Real-time quantitative PCR (qPCR) is a key technique for elucidating the mechanisms underlying environmental stress responses in aquatic animals, and its accuracy relies heavily on stable reference genes for data normalization. In this study, histopathological examination revealed alterations in the gill, hepatopancreas, and muscle tissues of Procambarus clarkii following NH₃ stress. Based on transcriptomic screening and reference genes reported in the literature, 13 candidate reference genes were selected. Their expression levels in 8 different tissues before and after NH₃ stress were detected using qPCR, and their stability was systematically evaluated using multiple algorithms, including geNorm, NormFinder, BestKeeper, Delta CT and RefFinder. The results identified 60S ribosomal protein L13a (RPL13a), ribosomal protein S18 (RPS18) and eukaryotic translation initiation factor 4A (EIF4A) as the three most stable candidates under NH₃ stress, whereas the traditionally used reference genes beta-actin (β-actin) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed relatively low stability under these conditions. geNorm pairwise variation analysis indicated that the inclusion of a third reference gene could further improve normalization. Validation using four NH₃-responsive genes showed that (RPL13a + RPS18) and (RPL13a + RPS18 + EIF4A) yielded similar log₂ fold-change estimates, supporting their use as normalization options under the tested conditions. It is worth noting that RPL13a remains highly stable across four conditions: different embryonic developmental stages, different ovarian developmental stages, tissues from both males and females, and following infection with white spot syndrome virus (WSSV). These findings contribute to the selecting and validating reference genes for qPCR analyses in Procambarus clarkii under the specific experimental conditions evaluated in this study.

A 10-week growth experiment was conducted to evaluate the physiological effects of dietary phosphorus supplementation on red swamp crayfish (Procambarus clarkii) feeding diets with high Clostridium autoethanogenum protein (CAP) levels. Six isonitrogenous and isolipid diets were formulated: The FM diet contained 10% fishmeal, which is equivalent to a dietary phosphorus level of 1.41%, and the CAP, CAPSP1, CAPSP2, and CAPSP3 diets substituted all fishmeal with CAP and supplemented with 0, 2.5%, 3%, and 3.5% Ca(H₂PO₄)₂, respectively (corresponding to dietary phosphorus levels of 0.66%, 1.27%, 1.40%, and 1.52%). A total of 600 crayfish with an initial mean weight of (5.01 ± 0.02) g were selected and randomly assigned to 15 cages for feeding and sampled at the end of the experiment. Results indicate that high-dose CAP replacing fishmeal caused abnormal hepatopancreatic tissue structure in crayfish, exacerbating lipid deposition and oxidative stress. Compared with the CAP group, the specific growth rate (SGR) of crayfish in the CAPSP2 and CAPSP3 groups significantly increased (p < 0.05). The activities of antioxidant enzymes and lipid-degrading enzymes in the hepatopancreas, along with the relative expression of related genes, were significantly enhanced (p < 0.05). Metabolomic analysis demonstrated significant differences in major differential metabolites and metabolic pathways between the CAP group crayfish and the CAPSP2 group (p < 0.05). CAPSP2 group crayfish exhibited a higher content of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC), with significant enrichment in glycerophospholipid metabolism and fatty acid metabolism pathways (p < 0.05). Overall, supplementing dietary phosphorus levels to 1.40–1.52% effectively mitigated growth retardation, oxidative damage, and lipid metabolism disorders induced by high-proportion CAP replacement of fishmeal.