Molecular Characterization Of The Effects Of Cantharidin In Helicoverpa Armigera Hubner

Cotton bollworm, Helicoverpa armigera Hubner is a serious pest of many economiccrops. Its control has not been adequate due to its resistance to many groups of insecticides.Toxicity of cantharidin on armyworm and diamondback moth has already been reported.However, its toxicity on H. armigera has not been investigated previously. In this study, wereport lethal and sub-lethal effects of cantharidin on H. armigera under laboratory conditions.Our results showed gross abnormalities in population parameters of H. armigera, rangingfrom larvae to adults. Reduction in larval weight and wings malformation was observed incantharidin-treated population cohort. Comparatively, higher mortality at larval, pupal andadult stages was observed in cantharidin-treated, compared to control. Almost five times lessfecundity was recorded in treated population cohort. Fertility was also severely affected andreduction in all population parameters was observed. Cantharidin in our study caused larvalmortality and other serious abnormalities in H. armigera population parameters and therefore,may have positive implications for pest management decision making process. Moreinterestingly, our experiment revealed that cantharidin in sub-lethal dose mimicked IGR(Insect Growth Regulator) insecticides. Furthermore, cantharidin could be used as a precursorcompound for the synthesis of new analogues and compounds to replace ineffective oldercompounds.Previous investigations have implicated glutathione S-transferases (GSTs) as one ofthe major reasons for insecticide resistance. Therefore, effectiveness of the new candidatecompounds depends on their ability to inhibit GSTs to prevent metabolic detoxification byinsects. Cantharidin has been developed as a bio-pesticide in China that proves highly toxic toa wide range of insects, especially lepidopteran. In present studies we test cantharidin as amodel compound for its toxicity, effects on the mRNA transcription of a model H. armigeraglutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on thecatalytic activity of GSTs, both in vivo and in vitro in H. armigera employing molecular andbiochemical methods. Bioassay results showed that cantharidin was highly toxic to H.armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcriptranging from2.5to12.5folds while biochemical assays showed in vivo inhibition of GSTs inmidgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalizedby homology and molecular docking simulations using a model GST (1PN9) as a templatestructure. Molecular docking simulations also confirmed accurate docking of the cantharidinmolecule to the active site of HaGST impeding its catalytic activity. Earlier investigations have shown high toxicity of cantharidin to many insectsespecially lepidopteran. However, its use in a higher dose for pest management has raisedserious environmental concerns. Therefore, its biological potential in sub-lethal dose as asynergist was considered. In fact, it is essential for a synergist to be an effective inhibitor ofmetabolic enzymes especially those responsible for biotic and abiotic stress. As alkalinephosphatase is an important enzyme involved in numerous physiological processes and also ininsecticide resistance, so any impairment in its function may lead to serious physiologicaldisturbances and could compromise their catalytic activity. Results of the presentinvestigation showed that a sub-lethal dose of25μg g-1treated artificial diet fed to H.armigera showed inhibitory effects on catalytic activity of alkaline phosphatase in the insectmidgut. Furthermore, Kinetic data showed that cantharidin inhibited HaALPs competitivelywith respect to p-NPP. In addition, cantharidin did not induce Helicoverpa armigera alkalinephosphatase gene (HaALP) rather caused slight down regulation. The inhibitory effect ofcantharidin on catalytic activity of ALPs as a result of its binding to the putative catalytic sitewas also confirmed by using homology modeling, molecular dynamics and dockingsimulations.