Hucheng Jiang

To examine the gut microbial composition and functional attributes of red swamp crayfish ( Procambarus clarkii ) across different rearing environments, this experiment employed high‐throughput sequencing of the 16S rRNA gene of bacterial DNA isolated from the gut. Using an intensive monoculture system in ponds (NP) as the control, this research analyzed and compared the gut microbial communities of crayfish reared in integrated frameworks, including lotus and crayfish co‐culture (XH), rice and crayfish co‐culture (YZ), as well as polyculture systems involving crayfish and crabs (NG). The findings indicated that when examining phylum‐level taxonomy, the bacterial community structures in the gut remained relatively similar across the various cultivation modes; the most abundant phyla identified were Bacteroidetes, Proteobacteria, and Firmicutes. In terms of genus‐level classification, Bacteroides and Citrobacter were identified as the dominant taxa. Correspondingly, when analyzed at the species level, Bacteroides neonati and Citrobacter freundii emerged as the most prevalent species. Significant differences in microbial diversity were observed among the farming systems. Compared with the intensive monoculture group, the lotus‐crayfish and rice‐crayfish systems showed higher gut microbial diversity, whereas the crayfish‐crab polyculture system exhibited lower diversity. Predictive functional analysis indicated that the dominant metabolic functions across all four systems were chemoheterotrophy, fermentation, and aerobic chemoheterotrophy. The genus‐level interaction network showed that the microbial network in the NG group had a relatively simple structure, while those in the XH and YZ groups displayed a more complex microbial structure. In summary, the gut microbial community of red swamp crayfish features a core profile primarily composed of Bacteroidetes, Proteobacteria, and Firmicutes, while its taxonomic diversity exhibits considerable variation across different rearing systems. These results deepen the comprehension of microbial ecology within the digestive tract of this species and offer important implications for the improvement of sustainable aquaculture strategies.

QRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all experimental conditions, and research on suitable reference genes in red swamp crawfish (Procambarus clarkii) is particularly scarce. In this study, eight commonly used crustacean reference genes were chosen from P. clarkii transcriptome data and investigated as potential candidates for normalization of qRT-PCR data. Expression of these genes under different experimental conditions was examined by qRT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, geNorm, NormFinder and BestKeeper. A final comprehensive ranking determined that EIF and 18S were the optimal reference genes for expression data from different tissues, while TBP and EIF were optimal for expression data from different ovarian developmental stages. To our knowledge, this is the first systematic analysis of reference genes for normalization of qRT-PCR data in P. clarkii. These results will facilitate more accurate and reliable expression studies of this and other crustacean species.