A Biofilm-State Bacillus thuringiensis Formulation Drives Midgut Structural Disruption and Transcriptomic Reprogramming in Ectropis grisescens

<i>Bacillus thuringiensis</i> (Bt) is one of the most extensively used microbial insecticides, attributed to the action of insecticidal crystal proteins (ICPs), primarily Cry toxins, which mediate damage to the insect midgut epithelium. Recent evidence suggests that Bt toxicity is also strongly influenced by its physiological state and interactions with the host gut environment. Biofilm formation represents an important adaptive strategy that enhances bacterial stress tolerance and may modulate insecticidal performance, although the underlying mechanisms remain unclear. However, it is still unclear how Bt in the biofilm state alters host responses at the structural and transcriptomic levels. Using the tea plantation pest <i>Ectropis grisescens</i> as a model, we systematically evaluated the insecticidal efficacy of biofilm-state Bt formulations and their synergistic effects with a biofilm inducer system composed of Tween-80, tea saponin, matrine, and tea polyphenols. Bioassays showed that the biofilm-state Bt supplemented with composite inducers achieved the highest corrected mortality and reduced the LC₅₀ against neonate larvae by 2.88-fold compared with conventional planktonic Bt. Histopathological, biochemical, and transcriptomic analyses further revealed that biofilm-state Bt caused more severe midgut damage and induced extensive remodeling of detoxification- and stress-response-related pathways. These findings highlight Bt physiological state as a critical determinant of formulation efficacy and provide a novel framework for Bt optimization through microbial physiological regulation.