Ecological Impact Assessment Of Transgenic Cry2A, Cry1C,Vip3H/Crylab Bt Rice For Their Non-target Different Thrips And Predator Species

Bt maize and Bt cotton, containing genes encoding insecticidal proteins from the bacterium Bacillus thuringiensis are already on the commercial scale. These Bt crops were developed to control the lepidopterans and other chewing insects pests but besides that their status has also been controversial as they can also interact with eco-system in terms of their effects on non-target arthropods including insects, natural enemies, soil dewelling organisms.Rice stem borers and leaf-folders cause less yield loss in rice than do lepidopteran pests of cotton and are the targets of fewer insecticide applications. Bt rice in China was developed to control these insects. But large number of experiments are needed to test its feasibility to bring it in the field but besides that, the effect on natural enemies needs some tier based studies including laboratory and other studies if needed. In the past several experiments have been conducted to observe the effects of Bt toxin on lepidopterans and other target insects for transgenic rice and has been found successful in the laboratory as well as field trials. So till date, two transgenic rice cultivars have been approved to further testing at large scale for further commercialization. But its effect on non-target insects really needs a lot of research and approach to understand exactly the phenomena. So in our laboratory long term experiments are going on since start of trials and this study is tier based study including the effects of Bt rice on non-target thrips and their potential predator.1-Mortality, life parameters, reproduction and ELISA of S. biformis observed under laboratory conditions for Bt H2A, H1C and non-Bt HCK rice linesThe mortality tests including three times larval and adult testing at both Bt rice cultivars were carried out as compared to their control cultivar. The development parameters including nymphal instars, pre-pupal and pupal development duration and reproduction included fecundity, duration of egg laying, total number of eggs of Stenchaetothrips biformis, the most important rice seedling insect pests and one of nontarget insect species of Bt rice, were evaluated on two transgenic Bt rice lines including H2A, H1C (expressing the Cry2A and Cry1C protein respectively) and their non-Bt correspondent parental cultivars including HCK under laboratory conditions. ELISA was performed to observe the presence of Bt toxin in the thrips and its possible effects on the thrips although the thrips as sucking insects have no specific site of action of Bt toxin in digestive system. Laboratory results for H2A rice revealed higher mortality for three times testing at the beginning of second larval instars while H1C did not reveal any higher larval mortality for all three times tests. On the other hand no higher mortality was observed in any of Bt rice cultivar for adults as compared to control. However the larval and pupal development duration as well as pre-oviposition period were longer, oviposition period and female adult longevity were shorter, and total number of eggs laid were lower when thrips were fed on H2A tested Bt rice varieties while there was no significant difference in H1C rice cultivars as compared to their respective controls. Higher levels of Bt protein were detected in case of H2A in which case higher concentration was observed in case of larvae as well as adult thrips. While higher concentration of Bt protein in case of H1C was also observed. While no Bt protein was detected in case of control cultivars.2-Life cycle, reproductive parameters and ELISA of S. biformis under laboratory conditions for Bt rice line E1and non-Bt rice line Xiushui110The one time mortality test, life cycle, reproductive parameters of S. biformis on Bt rice line E1(expressing the combined CrylAb and Vip3H) and its non-Bt correspondent parental cultivars including Xiushui110under laboratory conditions. ELISA was performed to observe the presence of Bt toxin in the thrips and its possible effects on the thrips although the thrips Laboratory results for E1rice mortality showed none of the difference in terms of mortality of E1as compared to control. The larval and pupal development duration as well as preoviposition period were same as control. Oviposition period and female adult longevity were similar in Bt and non-Bt rice lines, and total number of eggs laid were same when thrips were fed on E1tested Bt rice varieties while there was no significant difference in El Bt rice cultivars as compared to its respective control. Normal levels of Bt protein were detected in case of Cry1Ab concentration which was observed in case of larvae as well as adult thrips. While normal concentration of Bt protein in case of leaf was also observed. While no Bt protein was detected in case of control cultivars.3-Leaf dip bioassay of Bt proteins Cry2A, Cry1C, CrylAb at different concentrationsA leaf dip bioassay was carried using the leaf from control cultivar. Concentration of Cry2A, Cry1C and Cry1Ab proteins were used to observe the effects on mortality of S. biformis at different concentrations. For cry2A concentration of10,20,50and100mg/ml were used while for cry1C and crylAb were used at concentrations of3,9,27and54mg/ml. Adults as well as nymphs were observed for the effects on their morality. A higher mortality was observed in case of cry2A even at its lowest concentration while crylC and crylAb were lethal only at very high concentrations. The experiments were carried out upto eight days of the experiment.4-Semi-field population dynamics observations of H2A and H1C as compared to their controlA semi-field observation upto30days was carried out under semi-field conditions in the laboratory using glass cages on the plants. Plants of H2A, H1C and HCK were6-8tillers stage. Five adult S. biformis (including four females and one male), five larvae were released in the cages of respective cultivar. Lowest population of thrips was observed in case of H2A while higher population dynamics was observed in case of H1C and HCK. Number of larvae and adults were found at its lowest level in case if H2A as compared to its control HCK. These changes in pot conditions in laboratory reveal that H2A will not be ulikely favored by S. biformis for feeding in the field.5-Two years field observations of population abundance of S. biformis in H2A, H1C and their control cultivars The population abundance of Stenchaetothrips biformis (Bagnall), the most important rice seedling insect pests and one of nontarget insect species of Bt rice, were evaluated on H2A and H1C(expressing the Cry2A and Cry1C respectively) and their non-Bt correspondent parental cultivars under field conditions for two years of2011and2012. In the field, population of the thrips in Bt and non-Bt plots was monitored using the beat plate and plastic bag methods. The patterns of population dynamics of thrips, in the field, were similar on all tested Bt rice lines and their respective control, however, thrips individuals collected in the Bt plots were numerically less than those in the non-Bt plot almost at the entire rice growth stages in2011. While in2012, there was significant difference in population dynamics as well as population densities of S. biformis nymphs but not for adults. A numerical and even statistical lower average density of all individuals of thrips larvae and adults occurred across the entire sampling period in Bt rice plots, and thrips individuals in Bt rice plots, at some sampling dates. Differential population changes in Bt and non-Bt cultivars suggest that there can be some other reason of Bt rice including biochemical, physiological or semio-chemical changes which can also result in these kind of changes.6-Two years field observations of population abundance of S. biformis in E1and its control cultivarStenchaetothrips biformis (Bagnall) were also evaluated in El (expressing the combined Cry1Ab and vip3H protein) and its non-Bt correspondent parental cultivars under field conditions for two years. In the field, population of the thrips in Bt and non-Bt plots was monitored using the plastic bag and beat plant methods. The patterns of population dynamics of thrips, in the field, were similar on all tested Bt rice lines and their respective control during both years of sampling. There was none of the date where the density or dynamics of S. biformis was found more in Bt rice as compared to control. Thus it proves that Bt rice can be more safer for commercial release but a case by case study is needed before its commercialization. 7-Population densities of three thrips species attacking panicle of rice and their predator Orius tantilus in H2A, H1C and their control cultivar over two years periodThe population abundance of thrips attacking panicles of rice such as Haplothrips aculeatus (Fabricius), Frankliniella intonsa (Trybom) and Haplothrips tritici were observed on H2A, H1C(expressing the Cry2A and Cry1C protein respectively) and their non-Bt correspondent parental cultivars under field conditions. In the field, population of all thrips in Bt and non-Bt plots was monitored using the beat plate and plastic bag methods. The patterns of population dynamics of all thrips, in the field, were similar on all tested Bt rice lines and their respective control. For population abundance of Orius tantilus overall no significant or non-significant difference was observed in Bt and non-Bt fields and their population was found more or less same in Bt and non-Bt rice fields.8-Population densities of three thrips species attacking panicle of rice and their predator Orius tantilus over two years period in E1and its control cultivarThe population abundance of thrips attacking panicles of rice such as Haplothrips aculeatus (Fabricious), Frankliniella intonsa (Trybom) and Haplothrips tritici were observed on E1(expressing the combined Cry1Ab or Vip3H protein respectively) and their non-Bt correspondent parental cultivars under field conditions. In the field, population of all thrips in Bt and non-Bt plots was monitored using the plastic bag and beat plant methods. The patterns of population dynamics of all thrips, in the field, were similar on all tested Bt rice lines and their respective control. The population abundance of Orius tantilus in the field were observed by using two sampling methods such as taking the whole plant into plastic bag and beat plate methods. Overall no significant or non-significant difference was observed in Bt and non-Bt fields and their population was found more or less same in Bt and non-Bt rice fields. 9-Population dynamics of five thrips species and their predator in paddy rice from seedling to harvesting using color trap method in KMD cultivars in2011One year field experiment was conducted to observe the field population dynamics and densities of S. biformis which attacks during seedling stage while F. intonsa, F. tenuicornis, H. aculeatus and H. tritici which attacks during panicle development stages and cause severe losses at that time using color traps sampling methods in2011. Color traps used for this assessment were white, blue and yellow. The results suggested that the thrips population was similar across whole sampling season and there was no difference in their population dynamics as well as population densities. Besides, O. tantilus patterns were also similar across the whole sampling season proving that transgenic rice does not affect the second and third trophic level of paddy rice fields.10-ELISA and population dynamics of Orius predator in paddy rice from seedling to harvesting using three sampling methods in KMD cultivars in2011Protein test ELISA and one year field experiment was conducted in2011for Orius tantilus population dynamics and population densities using three sampling methods. There was no Bt protein found in the predator O. tantilus when feeding on thrips or when fed with pollens from KMD1and KMD2Bt rice as compared to control..All three sampling methods found no significant difference in population dynamics and population densities of predator. Patterns of predators were same across whole sampling season. These experiments results prove that Bt rice is safe for predator O. tantilus in rice paddy fields and can not result in harmful effects for this predator.