Macrobrachium rosenbergii is an essential species for freshwater economic aquaculture in China, but in the larval process, their salinity requirement is high, which leads to salinity stress in the water. In order to elucidate the mechanisms regulating the response of M. rosenbergii to acute low-salinity exposure, we conducted a comprehensive study of the response of M. rosenbergii exposed to different salinities’ (0‰, 6‰, and 12‰) data for 120 h. The activities of catalase, superoxide dismutase, and glutathione peroxidase were found to be significantly inhibited in the hepatopancreas and muscle following low-salinity exposure, resulting in oxidative damage and immune deficits in M. rosenbergii. Differential gene enrichment in transcriptomics indicated that low-salinity stress induced metabolic differences and immune and inflammatory dysfunction in M. rosenbergii. The differential expressions of MIH, JHEH, and EcR genes indicated the inhibition of growth, development, and molting ability of M. rosenbergii. At the proteomic level, low salinity induced metabolic differences and affected biological and cellular regulation, as well as the immune response. Tyramine, trans-1,2-Cyclohexanediol, sorbitol, acetylcholine chloride, and chloroquine were screened by metabolomics as differential metabolic markers. In addition, combined multi-omics analysis revealed that metabolite chloroquine was highly correlated with low-salt stress.

Perfluorooctane sulfonate (PFOS) contamination poses a significant environmental threat due to its widespread distribution and persistence. However, the hepatotoxic effects of PFOS on key aquatic species, such as crucian carp, remain understudied. This study systematically investigated the hepatotoxicity and underlying molecular mechanisms associated with PFOS exposure in crucian carp over a 21 day period. We determined a 96 h 50% lethal concentration (LC₅₀) of 23.17 mg/L. Histopathological and transcriptomic analyses confirmed PFOS-induced liver damage in the carp, characterized by venous congestion, nucleolar dissolution and cellular vacuolation. Transcriptomic profiling further identified 1036 differentially expressed genes (DEGs), involving critical pathways related to lipid and energy metabolism, immunity, and endocrine regulation. These pathways are integral to the development of nonalcoholic fatty liver disease (NAFLD). Specifically, DEGs related to lipid metabolism showed significant changes, while those involved in energy metabolism indicated disrupted ATP production and mitochondrial function. Genes associated with immune response revealed an upregulation of pro-inflammatory markers, and hormone regulation genes highlighted alterations in endocrine signaling. Our findings emphasized that PFOS exhibits acute toxicity to crucian carp, potentially inducing hepatotoxicity by disrupting multiple physiological systems. This research provides a theoretical foundation for mitigating aquatic pollution and protecting eco-health, contributing to broader ecological and conservation biology discussions.

Functional gene expression is closely linked to an organism’s physiology and can be quantified using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). However, the stability of reference gene expression is not absolute, which may impact the accuracy of RT-qPCR results. In this study, we evaluated the suitability of nine genes including receptor for activated protein kinase c1 (rack1), ribosomal protein L6 (rpl6), ribosomal protein L9 (rpl9), ribosomal protein S2 (rps2), ribosomal protein S18 (rps18), ribosomal protein lateral stalk subunit P0 (rplp0), eukaryotic translation elongation factor 1β (eef1b), eukaryotic translation initiation factor 4a (eif4a), eukaryotic translation initiation factor 5a (eif5a) analyzed from RNA sequencing (RNA-Seq) data in addition to three genes including eukaryotic elongation factor 1α (eef1a), β-actin (actb), and glyceraldehyde 3-phosphate dehydrogenase (gapdh) selected from the literature to obtain the best internal controls in the RT-qPCR analysis of M. rosenbergii under overmating stress and natural aging. RefFinder was used to comprehensively evaluate the stability of the candidate reference genes. The initial results showed that three genes (eif5a, rps18, and rplp0) from the RNA-Seq data had relatively stable expression levels, which were more stable than those of the three commonly used reference genes. Eif5a and rps18 were the best combination for the RT-qPCR analysis of M. rosenbergii under overmating stress and aging. Further analysis indicated that eif5a might be the best reference gene for the study of M. rosenbergii.

During the breeding process of Macrobrachium rosenbergii, a male-to-female ratio of 1:3 or higher is typically adopted, so as a result, the quality of the male broodstock significantly influences the quality of the offspring. We observed that overused males exhibited notable changes in body color, particularly in the tail fan region, which turned orange or red due to the excessive accumulation of astaxanthin in the muscles and exoskeleton. Frequent mating also led to a significant decrease in male body weight, with histological analysis revealing disorganized muscle fiber patterns and increased tissue damage. To investigate the molecular mechanisms underlying these physiological changes, we performed transcriptomic and metabolomic analyses of muscle tissues. A total of 1069 differentially expressed genes (DEGs), 540 differentially expressed proteins (DEPs), and 385 differentially expressed metabolites (DEMs) were identified. Pathway analysis revealed that the DEGs were significantly enriched in pathways related to energy metabolism and degenerative diseases, while the DEMs were notably associated with cancer metabolism, signal transduction, substance transport, energy metabolism, nucleic acid metabolism, neurotransmission, immune response, and metabolic diseases. Proteome analysis showed that proteins and lipids were involved in muscle energy supply. These findings suggest that male M. rosenbergii upregulate energy metabolism in muscles to cope with frequent mating stress, but this adaptation leads to physiological damage. This study provides valuable insights for optimizing male broodstock selection and mating frequency in M. rosenbergii breeding practices.

Decapod Iridescent Virus 1 (DIV1) causes severe economic losses in Macrobrachium rosenbergii farming. Serum metabolic biomarkers are valuable tools for disease-resistant breeding, yet most existing studies employ mixed-sex samples, and the impact of sexual dimorphism on screening reliability remains poorly quantified. In this study, non-targeted serum metabolomics was performed on 28 healthy M. rosenbergii (14 females and 14 males), which were divided into marginally resistant and susceptible groups after DIV1 challenge. We compared results from mixed-sex and sex-specific screening strategies, and separately analyzed the overlap of upregulated and downregulated differential metabolites. Significant basal sexual dimorphism was observed in serum metabolism. Mixed-sex analysis identified 247 differential metabolites, while male-specific and female-specific analyses identified 121 and 161, respectively. Only 26 metabolites showed consistent directional changes across all three comparisons, accounting for 10.5% of total mixed-sex differential metabolites. Nearly 90% of mixed-sex results were either sex-specific signals or independent of sex. Analyses that do not account for sex introduce a large number of sex-unrelated differential markers and mask sex-specific metabolic features, and sex-specific screening is a necessary strategy to obtain reliable metabolic biomarkers for crustacean disease-resistant breeding.

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.