Risk Assessment Of Insect-resistant Transgenic Bt Cotton On Non-target Insects

Transgenic insect-resistant Bt cotton have contributed to the management of target insect pests,reduced the use of insecticide sprays.However,the environmental safety of Bt cotton has become a major concern.Thus,the environmental safety evaluation of Bt cotton need to be addressed before the commercialization of Bt cotton.The effect of transgenic insect-resistant cotton on non-target insects is an important part of environmental safety assessments.In the present study,we studied the ecological safety of transgenic Bt cotton ZMSJ and ZMKCKC(expressing Cry1Ac/Cry2 Ab and Cry1Ac/EPSPS,respectively)on non-target insects Adelphocoris suturalis,Bombyx mori,Apis mellifera and Haptoncus luteolus from these aspects as follows:1 Transmission of Bt protein from cotton to non-target insectsWe used ELISA method to detect the transmission of Bt protein from Bt cotton to non-target insects.The results indicated a high variability in levels of Cry1 Ac among samples collected in different dates for both ZMSJ and ZMKCKC,and the highest expression level was detected in July.The amount of Cry2 Ab protein in ZMSJ pollen was statistically steady throughout the season.The content of Cry1 Ac protein in ZMSJ leaves was higher than Cry2 Ab.After being fed with two transgenic Bt cotton varieties,trace amounts of Cry1 Ac and Cry2 Ab proteins were detected in non-target pest A.suturalis,and pollinating insects A.mellifera and H.luteolus.Thus,Cry1 Ac content was higher than Cry2 Ab in both transgenic Bt cotton varieties,and both Cry1 Ac and Cry2 Ab proteins could transmit from the transgenic Bt cotton to these three non-target insects through feeding.2 Effects of transgenic Bt cotton on the survival and growth of non-target insectsIn the present study,the biological effects of two transgenic cotton varieties ZMSJ and ZMKCKC on A.mellifera and B.mori were studied.The results showed that feeding on pollen from ZMSJ and ZMKCKC cotton did not affect the survival rate and the cumulative consumption of A.mellifera.Furthermore,feeding high concentration(10 mg/ml and 100 mg/ml)of transgenic cotton pollen on mulberry leaf had no adverse effects on survival rate and development time of 1st to 5th instar larvae,or body weight of 1st to 4th molters larvae.Therefore,transgenic Bt cotton ZMSJ and ZMKCKC had no significant effects on the survival and growth of A.mellifera and B.mori.3 Effects of transgenic Bt cotton on total hemocyte count and phenoloxidase activity of non-target insectsIn the present study,the physiological effects of Bt cotton on A.mellifera and B.mori were studied by total hemocyte count(THC)and enzyme activity test.Our results showed that feeding on pollen from ZMSJ and ZMKCKC cotton did not significantly affect the THC and phenoloxidase(PO)activity of A.mellifera.Moreover,feeding high concentration(10 mg/ml and 100 mg/ml)of transgenic cotton pollen on mulberry leaf had no adverse effects on the THC,the percentage of plasmatocytes and granulocytes cells,and the PO activity of B.mori 5th instar larvae.Thus,both transgenic Bt cotton had no significant effects on the total hemocyte count and PO activity of A.mellifera and B.mori.4 Effects of transgenic Bt cotton on gene expression of non-target insectIn the present study,the effects of ZMSJ and ZMKCK on the gene expression of A.mellifera were performed by real-time quantitative PCR.The results showed that ZMSJ and ZMKCKC pollen did not significantly alter the expression of detoxification genes Cytochrome P450 302A1(CYP302A1),Cytochrome P450 4G11(CYP4G11),Cytochrome P450 6AS14(CYP6AS14),carboxylesterase(CEST),juvenile hormone esterase(Jhe),esterase FE4-like(Cest04),Glutathione S-transferase delta 1(GSTd1),Glutathione S-transferase S3(GSTS3),and antioxidation genes Catalase(CAT),superoxide dismutase 1(SOD1),Cu Zn superoxide dismutase 1(Cu Zn SOD1),and immune related genes peptidoglycan recognition protein S2(Pgrp S2),Abaecin,Defensin1,Defensin2 in A.mellifera.These results showed that the two transgenic Bt cotton varieties had no significant effects on detoxification,anti-oxidation and immune-related gene expression of A.mellifera.5 Potential Bt binding proteins detection in midgut of non-target insectsIn the present study,binding assays with biotinylated Cry toxins and Ligand blot assays were conducted to detect the Bt binding proteins in the midgut brush border membrane vesicle(BBMV)of A.suturalis,A.mellifera and H.luteolus.Binding assays with biotinylated Cry toxins indicated that midgut BBMV proteins from none of these three kinds of insects showed specific binding to Cry1 Ac or Cry2 Ab.Ligand blot assays also suggested that neither Cry1 Ac nor Cry2 Ab could bind to A.suturalis,A.mellifera or H.luteolus midgut BBMV proteins.Taken together,Cry1 Ac or Cry2 Ab binding proteins are not present in the midgut BBMV proteins of the three non-target insects A.suturalis,A.mellifera and H.luteolus.