Dose-dependent effects of CO2-modified atmospheres on physiology and gut microbiota of red flour beetle

CO₂-modified atmosphere storage is an eco-friendly strategy for controlling stored grain pests. However, the molecular and microbial mechanisms of insect responses to different CO₂ concentrations remain poorly understood. This study investigated the physiological, transcriptomic, and gut microbiota responses of Tribolium castaneum exposed to different CO₂ concentrations (35 % and 60 % CO₂). Transcriptomic profiling combined with enzymatic analysis revealed that 35 % CO₂ primarily activated genes involved in digestion and NADPH generation, such as DHDH upregulated 2.77-fold. In contrast, 60 % CO₂ markedly upregulated GST (1.52-fold) and pancreatic secretion genes, CTRL (2.63-fold), CTRB (1.46-fold), and CPA2 (2.65-fold), as well as elevated in trypsin (1.96-fold), lipase (2.86-fold), and GST (3.58-fold) activities. Microbial profiling demonstrated that 35 % CO₂ enriched Pseudomonadota, whereas 60 % CO₂ favored Bacillota. Functional prediction suggested microbial contributions to host signal transduction under 35 % CO₂ and stress mitigation under 60 % CO₂. Key genera, such as Enterococcus and Pseudomonas exhibited treatment-specific correlations with host gene expression and enzyme activity, suggesting a potential microbiota-mediated modulation of host stress responses. These findings provided new insights into the dose-dependent physiological adaptations of T. castaneum under CO₂-MAs and highlighted the interaction between host transcriptional reprogramming and gut microbial plasticity.