Biochemical, molecular cytogenetic and correlation analyses of Bacillus thuringiensis sublethal concentration toxicity on Spodoptera frugiperda larval Hemolymph.

Bacillus thuringiensis is considered one of the most environmentally safe tools for controlling various insect pests, including the fall armyworm, Spodoptera frugiperda (J.E. Smith). This pest remains one of the most serious threats to global food security. Although Bacillus thuringiensis var. kurstaki (Btk) is an essential long-term pest control agent, most research has examined its lethal effects only on larval midgut tissue as the primary target organ. Accordingly, this study explored Btk toxicity on the third-instar S. frugiperda larval hemolymph as a secondary consequence using a multifaceted approach. Our findings revealed that a sublethal concentration (LC25) of Btk induced high oxidative stress in larvae. The biochemical effects were characterized by a significant increase in reactive oxygen species (ROS) and a significant decrease in the insect's primary antioxidant enzymes, including superoxide dismutase, catalase, and glutathione S-transferase. Due to the cytogenetic toxicity of Btk, enhanced micronuclei formation was observed, and the gene expression of DNA methyltransferase 2 (SfDNMT2) and inhibitor of apoptosis 2 (SfIAP2) mRNA were reduced, while autophagy-related protein 8 (SfATG8) was significantly increased in S. frugiperda hemolymph bioassays. A PCA-based bioinformatics analysis illustrated an inverse functional correlation between oxidative damage and antioxidant defensive systems, with PC1 accounting for 95.63% of the total variance. Following PCA, the cluster heatmap and dendrogram provided visualization of similarities and hierarchical relationships among the parameters mentioned. These results collectively indicate that Btk's insecticidal effects are not limited to midgut cells but rather involve a broader toxic response against the hemolymph carrier for cellular and molecular components, even at lower concentrations.