Hormetic mitogenic effects of α-arylphorin determine midgut regeneration and reduce susceptibility to Cry1Ac toxin in Chloridea virescens.

Damage to the lepidopteran larval midgut by insecticidal Cry proteins from Bacillus thuringiensis (Bt) induces molecular signals that activate intestinal stem cell (ISC) proliferation and differentiation, enabling epithelial repair. We previously reported that exposing Chloridea (Heliothis) virescens (tobacco budworm) larvae to the Cry1Ac Bt protein increases production of midgut α-arylphorin, a hexamerin protein. Subsequent reports support a central role for α-arylphorin as a potent mitogen in the regenerative response to Cry intoxication. Here, we examined the effects of feeding purified α-arylphorin to C. virescens larvae on ISC activity and larval susceptibility to Cry1Ac. Purified α-arylphorin stimulated in vitro proliferation of a Helicoverpa zea cultured cell line of midgut origin. Histological analyses revealed that oral ingestion of purified α-arylphorin elicited a hormetic response in C. virescens midgut ISCs. Feeding on lower α-arylphorin concentrations induced dose-dependent ISC proliferation, resulting in a pseudostratified midgut epithelium and reduced C. virescens larval susceptibility to Cry1Ac. In contrast, increasing α-arylphorin concentration failed to stimulate ISC proliferation or reduce susceptibility to Cry1Ac. At the highest α-arylphorin concentrations tested, reduced larval susceptibility occurred in the absence of ISC proliferation. Binding assays suggest that α-arylphorin may interact with Cry1Ac in the midgut lumen and hinder toxin binding to epithelial receptors, explaining reduced larval susceptibility when ISC proliferation is not observed. Together, these findings support a model in which local α-arylphorin titers determine whether protection from Cry1Ac intoxication occurs through ISC proliferation or via α-arylphorin acting as a putative decoy toxin-binding factor.