DNA methylation is an important epigenetic modification that has been shown to be associated with responses to non-biological stressors. However, there is currently no research on DNA methylation in response to environmental signals in shrimp. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles and differentially expressed genes between two strains of Litopenaeus vannamei with significantly different cold tolerance through whole genome bisulfite sequencing (WGBS) and transcriptome sequencing. Between Lv-C and Lv-T (constant temperature of 28 °C and low temperatures of 18 °C and 10 °C) under cytosine-guanine (CG) environments, 39,100 differentially methylated regions (DMRs) were identified, corresponding to 9302 DMR-related genes (DMRGs). The DMRs were mainly located in the gene body (exons and introns). Gene Ontology (GO) analysis showed that these DMRGs were significantly enriched in cell parts, catalytic activity, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed significant enrichment of these DMRGs in pathways such as proteasome (ko03050), oxidative phosphorylation (ko00190), mTOR signaling pathway (ko04150), fatty acid metabolism (ko01212), and fatty acid degradation (ko00071). The comprehensive results suggested that L. vannamei mainly regulates gene expression in response to low temperatures through hypermethylation or demethylation of some genes involved in thermogenesis, glycolysis, the autophagy pathway, the peroxisome, and drug metabolism pathways. These results provide important clues for studying DNA methylation patterns and identifying cold tolerance genes in shrimp.

Procambarus clarkii is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 (Foxl2) gene has been found to be involved in the reproductive development of many crustaceans. To understand the role of the Foxl2 gene in the gonad development of P. clarkii, we designed CDS-specific primers for the P. clarkii Foxl2 (PcFoxl2) gene and cloned its CDS sequence using RT-PCR. The nucleotide and protein sequence information was then analyzed through bioinformatics analysis. The expression and subcellular localization of PcFoxl2 in various tissues were detected using qRT-PCR and in situ hybridization. The effects of PcFoxl2 knockdown on gonad development were investigated using RNA interference. The results showed that the CDS length of the PcFoxl2 gene was 1614 bp and encoded 537 amino acids. Protein sequence comparison and phylogenetic analysis showed that PcFoxl2 was the closest relative to Crayfish. qRT-PCR analysis indicated that the expression level of PcFoxl2 in the testis was significantly higher (>40 fold) than that in the ovary (p < 0.01). The in situ hybridization results showed that PcFoxl2 was expressed in both the cytoplasm and the nucleus of egg cells, and that the expression was strongest in egg cells at the early stage of yolk synthesis, while weak in the secondary oocytes. The positive signal was strongest in the spermatocyte nucleolus, while only a trace signal was observed in the cytoplasm. After interfering with the PcFoxl2 gene using dsRNA, the expression of PcFoxl2 in the RNA interference group was significantly lower than that in the control group, and this interference effect lasted for one week. Moreover, the gonad index of the experimental group was significantly lower than that of the control group (p < 0.05) after 10 days of P. clarkii cultivation following PcFoxl2 knockdown. The expression levels of the nanos and S3a genes, which are related to gonad development, decreased significantly after PcFoxl2 gene interference. The results suggest that the Foxl2 gene is involved in the growth and development of gonads, particularly in the development of testis, and is related to the early development of oocytes. This study provides a theoretical basis for the artificial breeding of P. clarkii.

Nitrite is a significant toxic substance that causes acute toxicity in aquatic animals. However, the molecular mechanisms underlying nitrite tolerance remain largely unidentified. Here, we investigated the physiological and transcriptomic profiles of nitrite-sensitive and nitrite-tolerant families of the white shrimp Penaeus vannamei under nitrite stress. Exposure to a lethal concentration of nitrite for 72 h caused higher mortality, higher nitrite accumulation, and more severe physiological damage in both the hemolymph and hepatopancreas of nitrite-sensitive families than in nitrite-tolerant families. This damage manifested as hepatic tubular deformation, hepatocyte necrosis, decreased hemocyte counts, lowered phagocytic activity in hemocytes, elevated production of reactive oxygen species (ROS), and decreased AKP enzyme activity. Furthermore, transcriptomic analysis revealed an upregulation in protein processing in the endoplasmic reticulum (ER) in the nitrite-tolerant families, triggering apoptosis- and autophagy-related pathways in the hemocytes and hepatopancreas upon nitrite exposure. Additionally, TUNEL staining and transmission electron microscopy (TEM) experiments revealed that the tolerant families may mitigate nitrite toxicity by modulating autophagy and apoptosis. Collectively, our results provide a valuable foundation for exploring the molecular mechanisms underlying nitrite tolerance in shrimp, which could facilitate the targeted breeding of shrimp germplasm with enhanced nitrite tolerance in aquaculture.

Background: As a globally significant aquaculture species, elucidating the molecular mechanisms underlying the regulation of the Pacific White Shrimp (Litopenaeus vannamei) growth holds substantial scientific and industrial value. This study systematically investigates the role of the LvChia2 gene in governing growth and development through a cross-tissue metabolic network approach. Methods: RNA knockdown (RNAi)-mediated knockdown of LvChia2 significantly impaired growth performance and triggered a tissue-specific metabolic compensation mechanism. Results: This mechanism was characterized by reduced crude lipid content in muscle and adaptive modulation of lipase (LPS) activities in hepatopancreatic and intestinal tissues, suggesting inter-tissue metabolic coordination. Transcriptomic profiling identified 610 differentially expressed genes (DEGs), forming a three-dimensional regulatory network encompassing “energy metabolism, molt regulation, and nutrient utilization.” Key mechanistic insights revealed the following: (1) Enhanced mitochondrial energy transduction through the upregulation of ATP synthase subunits and NADH dehydrogenase (ND-SGDH). (2) The disruption of ecdysteroid signaling pathways via suppression of Krueppel homolog 1 (Kr-h1). (3) The coordinated regulation of nitrogen metabolism through the downregulation of glutamine synthetase and secretory phospholipase A2. These molecular adaptations, coupled with tissue-specific oxidative stress responses, reflect an integrated physiological strategy for environmental adaptation. Conclusions: Notably, this study provides the first evidence in crustaceans of chitinase-mediated growth regulation through cross-tissue metabolic interactions and identifies six core functional genes (ATP5L, ATP5G, ND-SGDH, Kr-h1, GS, sPLA2) as potential targets for molecular breeding. A novel “gut-hepatopancreas axis” energy compensation mechanism is proposed, offering insights into resource allocation during metabolic stress. These findings advance our understanding of crustacean growth regulation and establish a theoretical foundation for precision aquaculture strategies, including genome editing and multi-trait genomic selection.