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

## TL;DR

This study explores how crayfish prepare for molting by analyzing gene and microbiome changes after 20E injections, offering insights to improve crustacean farming.

## Contribution

The study provides tissue-specific insights into early and middle premolt regulation in crayfish using transcriptomic and microbiome data.

## Key findings

- Early premolt involves exoskeleton degradation and proteolysis, as shown by epidermal gene expression.
- Middle premolt promotes new exoskeleton formation and energy metabolism, with distinct gene activity in epidermis and hemocytes.
- 20E injection may cause dysbiosis in pathogenic bacteria like Escherichia-Shigella and Vibrio.

## Abstract

Molting is a crucial physiological process for crayfish and other crustaceans, requiring them to shed their hardened old exoskeleton for growth. However, the molting period is extremely risky, and many farmed crayfish fail to survive this stage, thus limiting crustacean production. This study investigated how crayfish prepare for molting by injecting them with different doses of 20-hydroxyecdysone, simulating the early and middle stages of the premolt process. This study found that in the early stages of premolt, the crayfish body primarily focuses on degradation and resorption of the old exoskeleton. As the molting process progresses into the middle premolt stages, the crayfish promotes the formation of a new exoskeleton and enhances energy metabolism and antioxidant defense capabilities. By identifying these specific biological changes, it provides guidance for improving the survival rate and growth rate of farmed crustaceans.

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.