunction with transcriptome sequencing results.We extracted the crude venom of T.radiata,isolated and identified its venom protein fractions,which provided research and application basis for the development and utilization of biological control agents.The main results were as follows:(1)The effect of T.radiata parasitism on D.citri.Under laboratory conditions(27±1°C,70±5%RH,photoperiod of L:D=14:10h),the parasitic rate of T.radiata was63.64%,egg production was 12.23 eggs/day,and emergence rate was 73.11%.After the treatment of de-oviposition,the development of the nymph was hindered and the age of the nymph increased significantly and they were unable to eclosion,with an eclosion rate of 0.After the treatment of in vitro injection,the development of the nymph was hindered and only some individuals successfully eclosion,with an eclosion rate of 20.57%,while the eclosion rate of the control group(injected with PBS)was 58.72%,and there was a significant difference between the two.In addition,the horizontal crawling distance of the nymph was less than 1/2 of that of the control group within 10 s after de-oviposition,while the horizontal crawling distance of the nymph was about 2/3 of that of the control group after envenomate.After natural parasitism,immune processes such as recognition receptors,Toll,JNK,Jak/STAT pathway and melanization in the nymph were differentially responded.Dscam2 was persistently upregulated at 48h;Spaetzle 3-like was significantly downregulated at 0h and upregulated at 24h;TBK1,Jak2 and phenoloxidase 1-like were significantly highly expressed at 24h;the expression level of dual oxidase was inhibited at all periods.The venom-injected treatment and the parasitic treatment maintained a high consistency in the first 24 h,but the difference was larger at 48 h.These results suggest that venom,as a key parasitic factor,is involved in the regulation of physiological processes such as growth and development,immunity and behavior of the host(P<0.05).(2)Transcriptome analysis of T.radiata after parasitizing D.citri.The results showed that at any time point,the number of DEGs produced by the parasitic treatment was always much higher than that of the venom-injected treatment when compared to the normally developing samples,and the number of DEGs was proportional to time.Using a specific comparison method,332 and 80 DEGs were screened from the difference between parasitic and venom-injected treatment,respectively,and 18 DEGs were shared between the two(P<0.05).The 332 DEGs were classified according to their expression patterns,and 35 specific DEGs were screened,among which Hemocyanin-1 and Galectin-4 in the "continuously ascending" type,and pro-resilin-like in the "ascending and then descending" type were identified,which are involved in immune regulation and metamorphosis of the host.(3)Isolation and characterization of venom proteins from T.radiata.The results of proteomic analysis showed that 55 putative venom proteins in the venom of T.radiata were obtained by screening,containing 40 proteins of known function and 15 proteins of unknown function.The known functional proteins were composed of 25 serine proteases,2 metalloproteases and 13 other functional proteins.25 serine proteins were mostly trypsin or chymotrypsin,and a few proteins were not classified at this time.In summary,this thesis presents a preliminary investigation of the interaction mechanism between T.radiata and D.citri,and clarifies the regulation of host growth and development,immunity and behavior by the venom of T.radiata through a twoway approach of natural parasitism and in vitro venom injection;At the same time,35 functional DEGs with certain research significance were screened in the transcriptome database for predictive analysis of the action targets in the parasitic process;in addition,the first isolation and identification of the venom fractions of T.radiata filled the relevant theoretical gaps,and also provided new ideas to the development and utilization of biological control agents for D.citri. |
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