The influence of inactivated entomopathogenic bacterium Bacillus thuringiensis on the immune responses of the Colorado potato beetle
Authors: Polenogova Olga V. , Kryukova Natalia A. , Klementeva Tatyana , Artemchenko Anna S. , Lukin Alexander D. , Khodyrev Viktor P. , Slepneva Irina , Vorontsova Yana , Glupov Viktor V. 
Published: October 2024

Background
Invasion of microorganisms into the gut of insects triggers a cascade of immune reactions accompanied by increased synthesis of effectors (such as antimicrobial peptides, cytokines, and amino acids), leading to changes in the physiological state of the host. We hypothesized that even an inactivated bacterium can induce an immune response in an insect. The aim of this study was to compare the roles of reactive oxygen species (ROS) formation and of the response of detoxification and antioxidant systems in a Colorado potato beetle (CPB) larval model in the first hours after invasion by either an inactivated or live bacterium.

Methods
The influence of per os inoculation with inactivated entomopathogenic bacterium Bacillus thuringiensis var. tenebrionis (Bt) on the survival and physiological and biochemical parameters of CPB larvae was assessed as changes in the total hemocyte count (THC), activity of phenoloxidases (POs), glutathione-S-transferases (GSTs), nonspecific esterases (ESTs), catalase, peroxidases, superoxide dismutases (SODs) and formation of reactive oxygen species (ROS).

Results
A series of changes occurred within the hemolymph and the midgut of CPBs inoculated with inactivated Bt at 12 h after inoculation. These physiological and biochemical alterations serve to mediate generalized resistance to pathogens. The changes were associated with an increase in the THC and a 1.4–2.2-fold enhancement of detoxification enzymatic activities (such as GST and EST) as well as increased levels of antioxidants (especially peroxidases) in hemolymph in comparison to the control group. Suppressed EST activity and reduced ROS formation were simultaneously detectable in the larval midgut. Inoculation of beetle larvae with active Bt cells yielded similar results (elevated THC and suppressed PO activity). A fundamental difference in the immune activation processes between larvae that ingested the inactivated bacterium and larvae that had consumed the active bacterium was that the inactivated bacterium did not influence ROS formation in the hemolymph but did reduce their formation in the midgut. At 24 h postinfection with active Bt, ROS levels went up in both the hemolymph and the midgut. This was accompanied by a significant 5.7-fold enhancement of SOD activity and a 5.3-fold suppression of peroxidase activity. The observed alterations may be due to within-gut toxicity caused by early-stage bacteriosis. The imbalance in the antioxidant system and the accumulation of products toxic to the “putative” pathogen can activate detoxification mechanisms, including those of an enzymatic nature (EST and GST). The activation of detoxification processes and of innate immune responses is probably due to the recognition of the “putative” pathogen by gut epithelial cells and is similar in many respects to the immune response at early stages of bacteriosis.