Transcriptome and metabolome integration reveals molecular mechanisms underlying divergent cold tolerance in geographically distinct Procambarus clarkii populations

Low temperature is a major environmental stressor that constrains the survival and distribution of Procambarus clarkii. To explore the cold adaptation mechanisms of geographically differentiated populations, we exposed P. clarkii from Northeast China, Hubei and Anhui to continuous cold stress at 4 °C, while crayfish maintained at 20 °C serving as the control group. After 10 days of stress exposure, tail-muscle tissues were collected for transcriptomic and metabolomic analyses. Phenotypic observation revealed distinct cold tolerance among populations: the Northeast population had the highest cold tolerance while the Anhui population was the most cold-sensitive. Transcriptomic screening identified population-specific differentially expressed genes (DEGs), with 598, 113, and 193 DEGs detected in the Anhui, Hubei, and Northeast populations, respectively. Meanwhile, metabolomic analysis yielded 76, 456, and 176 differentially expressed metabolites (DEMs) in the Anhui, Hubei, and Northeast populations respectively. In this study, we performed integrated KEGG enrichment analysis of transcriptomes and metabolomes to delineate cold adaptive signatures across three geographically distinct P. clarkii populations. The Northeast population exhibited the most distinct adaptive reprogramming, with specific enrichment of pathways related to cell junction, phagocytosis, estrogen signaling, glutathione metabolism and D-amino acid metabolism, which may confer superior cold tolerance by reinforcing cellular structural integrity, enhancing innate immune defense and improving antioxidant capacity. The Hubei population was characterized by enriched tricarboxylic acid cycle, AMPK-mediated energy signaling, MAPK signaling and protein processing pathways, reflecting a strategy of energy homeostasis coupling with cellular stress response. The Anhui population mainly enriched basal metabolic pathways including carbohydrate metabolism, purine metabolism and aminoacyl-tRNA biosynthesis, suggesting a relatively basal metabolic maintenance strategy. Notably, basal energy and substance metabolism pathways such as sulfur metabolism, HIF-1 signaling and purine metabolism were conserved across all three populations. Collectively, the differential metabolic and transcriptional pathway reprogramming contributes to the cold adaptive differentiation of geographically separated P. clarkii populations, providing a comprehensive theoretical basis for exploring stress resistance mechanisms in crustaceans.