Expression Analyses, Efficacy Studies and Environmental Risk Assessment of Insecticidal Proteins Produced in Transgenic Plants

Insect pests are a serious threat to agricultural crops all over the world as they cause huge losses to crop yield and economy. Due to low innate immunity in plants, synthetic insecticides have been used to cope with the pest problem since decades. These chemicals are expensive, broad spectrum and hazardous to health and environment. Moreover, most of the target insect species have developed resistance against the available insecticides. Therefore, search for alternative methods of insect pest control has been a priority in agricultural research. One of the successful methods is to develop Insect Resistant, Genetically Modified Crops (IR-GMCs) expressing insecticidal proteins. IR-GMCs have been rapidly adopted around the world as they are target specific, environment friendly, cost effective and easy to use. However, two major risks of growing IR-GMCs have been proposed; resistance development against insecticidal toxins (e.g. Cry proteins) expressed in plants and adverse effects to beneficial, non target arthropods (NTAs). NTAs include important species that contribute directly to agricultural ecology via biological control, pollination and decomposition of organic materials. Pakistan officially adopted Bt-cotton in 2010 and became one of the leading countries in terms of area under cultivation by 2013. Variation in the expression of Bt proteins have been associated with reduced efficacy of the IR crops and also resistance development in the target insect pests. Therefore, one of the objectives of this study was expression profiling of first batch of approved, locally bred Bt-cotton varieties through ELISA and efficacy analysis against Helicoverpa armigera. While Bt cotton varieties are effective in controlling several key lepidopteran pests, they are less effective against some Lepidoptera species and provide no protection against non-lepidopteran pests. In addition, the initial cases of resistance to Cry1Ac have been observed in target insect pests in many countries. It is thus indicated to explore alternative source for toxins that can be incorporated in future transgenic plants. The peptide ? -Hexatoxin-Hv1a (Hvt) is one of the most studied spider venom toxins. Its insecticidal potential has been reported for species belonging to different arthropod orders including Lepidoptera, Diptera and Orthoptera. The gene encoding for Hvt has thus been transformed into cotton and tobacco to protect the plants from damage by Lepidopteran pests. The second objective of the study therefore, was to evaluate the expression of ? -Hexatoxin-Hv1a gene in transgenic plants, and the toxicity of plant expressed and purified Hvt against target lepidopteran insects and four non-target arthropod species. Bollgard IITM cotton plants and purified Cry2Ab2 protein were included in the study as comparators. In studies with locally bred Bt cotton, expression of Cry1Ab/1Ac protein were found to vary significantly (P<0.05) among varieties and across different sampling dates. The highest mean expression was recorded in GN-31 and Sitara-008 and the lowest in FH 113 and MG-6 while across sampling dates the highest mean expression was recorded at 30 days after emergence (DAE) which decreased along the season with lowest mean at 120 DAE. The control of the H. armigera was directly proportional to the amount of protein expressed in the leaves. Critical level of Cry1Ab/1Ac in leaves was found to be 770±25 ng g-1 for 95% control of the target insect pests. In studies with Hvt plants, 100% larval mortality was observed on Hvt-transgenic tobacco line T-21 and cotton line T-7 plants but not on cotton line T-10. The likely reason was the significantly lower expression level in the transgenic cotton line T-10. When fed on artificial diet containing purified Hvt protein, LC95 values for Spodoptera littoralis and Heliothis virescens were found to be 28.31 and 27.57 µg mL-1, respectively. Non-target studies were performed with two predatory species, larvae of Chrysoperla carnea and Coccinella septempunctata, adult females of the aphid parasitoid Aphidius colemani, and adult workers of honey bees Apis mellifera. Orally administered Hvt protein even at a concentration of 40 µg mL-1 did not affect a number of life-table parameters of tested NTAs. Similarly purified Cry2Ab2 did not cause adverse effects on any of the non-target species tested at a concentration of 10 µg mL-1. However, detailed molecular work is required to determine the fate of orally administered Hvt protein in these species. Hence, Hvt might be an interesting candidate for developing insecticidal plant varieties to control pest Lepidoptera. However, the concentration at which the protein is available in the plants appears to be critical in determining its ef? cacy against target pests. Therefore, breeders and biotechnologists must combine genotypes and promoters promising higher expression of insecticidal proteins. Furthermore, detailed risk assessment studies may be conducted before commercial release of Hvt cotton and tobacco.