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.

Grass carp in aquaculture exhibited symptoms of bacterial infection leading to mortality. To investigate the cause of the disease and control grass carp infections, samples from diseased grass carp were collected, and a bacterial strain named XH-1 was isolated from the internal organs of the infected fish. Artificial infection experiments were conducted to determine whether the isolated strain XH-1 was the pathogenic bacterium. The biological characteristics of the isolated strain were studied through a 16S rRNA sequence analysis, physiological and biochemical identification, and phylogenetic tree construction. Extracts from 14 traditional Chinese herbs were tested to evaluate their bacteriostatic and bactericidal effects on the isolated strain. The regression infection experiment confirmed that the isolated strain XH-1 was the pathogenic bacterium causing the grass carp disease. Biological characterization studies identified the bacterium as Aeromonas veronii, which is clustered with A. veronii MW116767.1 on the phylogenetic tree. Among the 14 Chinese herbal extracts, Lignum sappa, Pericarpium granna, Artemisia argyi, Scutellaria baicalensis Georgi, Coptis chinensis, and Artemisiacapillaris thunb exhibited significant bacteriostatic effects on XH-1. Lignum sappa showed the highest sensitivity to A. veronii, with the largest inhibition zone diameter, and its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 7.813 mg/mL and 15.625 mg/mL, respectively. As the concentration of Lignum sappa extract increased, its bacteriostatic and bactericidal effects strengthened. When the concentration exceeded 14 mg/mL, it maintained strong bactericidal activity over 32 h. This study on A. veronii XH-1 provides theoretical insights for the prevention of grass carp aquaculture diseases and the use of traditional Chinese herbs for treatment.