Assessing the impact of cry insecticidal proteins from Bacillus thuringiensis on the midgut of Spodoptera littoralis (Boisduval).

Bacillus thuringiensis (Bt) Cry toxins exert their insecticidal activity by targeting the larval midgut epithelium, yet integration of physiological, ultrastructural, and microbiological responses under near-native conditions remains limited. In this study, we investigated the effects of Cry1Ca on Spodoptera littoralis larvae using an integrative approach combining physiological measurements with high-resolution scanning and transmission electron microscopy (SEM and TEM). Building on previous whole-organism imaging approaches, we analyzed intact larvae without prior dissection, an approach that preserves tissue architecture and the spatial relationships between gut compartments. Short-term exposure to sublethal toxin concentrations resulted in significant growth inhibition without mortality, accompanied by extensive disruption of the midgut epithelium, including microvilli loss, columnar cell lysis, and degradation of the peritrophic matrix. These alterations were accompanied by the presence of bacteria within normally protected gut compartments. Notably, partial recovery of epithelial organization, microvilli, and peritrophic matrix structure was observed 48 h after transient exposure, suggesting a strong regenerative capacity. In contrast, continuous exposure to a lethal concentration caused irreversible epithelial destruction, widespread bacterial invasion, and larval death. The absence of qualitative differences in epithelial damage across sublethal concentrations is compatible with a saturating, threshold-type response, although this was not quantified across concentrations. Overall, our findings provide direct ultrastructural visualization of epithelial disruption and barrier breakdown, together with the presence of bacteria within normally protected compartments, consistent with the toxin-induced bacterial translocation previously demonstrated in this species, while highlighting the importance of exposure dynamics. Moreover, the methodological approach presented here offers a useful, complementary framework for studying the mode of action of Cry proteins and other gut-targeting toxins under conditions that closely approximate the in vivo state.