The Pacific white shrimp (Litopenaeus vannamei) is the world’s most prevalent cultured crustacean species. However, the supply of high-quality broodstocks is limited and baseline information related to its reproductive activity and molecular issues related to gonad development are scarce. In this study, we performed transcriptome sequencing on the gonads of adult male and female L. vannamei to identify sex-related genes. A total of 25.16 gigabases (Gb) of sequences were generated from four L. vannamei gonadal tissue libraries. After quality control, 24.11 Gb of clean reads were selected from the gonadal libraries. De-novo assembly of all the clean reads generated a total of 65,218 unigenes with a mean size of 1021 bp and a N50 of 2000 bp. A search of all-unigene against Nr, SwissProt, KEGG, COG and NT databases resulted in 26,482, 23,062, 20,659, 11,935 and 14,626 annotations, respectively, providing a total of 30,304 annotated unigenes. Among annotated unigenes, 12,320 unigenes were assigned to gene ontology categories and 20,659 unigenes were mapped to 258 KEGG pathways. By comparing the ovary and testis libraries, 19,279 testicular up-regulated and 3,529 ovarian up-regulated unigenes were identified. Enrichment analysis of differentially expressed unigenes resulted in 1060 significantly enriched GO terms and 34 significantly enriched KEGG pathways. Nine ovary-specific, 6 testis-specific, 45 testicular up-regulated and 39 ovarian up-regulated unigenes were then confirmed by semi-quantitative PCR and quantitative real-time PCR. In addition, using all-unigenes as a reference, a total of 13,233 simple sequence repeats (SSRs) were identified in 10,411 unigene sequences. The present study depicts the first large-scale RNA sequencing of shrimp gonads. We have identified many important sex-related functional genes, GO terms and pathways, all of which will facilitate future research into the reproductive biology of shrimp. We expect that the SSRs detected in this study can then be used as genetic markers for germplasm evaluation of breeding and imported populations.

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.