Interaction of proteinase inhibitors with Cry1Ac toxicity and the presence of 15 chymotrypsin cDNAs in the midgut of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae).

The potential development of resistance to Bacillus thuringiensis (Bt) cotton and surging of non-targeted insects is a major risk in the durability of Bt plant technology. Midgut proteinases are involved in Bt activation and degradation. Proteinase inhibitors may be used to control a wide range of insects and delay Bt resistance development. Proactive action to examine proteinase inhibitors for synergistic interaction with Bt toxin and cloning of proteinase cDNAs for RNAi is necessary to make transgenic cotton more versatile and durable. A sublethal dose (15 ppb) of Cry1Ac, 0.5% benzamidine and 0.02% phenylmethylsulfonyl fluoride significantly suppressed midgut azocaseinase, tryptic and chymotryptic activities, and resulted in reductions in larval and pupal length and mass of Heliothis virescens. The combination of proteinase inhibitor and Bt suppressed 20-37% more larval body mass and 26-80% more enzymatic activities than the inhibitor only or Bt only. To facilitate knockdown-resistance-related proteinase genes, 15 midgut chymotrypsin cDNAs were sequenced. Most predicted chymotrypsins contained the conserved N-termini IVGG, three catalytic center residues (His, Asp and Ser), substrate specificity determinant (Ser or Gly) and cysteines for disulfide bridges. These putative chymotrypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. H. virescens has evolved diverse midgut proteinase genes. Proteinase inhibitors have potential insecticidal activity, and the interaction of Bt with proteinase inhibitors is desirable for enhancing Bt toxicity and delaying resistance development. Intensive sequencing of chymotrypsin cDNAs will facilitate future functional examinations of individual roles in Bt toxicity and resistance development and facilitate targeted control using RNAi and/or proteinase inhibitors.