Key Differences in the Gut Microbiota of Red-Claw Crayfish with Different Sizes and Genders Under Consistent Farming Conditions.

The red-claw crayfish has been widely introduced and cultured in China and has become a crucial economic freshwater species. However, individuals reared from the same batch of seedlings in uniform aquaculture systems exhibit significant size variation within and between genders, which notably impedes the optimization of both their quality and yield. Gut microbiota plays an important role in the metabolism, development, and immunity of aquatic animals. However, the knowledge on the intestinal microbiota of red-claw crayfish with various sizes and genders is poor. In this study, the intestinal microbiota of red-claw crayfish cultured in consistent farming conditions were separated to larger-sized female (GUBF), larger-sized male (GUBM), smaller-sized female (GUSF), and smaller-sized male (GUSM) groups based on their body size (weight) and gender, before being analyzed via high-throughput 16S rRNA gene sequencing. The intestinal microbiota results showed that alpha diversity tended to generally decrease in the order of GUBF, GUBM, GUSF, and GUSM, indicating that the richness and evenness of the gut flora were gradually improved with the increase in body weight or from male to female. Community richness and diversity were highest in the GUBF group, followed by the GUBM, GUSF, and GUSM groups, respectively. Beta diversity indicated significant differences in gut microbiota between the GUBF and GUSF, GUBM and GUSM, GUBF, and GUBM groups. Further analysis showed that the dominant phyla in the intestine of the red-claw crayfish were Firmicutes, Proteobacteria, Fusobacteriota, Bacteroidota, and Deinococcota, and the dominant genera were Vibrio, Tyzzerella, Candidatus Bacilloplasma, Citrobacter, and Candidatus Hepatoplasma. Moreover, nine phyla and 106 genera were identified to be significantly different in abundance among all four groups. Pairwise comparisons revealed that the phylum Dependentiae and Planctomycetota and genus Babeliaceae_unclassified were significantly abundant in the gut of female crayfishes, regardless of body size. On the other hand, irrespective of genders, the abundance of Novosphingobium, Piscinibacter, and Citrobacter was significantly increased or declined in the larger or smaller crayfishes, respectively. PICRUSt2 analysis based on the KEGG database suggested that the pathway bacterial secretion system, isoflavonoid biosynthesis, and pathway glycerolipid metabolism were significantly up- and down-regulated in female individuals, respectively, regardless of body sizes. Meanwhile, the adipocytokine signaling pathway, pyruvate metabolism, and pathway electron transfer carriers were significantly up- and down-regulated in larger individuals, respectively, regardless of gender. Gender differences may induce gut microbiota to exert a greater impact on hormonal regulation, whereas differences in individual size seem to lead gut microbiota to develop a preference for food intake and energy sources. In summary, this study revealed key differences in the intestinal microbiota of the crayfish with different sizes and genders, even in those which were cultured in the same environment and period, which potentially suggest that the intestinal microbiota may be influenced by some other factors in the culture system, such as hormone secretion, metabolism, and immunity. This study will contribute to improving growth performance and animal quality in the aquaculture of .