Influences of Diurnal Rhythms on Gut Microbiota and Clock Gene Expression in Procambarus clarkii

This study investigated the diurnal dynamics of the gut microbiota and core clock gene expression in the red swamp crayfish, Procambarus clarkii. Samples were collected at four time points (06:00, 12:00, 18:00, and 24:00) over a 24 h period. Gut microbiota characteristics were analyzed using 16S rRNA gene sequencing, while quantitative real-time polymerase chain reaction (qPCR) was used to examine the expression patterns of core clock genes, including Cycle (Cyc), Clock (Clk), and cryptochrome type 1 (Cry1), in the hepatopancreas and eyestalk. The results showed that Kruskal-Wallis tests for α-diversity indices (Shannon, Simpson, ACE, Chao1) and PERMANOVA for β-diversity (Bray-Curtis) of the gut microbiota revealed no significant diurnal variation across the four time points (all p > 0.05). Firmicutes, Proteobacteria, and Bacteroidota were the dominant phyla, with norank_o_RsaHf231, ZOR0006, and Bacteroides as the predominant genera. Although the overall microbial structure remained stable, several taxa, including unclassified_c_Bacilli, unclassified_f_Caulobacteraceae, Gemmobacter, unclassified_f_Rhodobacteraceae, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Lactobacillus, and unclassified_f_Vibrionaceae exhibited time-dependent fluctuations. In addition, the relative mRNA expression levels of Cyc, Clk, and Cry1 in the hepatopancreas and eyestalk showed significant diurnal variation. This study reveals the diurnal dynamic characteristics of the gut microbiota and core clock gene expression in P. clarkii, providing a foundation for further investigation of diurnal regulatory mechanisms and physiological adaptations.