Transcriptomic and Microbiome Analyses of Procambarus clarkii Exposed to Different Doses of 20E

Molting determines survival and growth in cultured crustaceans, yet its specific regulatory mechanisms remain complex. This study integrated transcriptomics and microbiome analyses to elucidate molting regulation in crayfish (Procambarus clarkii). Crayfish were injected with 20-hydroxyecdysone (20E) at 20 and 250 ng/g to simulate early premolt and middle premolt, respectively. The comprehensive upregulation of nuclear receptor family genes confirmed the reliability of the in vivo 20E injection simulation. The results showed that 20 ng/g 20E stimulation induced 13,253 unique DEGs in the epidermis, mainly enriched in protein catabolism (promoting proteolysis to degrade the old exoskeleton), and induced 137 unique DEGs in hemocytes, mainly linked to ribosomal biosynthesis, while the 250 ng/g group showed 2395 unique DEGs in the epidermis, enriched in metabolic processes and biosynthetic processes (supporting the biosynthesis of the new stratum corneum), and 99 unique DEGs in hemocytes enriched in mitochondrial pathways, concomitantly enhancing energy metabolism and antioxidant defense capabilities. Notably, 20E upregulation potentially leads to the dysbiosis of pathogens, specifically Escherichia-Shigella and Vibrio. This study elucidates key biological events in the early and middle premolt of crayfish, clarifies tissue-specific regulatory mechanisms during premolt, and provides molecular-level insights into the growth regulatory network of crustaceans.