Low temperatures can induce adaptive responses in ectothermic animals, the grain pest Cryptolestes ferrugineus has exhibited a remarkable adaptability to cold environment. Mitochondrial protein-coding genes are crucial for maintaining respiration metabolism and ATP synthesis in organisms, and they play various functions in insects. However, the role of these genes in cold adaptation is poorly understood.

The purpose of this study is to elucidate the roles of mitochondrial protein-coding genes in the formation of cold tolerance of C. ferrugineus.

The cold tolerance of C. ferrugineus ST and CK populations was determined at a lethal low temperature (-20 ℃). The respiration rate and ATP content of C. ferrugineus ST and CK populations were measured using a CO₂ detector and an ATP content assay kit. The relative expression levels of 13 mitochondrial protein-coding genes between the ST and CK populations were assessed by using RT-qPCR. RNA interference (RNAi) technology was employed to knock down the key mitochondrial protein-coding genes ND6 and ATP6 in C. ferrugineus, and then the expression levels of the remaining 12 mitochondrial protein-coding genes, respiration rate, ATP content, and changes in cold tolerance were explored after effective silencing of ND6 and ATP6.

The cold tolerance of C. ferrugineus CK population was higher than that of the ST population, while the respiration rate and ATP content of the CK population were only 58.68% and 62.54% of those in the ST population, respectively. Additionally, the expression levels of 12 mitochondrial protein-coding genes (except ND3) in the CK population were significantly lower than those in the ST population. These results suggested a negative correlation between cold tolerance and physiological indicators (respiration rate, ATP content, and the expression levels of mitochondrial protein-coding genes). When the key mitochondrial protein-coding genes ND6 and ATP6 were effectively silenced via dsRNA feeding, the respiration rate and ATP content were significantly reduced, while cold tolerance in C. ferrugineus was significantly enhanced.

The mitochondrial protein-coding genes ND6 and ATP6 are involved in the formation of cold tolerance by regulating energy metabolism in C. ferrugineus.