In Vivo High-Resolution MSSR Microscopy Reveals Cry Toxin-Induced Actin Disruption, Endocytic Activity, and Vesicle Release in Insect Midgut Cells

Introduction: Pore-forming toxins (PFTs) play a pivotal role in bacterial pathogenicity, yet the cellular responses they elicit remain incompletely understood.

Methods: Using in vivo Mean Shift Super-Resolution (MSSR) microscopy, we analyzed mosquito larval midgut cells exposed to Bacillus thuringiensis Cry11Aa toxin, achieving unprecedented nanoscale visualization of the intoxication process. Transmission electron microscopy was also used in this study.

Results: Our observations uncovered a sequential cascade beginning with toxin binding to brush border microvilli (BBM), followed by membrane pore formation that disrupts actin cytoskeletal integrity and stimulates massive endocytosis. An additional observed response was the release of large (5–8 µm) membrane-enclosed vesicles into the midgut lumen. These vesicles, segmented by endoplasmic reticulum (ER) membranes and actin filaments, carried damaged organelles—such as mitochondria, lysosomes, and endosomes—yet lacked nuclear material. Notably, the toxin was found in close association with ER membranes within these vesicles, suggesting this process may represent a toxin-clearing defense mechanism. Transmission electron microscopy confirmed vesicle morphology and contents. A comparison with a non-toxic Cry11E97A mutant, defective in oligomerization and membrane insertion, confirmed that pore formation is essential for triggering the observed cellular events. Vesicles of similar morphology were rarely detected in untreated controls, implying that their production may reflect an amplified version of a natural epithelial renewal process.

Conclusions: These findings shed light on a previously unrecognized aspect of midgut epithelial defense against Cry toxins, emphasizing the complexity of the insect cellular response to PFTs. Understanding this pathway offers new perspectives for enhancing the effectiveness of Bt-based pest control and managing resistance in insect populations.