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.

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.

We previously reported on the comparison of proteomic data between seven tissue types of a novel “iron prawn” species. However, no transcripts or metabolic information are available for this species. We therefore performed shotgun LC–MS/MS metabonomic and RNA-seq analyses of the total protein from “iron prawns”. KEGG analysis revealed that the largest group consisted of a total of 114 KEGG pathway proteins, comparing the “iron prawns” with the normal prawns. A total of 423 peptides, corresponding to metabolic pathways, ABC transporters, starch and sucrose metabolism, insulin resistance/secretion, fatty digestion and absorption, and lipid metabolism, were identified. The pathways of carbohydrate and amino acid metabolism decreased in female iron prawns, while organic acid and its derivatives increased. However, the pathway of organic acid and its derivatives decreased and lipid metabolism increased in the male iron prawns. The pathways of choline metabolism in cancer and glycerophospholipid/histidine/propanoate metabolism have been significantly affected in iron prawns. Our work provides insight into the understanding of the formation mechanism of the “iron prawn”.

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.