Tea tree oil (TTO) is an essential plant oil with diverse antibacterial and antioxidant properties; however, whether the role played by TTO in low fish meal (LF) diets induced the observed effects in the farmed crustaceans remains unclear. Therefore, this study used Macrobrachium rosenbergii as the model crustacean, and an 8-week feeding experiment with NF (normal fish meal), LF (soybean meal replacing 40% fish meal), and LFT (LF with 200 mg/kg TTO) diets was conducted to evaluate the positive effects of TTO under the LF diet. Compared to the NF diet, the LF diet reduced hemolymph antioxidant capacity and non-specific immunity, and induced hepatopancreas apoptosis and damage. However, in comparison with LF, LTF significantly ameliorated morphological impairment in the hepatopancreas, improved hepatopancreas energy metabolism by upregulating the Bcl-2/Bax and Akt/mTOR pathways, and enhanced antioxidant and non-specific immune capacity by activating the NF-κB/NO pathway. In addition, LFT repaired intestinal barrier injury and the imbalance of intestinal microbiota induced by the LF diet. Moreover, the Pearson correlation revealed the variations of the above indicators, which were related to the abundance changes of Klebsiella, Clostridium sensu stricto 12, Thermobifida, Bifidobacterium, and Alistipes, indicating that these microbes might serve as prospective targets for the intestine–hepatopancreas axis to affect hepatopancreas apoptosis, metabolism, and non-specific immunity. In summary, 200 mg/kg TTO supplementation mediated gut microbiota and positively improved energy metabolism and non-specific immunity, thereby alleviating hepatopancreas dysplasia and damage induced by the LF diet in M. rosenbergii.

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.

Macrobrachium rosenbergii is an economically important freshwater prawn species in China. The X-organ–sinus gland complex in crustacean eyestalks regulates reproduction through neurohormones, with ablation significantly promoting ovarian maturation. However, the miRNA mediated molecular mechanisms underlying this eyestalk ovary regulatory axis remain poorly characterized in M. rosenbergii. RNA sequencing of 9 ovarian samples produced 207 million paired-end reads (62.25 Gb total), averaging > 6 Gb per sample with Q30 scores exceeding 94.42%. The reads showed 84.32% alignment efficiency to the reference transcriptome. Transcriptomic analysis identified 769 differentially expressed genes (DEGs) and 176 differentially expressed miRNAs (DEMs), which were significantly enriched in reproduction related pathways including steroid biosynthesis and Wnt signaling.​ Integration analysis revealed 11 functionally important miRNA-mRNA regulatory pairs targeting key ovarian development genes. qRT-PCR validation confirmed the high reliability of sequencing data. Transcriptomic analysis of ovarian mRNA and miRNA in eyestalk-ablated M. rosenbergii revealed the molecular mechanisms of eyestalk mediated ovarian regulation.​​ Eyestalk ablation significantly upregulated vitellogenin genes and ecdysteroid biosynthesis genes, ​​demonstrating its dual regulatory role in reproduction and molting.​​ The findings indicate potential roles of the eyestalk in reproductive regulation via specific pathways, with the identified DEGs and DEMs serving as a valuable resource for future investigations.

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.