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.

Red swamp crayfish (Procambarus clarkii) is one of the most important aquaculture species in China. Frequent outbreaks of diseases seriously threatened the sustainable development of the industry. It is necessary to understand the causes of disease and study the mechanism of disease resistance in P. clarkii. In this paper, the pathogenic bacteria causing head ulcers in juvenile P. clarkii were found and identified as Citrobacter freundii, which can cause severe pathological changes in the hepatopancreas and intestines of juvenile P. clarkii. Detection of humoral immune factors revealed that PO activity and lysozyme activity of infected P. clarkii were significantly enhanced at 15 and 20 dpi, respectively. Transcriptome analysis was conducted of hepatopancreas from normal and diseased P. clarkii after C. freundii injection, as well as bacteria-free control of P. clarkii. It was found that DEGs are rich in NF-κB, oxidative phosphorylation, JAK/STAT, Leukocyte transendothelial migration, MAPK, and PPAR signaling pathway. These pathways are related to immune modulation, metabolism, and pathogen clearance. Meanwhile, immune-related genes such as Gip, nfyA, psmD13, and FGFR were significantly highly expressed in the normal group, which was verified by qRT-PCR results, suggesting that they may be the key regulatory genes for juvenile P. clarkii resistance to C. freundii. This study will help to elucidate the molecular mechanism of the immune response of P. clarkii to C. freundii. The results are instructive for the prevention and treatment of P. clarkii diseases and for further understanding of the invertebrate immune system.

Background/Objectives: Procambarus clarkii is an important freshwater aquaculture species in China which has the characteristics of rich nutrition and delicious taste. However, the expansion of aquaculture scale, germplasm degradation, and other problems that have become increasingly prominent seriously restrict the sustainable development of the crayfish industry. Genetic improvement is an urgent need for the crayfish aquaculture industry, and selective breeding is an important way to improve the crayfish varieties. Methods: We established full-sibling family populations of the red swamp crayfish and performed whole-genome resequencing of the F3 family-selected red swamp crayfish population and wild red swamp crayfish populations from four regions of Hunan Province (Nanx, Mil, Caish, and Wangc). Results: The results showed that there was a clear separation between the wild population and the family population, and the decline rate was slightly faster in the wild population than that of the family breeding population. There was local gene flow between family populations, as well as gene flow between Mil, Caish, and families. In addition, 52 SNP loci related to body weight traits were identified by genome-wide association analysis, and the candidate gene WIPF1 related to growth was screened out. Conclusions: We established a line selection population of red swamp crayfish and obtained more stable candidate lines. In addition, this study identified Wiskott–Aldrich syndrome protein-interacting protein family member 1 (WIPF1) as a candidate gene related to body weight for the first time. The results provide a theoretical basis for exploring the growth mechanism of P. clarkii and carrying out in-depth genetic improvement.

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.

Dynamic developmental states of follicles are regarded to be determinants of sexual maturation in fish ovaries. However, it is still a challenge to identify the critical points at which the developmental processes of different types of follicles interact and affect the ovarian development. In this study, four subtypes of the primary follicle (PF) in the ovarian folliculogenesis of zebrafish, i.e., the so-called PF-i, PF-ii, PF-iii, and PF-iv, are first identified by discontinuous NaCl-Percoll gradient centrifugation, as well as their respective morphological features. Then, for the four subtypes of PFs, stage-specific comparative analysis is employed to identify the differentially expressed genes and the differentially methylated regions, which have been validated to be significantly enriched in biological processes encompassing ribosomal biogenesis, meiotic progression, transcriptional regulation, and mitochondrial respiration. Results from transcriptional analysis further demonstrate significant changes in the expression profiles at different developmental stages from the PF-ii to the PF-iii. By molecular biology identification, it is shown that the enhancement of Notch and mTOR pathways can significantly regulate the ovarian development through the pacing effect of primary follicles. Clearly, all these uncovered results could provide a deeper understanding of the initial regulation of ovarian maturation, as well as a new multidisciplinary analytic tool to study follicle candidate regulators in the developmental process of other fish.