Mechanisms of feeding cessation in Helicoverpa armigera larvae exposed to Bacillus thuringiensis Cry1Ac toxin

Abstract

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been applied in sprayable formulations and expressed in transgenic crops for the control of pests in the field. When exposed to Bt proteins insect larvae display feeding cessation, yet the mechanism for this phenomenon remains unknown. In this study, we investigated the feeding behavior and underlying mechanisms of cotton bollworm (Helicoverpa armigera) larvae after exposure to the Cry1Ac protein from Bt. Three H. armigera strains were studied: the susceptible SCD strain, the C2/3-KO strain with HaABCC2 and HaABCC3 knocked out and high-level resistance to Cry1Ac (>15,000-fold), and the SCD-KI strain with a T92C point mutation in tetraspanin (HaTSPAN1) and medium-level resistance to Cry1Ac (125-fold). When determining the percentage of insects that continued feeding after various exposure times to Cry1Ac, we observed quick cessation of feeding in larvae from the susceptible SCD strain, whereas larvae from the C2/3-KO strain did not display feeding cessation. In contrast, larvae from the SCD-KI strain rapidly recovered from the initial feeding cessation. Histopathological analyses and qRT-PCR in midguts of SCD larvae after Cry1Ac exposure detected serious epithelial damage and significantly reduced expression of the neuropeptide F gene (NPF) and its potential receptor gene NPFR, which are reported to promote insect feeding. Neither epithelial damage nor altered NPF and NPFR expression appeared in midguts of C2/3-KO larvae after Cry1Ac treatment. The same treatment in SCD-KI larvae resulted in milder epithelial damage and subsequent repair, and a decrease followed by an initial increase in NPF and NPFR expression. These results demonstrate that the feeding cessation response to Cry1Ac in cotton bollworm larvae is closely associated with midgut epithelial damage and downregulation of NPF and NPFR expression. This information provides clues to the mechanism of feeding cessation in response to Bt intoxication and contributes to the mode of action of the Cry1Ac toxin in target pests.

Keywords: Bacillus thuringiensis; Feeding cessation; Helicoverpa armigera; Midgut epithelial damage, Neuropeptide F.