Discovery of Sex-Determining Genes in Portunus trituberculatus: A Comparison of Male and Female Transcriptomes During Early Developmental Stages

Portunus trituberculatus is one of the main mariculture crabs of high economic value. To identify genes involved in sex determination, we first performed sex-specific transcriptome sequencing at six larval development stages using a DNA/RNA co-extraction method. A total of 907,952,938 and 828,774,880 reads were obtained from female and male crabs, respectively. 2,379 differentially expressed genes (DEGs) were found between females and males, and were mainly enriched in DNA replication, folate biosynthesis, and retinol metabolism pathways. Furthermore, transcription patterns of genes in the sex-determining region (SD) were analyzed based on the transcriptome data, and one Dmrt gene (PtDMY) was found to be exclusively expressed in males during early developmental stages. Notably, some known sex-related genes, including IAG, Dmrt11E, DmrtB1, and DmrtC2 were significantly down-regulated after knocking down PtDMY. Our results suggested that PtDMY is involved in sex determination and may be one of the key upstream regulators of the sex determination pathway. In addition, the massive volume of transcriptome data obtained in this study provided an important basis for the systematic study of sex determination mechanisms in P. trituberculatus.

Molecular Cloning of Dynein Heavy Chain and the Effect of Dynein Inhibition on the Testicular Function of Portunus trituberculatus

Dynein is a motor protein with multiple transport functions. However, dynein’s role in crustacean testis is still unknown. We cloned the full-length cDNA of cytoplasmic dynein heavy chain (Pt-dhc) gene and its structure was analyzed. Its expression level was highest in testis. We injected the dynein inhibitor sodium orthovanadate (SOV) into the crab. The distribution of Portunus trituberculatus dynein heavy chain (Pt-DHC) in mature sperm was detected by immunofluorescence. The apoptosis of spermatids was detected using a TUNEL kit; gene expression in testis was detected by fluorescence quantitative PCR (qPCR). The expression of immune-related factors in the testis were detected by an enzyme activity kit. The results showed that the distribution of Pt-DHC was abnormal after SOV injection, indicating that the function of dynein was successfully inhibited. Apoptosis-related genes p53 and caspase-3, and antioxidant stress genes HSP70 and NOS were significantly decreased, and anti-apoptosis gene bcl-2 was significantly increased. The activities of superoxide dismutase (SOD) and alkaline phosphatase (AKP) were significantly decreased. The results showed that there was no apoptosis in testicular cells after dynein function was inhibited, but the cell function was disordered. This study laid a theoretical foundation for the further study of apoptosis in testis and the function of dynein in testis and breeding of P. trituberculatus.

Physiological and Molecular Responses in the Gill of the Swimming Crab Portunus trituberculatus During Long-Term Ammonia Stress

Ammonia is a common environmental stressor encountered during aquaculture, and is a significant concern due to its adverse biological effects on vertebrate and invertebrate including crustaceans. However, little information is available on physiological and molecular responses in crustaceans under long-term ammonia exposure, which often occurs in aquaculture practices. Here, we investigated temporal physiological and molecular responses in the gills, the main ammonia excretion organ, of the swimming crab Portunus trituberculatus following long-term (4 weeks) exposure to three different ammonia nitrogen concentrations (2, 4, and 8 mg l–1), in comparison to seawater (ammonia nitrogen below 0.03 mg l–1). The results revealed that after ammonia stress, the ammonia excretion and detoxification pathways were initially up-regulated. These processes appear compromised as the exposure duration extended, leading to accumulation of hemolymph ammonia, which coincided with the reduction of adenosine 5′-triphosphate (ATP) and adenylate energy charge (AEC). Considering that ammonia excretion and detoxification are highly energy-consuming, the depression of these pathways are, at least partly, associated with disruption of energy homeostasis in gills after prolonged ammonia exposure. Furthermore, our results indicated that long-term ammonia exposure can impair the antioxidant defense and result in increased lipid peroxidation, as well as induce endoplasmic reticulum stress, which in turn lead to apoptosis through p53-bax pathway in gills of the swimming crab. The findings of the present study further our understanding of adverse effects and underlying mechanisms of long-term ammonia in decapods, and provide valuable information for aquaculture management of P. trituberculatus.