Assessing The Effects Of Cry2Aa Protein On Habrobracon Hebetor(Hymenoptera: Braconidae),A Parasitoid Of Indian Meal Moth,Plodia Interpunctella(Lepidoptera: Pyralidae)

The Bt protein expressed by transgenic rice shows high resistance to target pests,and its safety to non-target organisms has also received widespread public attention.In the rice field ecosystem,the Bt protein expressed by the Bt transgenic rice may be transmitted from the transgenic rice to the herbivorous insects through the food chain,and then passed from the herbivorous insects to the parasitic natural enemies.Based on the tertiary nutritional relationship of Bt transgenic rice-target pests-parasitic natural enemies,when evaluating the safety of Bt protein to target insect,because the growth and development of target insect is negatively affected by Bt protein,it may lead to the decline of host quality of parasitoids,and finally lead to the adverse impact on the development of parasitoids,that is,"host quality mediated effect".Because of the"host quality mediated effect",the accuracy of safety evaluation of parasitic natural enemies is reduced.The resistant strains to Bt protein can avoid this effect,but the period of obtaining resistant strains is long,time-consuming and low efficiency.It is suggested that Bt protein may be transmitted to Habrobracon hebetor,which is the parasitoid of Plodia interpunctella.Therefore,it is necessary to evaluate the safety of Bt protein to the H.hebetor.In this study,Cry2Aa protein was injected into the hemolymph of the P.interpunctella by microinjection,and the hemolymph of the P.interpunctella was used as the carrier to transfer Cry2Aa protein to the H.hebetor,avoiding the"host quality mediated effect",accurately evaluating the effect of Cry2Aa protein on the growth,reproduction and protective enzyme activity of the H.hebetor,so as to provide reliable data for the safety evaluation of Cry2Aa transgenic rice.At the same time,the safety of Bt protein to parasitic natural enemies was analyzed by meta-analysis.The main results are as follows:After P.interpunctella eating T2A-1 rice for two days,the content of Cry2Aa protein in the hemolymph was 176.0 ng/g,which was the actual exposure of the H.hebetor.After200 ng Cry2Aa protein?400 nl*0.5 ng/nl?was injected into the 4^th instar larvae of P.interpunctella,the Cry2Aa protein could be evenly distributed in the systemic hemolymph within 2 minutes.We found that the degradation rate of Cry2Aa protein in hemolymph of P.interpunctella was slow under parasitism,and the concentration of Cry2Aa protein in hemolymph?2028 ng/g?was always more than 10 times higher than the actual exposure in three days.Western Blot assay revealed that the Cry2Aa protein was the same as that in the natural state,and biological assay experiments showed that the Cry2Aa protein in the hemolymph was still bioactive.The female adults of the H.hebetor were introduced into the newly injected 4^th instar larvae of the P.interpunctella to lay eggs for 6 h,and the parasitic wasps and excess eggs were removed to ensure that only one egg was present on the body surface of each larva.Three days later,the larvae of the H.hebetor were transferred to the newly injected and parasitized 4^th instar larvae of the P.interpunctella,and allowed to continue feeding until pupation.Bovine serum albumin?BSA?was selected as a negative control,and Galanthus nivalis agglutinin?GNA?was used as a positive control.The results showed that high concentration of Cry2Aa protein had no significant effect on the larval development time,sex ratio,weight of pupa,weight of adults,adult longevity,fecundity and enzymatic activity?SOD,CAT,TPS,t-Pro and GR?of H.hebetor,while positive control GNA had significant negative effect on the larval development time,weight of pupa,weight of adults,adult longevity,fecundity and enzymatic activity?SOD,CAT,TPS,t-Pro and GR?of H.hebetor.Search the published literature about the effect of Bt protein on the parasitic natural enemies from the database,and make meta analysis after sorting and screening.In the study on the safety evaluation of parasitical natural enemies by Bt protein transmitted by transgenic plants-target pests?strain sensitive to Bt protein?-parasitical natural enemies,Bt protein significantly prolonged the development time of parasitical natural enemies,and reduced the survival rate,fertility and longevity of parasitical natural enemies.In the study of the safety evaluation of the parasite natural enemies by Bt protein transmitted by transgenic plants-target pests?anti Bt protein strains?-parasite natural enemies,Bt protein had no significant negative effect on the development time,survival rate,fertility and longevity of the parasite natural enemies.In the study on the safety evaluation of parasitical natural enemies by Bt protein delivered by artificial feed,Bt protein has no significant negative effect on the fertility and longevity of parasitical natural enemies.By microinjecting Cry protein into the hemolymph of host,we established a new method to avoid host-mediated quality effects in biosafety evaluation of Cry protein on ectoparasitoids.The results indicated that Cry2Aa protein has no negative effect on H.hebetor.At the same time,our experiment provides new ideas for the biosafety evaluation of Cry protein on ectoparasitoid of target pests.