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Dually Functional Recombinant Baculovirus Expressing Both the Excitatory and Depressant Insect Selective Neurotoxins

The present project is aimed to improve the insecticidal potency of baculoviruses, to American and Israeli lepidopterous pests of Spodoptera and Heliothis species, by engineering recombinant baculoviruses expressing anti-insect toxins derived from scorpion venom. Through this study were isolated recombinant Autographa california M Nucleopolyhedroviruses (AcNPVs) which expressed alpha (LqhaIT), excitatory (AaIT, LqhIT1 and LqhIT3) and depressant (LqhIT2) anti-insect neurotoxins. Bioassays on Heliothis species (Helicoverpa armigera and Heliothis virescens) were employed to assess the potency of the viruses. The recombinant viruses possessed an enhanced speed of kill compared to wild type AcNPV. Recombinant AcNPVs expressing the depressant toxins emerged as appealing improved baculoviruses. Applied combinations of alpha, excitatory and depressant toxins enhanced their insecticidal activity against blowfly and lepidopterous larvae. Moreover, combined application of recombinant AcNPVs expressing LqhaIT and AaIT possessed increased insecticidal activity compared to single applications of them. A reduced growth rate of H. virescens larvae was obtained by comparing the larvae infected with recombinant AcNPV expressing AaIT under the control of the AcNPV early ie1 to the very late p10 promoters. Through this project improved protocols and methods were developed to purify and bioassay the anti-insect toxins and their correspondent recombinant baculoviruses. A novel highly potent anti-insect toxin Aa IT5 was isolated and characterized. Finally, the impact of use of recombinant baculoviruses, expressing anti-insect scorpion neurotoxins to non-target insects, was evaluated.