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.

As a non-essential and toxic element, cadmium poses an important threat to aquatic organisms and human food safety. In this study, the effects of cadmium on antioxidant and non-specific immunity of Macrobrachium nipponense were studied from the physiological and biochemical indexes, histology and expression of related genes. These results showed that low concentrations (0.01, 0.02 mg/L) of cadmium have a positive effect on the non-specific immunity of M. nipponense, but high concentration (0.04 mg/L) of cadmium could inhibit or even damage the non-specific immunity of M. nipponense. The cadmium could induce oxidative stress in M. nipponense, and M. nipponense actived the antioxidant defense system to deal with oxidative stress, but high concentration (0.04 mg/L) of cadmium could inhibit the antioxidant defense system of M. nipponense, leading to oxidative damage, and may induce apoptosis in severe case. At the same time, the results of histology showed that cadmium can damage the structure of gill and hepatopancreas tissues of M. nipponense. This study provides theoretical data for evaluating the influences of heavy metal cadmium on M. nipponense and the toxic mechanism of heavy metal cadmium.

The copper, as heavy metal has important impacts on the antioxidant and immune defense systems in aquatic organisms, and the toxic effects of copper can be accumulated and magnified with the food chain, thus posing a threat to food safety as well as ecosystems. This study explored the response of the antioxidant system and non-specific immunity in M. nipponense to copper stress. Low concentration of copper (0.05, 0.1 mg L^-1) had positive effects on the non-specific immunity in M. nipponense, while the non-specific immunity in M. nipponense could be affect negatively or even be inhibited by high copper concentration (0.15 mg L^-1). Even low concentrations of copper could cause oxidative stress, and high copper concentration (0.15 mg L^-1) could induce oxidative damage and even apoptosis, and thus causing damage to the antioxidant defense system in M. nipponense. Low concentration of copper could affect the gill and hepatopancreas structure in M. nipponense, but high level oxidative stress caused by high copper concentration could cause oxidative damage to these tissue, resulting in the destruction of gill and hepatopancreas. This study provides the safety concentration for using copper-containing fish drugs in the actual culture of M. nipponense and provides basic data for the toxicity mechanism of copper to M. nipponense.

Based on Box–Behnken experimental design and response surface method, the joint effect of temperature (16–36°C), salinity (0–22 ppt) and rearing density (200–1000 fish.m⁻³) on the specific growth rate (SGR) and feed conversion rate (FCR) of Nile tilapia juveniles were studied under laboratory conditions. The entire experiment lasted for 1 month (30 days). Results showed that the linear and quadratic effects of temperature, salinity on both growth and feed utilization were highly statistically significant (P < 0.01). The linear and quadratic effect of rearing density on the growth was highly significant (P < 0.01); the linear effect of rearing density on feed utilization was significant (P < 0.05), but the quadratic effect nonsignificant (P > 0.05). Interactions between temperature and salinity, and between salinity and rearing density on the growth statistically differed from zero (P < 0.05). Interactions between temperature and salinity, between temperature and density on feed utilization was significant (P < 0.05). Model equations of the growth and feed utilization on temperature, salinity and density were established, with the coefficient of determination being 98.34% for growth and 98.11% for feed utilization, and could be applied to projection. The optimal temperature/salinity/density combination was obtained utilizing statistical optimization approach: 29°C/6 ppt/500 fish.m⁻³, at which the maximal specific growth and feed utilization reached 4.228%.d⁻¹ and 0.520 respectively, with the desirability being 0.989.

Fish sex-determining mechanisms can be classified as genotypic (GSD), temperature (TSD), or genotypic plus temperature effects (GSD+TE). Previous studies have shown that culturing water temperature during thermosensitive periods (TSP) could affect the expression of many genes in the gonad in some fish. However, few studies have focused on gene expression changes in the brain after temperature treatment during TSP in fish species. In this study, three families were developed by crossing XX neomales with XX females and one of them was used for transcriptome analysis. The results showed that a total of 105, 3164 and 4666 DEGs were respectively obtained in FC (female control) vs FT (high temperature treated females at TSP), FC vs MC (male control) and MC vs FT comparison groups. By profiling analysis, we identified that the mRNA expression levels of sixteen DEGs exhibited significant downregulation or upregulation after high temperature treatment and reached a similar level as that in MC. Among the 16 DEGs, LOC100699848 (lysine specific demethylase 6A) and Jarid2 contained JmjC domain, showing the possible important role of JmjC domain in response to temperature treatment in Nile tilapia. Kdm6b (lysine demethylase 6B) and Jarid2 have been shown to play important roles in reptile TSD, showing the relative conservation of regulation mechanisms underlying between TSD in reptile and TSD or GSD+TE in fish species. Finally, the transcriptome profiling was validated using quantitative real time PCR in 9 selected genes. These results will give a hint for investigating the GSD+TE molecular mechanism in fish species.