The swimming crab Portunus trituberculatus is a commercially important crab species in East Asia countries. Gonadal development is a physiological process of great significance to the reproduction as well as commercial seed production for P. trituberculatus. However, little is currently known about the molecular mechanisms governing the developmental processes of gonads in this species. To open avenues of molecular research on P. trituberculatus gonadal development, Illumina paired-end sequencing technology was employed to develop deep-coverage transcriptome sequencing data for its gonads. Illumina sequencing generated 58,429,148 and 70,474,978 high-quality reads from the ovary and testis cDNA library, respectively. All these reads were assembled into 54,960 unigenes with an average sequence length of 879 bp, of which 12,340 unigenes (22.45% of the total) matched sequences in GenBank non-redundant database. Based on our transcriptome analysis as well as published literature, a number of candidate genes potentially involved in the regulation of gonadal development of P. trituberculatus were identified, such as FAOMeT, mPRγ, PGMRC1, PGDS, PGER4, 3β-HSD and 17β-HSDs. Differential expression analysis generated 5,919 differentially expressed genes between ovary and testis, among which many genes related to gametogenesis and several genes previously reported to be critical in differentiation and development of gonads were found, including Foxl2, Wnt4, Fst, Fem-1 and Sox9. Furthermore, 28,534 SSRs and 111,646 high-quality SNPs were identified in this transcriptome dataset. This work represents the first transcriptome analysis of P. trituberculatus gonads using the next generation sequencing technology and provides a valuable dataset for understanding molecular mechanisms controlling development of gonads and facilitating future investigation of reproductive biology in this species. The molecular markers obtained in this study will provide a fundamental basis for population genetics and functional genomics in P. trituberculatus and other closely related species.

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.

Ammonia is the most common contaminant in aquaculture systems. Due to the unexpected deterioration of natural seawater quality caused by harmful alga blooms in summer, the water exchange cycle for the culture ponds of the swimming crab Portunus trituberculatus, an important aquaculture species in China, is often much longer, and the crabs may be exposed to long-term ammonia stress. However, less information is available regarding the long-term effects of ammonia in marine decapod crustaceans. Therefore, it is of great significance to understand the toxic effects of prolonged ammonia in decapods. In this study, alteration of histology, cellular stress responses (CSRs), and apoptosis in the hepatopancreas of P. trituberculatus, an important aquaculture species, during ammonia stress (5, 15, and 45 mg⋅L^–1) for 1, 7, and 15 days and recovery at the normal condition for 7 days, were analyzed. Our results demonstrated a serious impact of long-term (15 days) ammonia stress by depressing crab CSR especially when ammonia concentration exceeds 15 mg⋅L^–1. Overall, short- (1 day) and medium-term (7 days) ammonia stress induced CSR, evidenced by upregulated expression of the genes involved in antioxidant defense (SOD, CAT, and GPX), apoptosis (p53, Bax, and Caspase-3), heat shock response (HSR) (Hsp70 and Hsp90), unfolded protein response (UPR) (IRE1, ATF6, and XBP1), and DNA damage response (DDR) (ATR and DNA-PKcs). However, long-term (15 days) ammonia stress, especially when exposed to the ammonia of 15 and 45 mg⋅L^–1, resulted in a higher level of apoptosis and severe damage of hepatopancreas, which may be related to the depressed CSR including antioxidant defense, HSR, UPR, and DDR. Notably, after recovery, the expressions of many genes involved in apoptosis, antioxidant response, HSR, UPR, and DDR in the groups exposed to ammonia at 15 and 45 mg⋅L^–1 were still significantly different to that of the control group. In summary, care should be taken when P. trituberculatus is exposed to ammonia over 15 mg⋅L^–1 especially when exposure duration is longer than 15 days, as the CSR could be compromised. This study provides a reference for a comprehensive understanding of CSR in decapod crustaceans under ammonia stress and will be beneficial for management in the intensive culture of the swimming crab.

Nitrite is a common pollutant encountered in aquaculture systems. During intensive hatchery, accumulation of nitrite can cause massive mortality of juvenile crustaceans. However, the nitrite toxicity and cellular stress responses in juvenile crustaceans is not clearly understood. Here, we investigate the survival, energy metabolism, and cellular stress responses in juvenile P. trituberculatus, an important aquaculture species in China, under acute nitrite stress. The results revealed nitrite resulted in a significant decrease in survival rate of juvenile swimming crab. After nitrite exposure, the activity of catabolic enzymes, such as HK, PK, CS, and CPT-1, were initially enhanced, and then they showed significant decrease at the late stage of exposure, accompanied by reduction in ATP and adenylate energy charge (AEC). The impaired energy homeostasis was possibly associated with disturbed AMPK signaling and enhanced anaerobic metabolism, which was indicated by the high levels of LDH activity and HIF-1α expression. Furthermore, we found that nitrite stress can depress antioxidant systems and unfold protein responses, causing oxidative damage and endoplasmic reticulum (ER) stress, and this, in turn, can trigger autophagy and apoptosis through both caspase-dependent and caspase-independent pathways. The results of the present study improve our understanding regarding adverse effects of nitrite on P. trituberculatus and provide valuable information for hatchery management.

Ammonia is a significant concern during hatchery culture in brachyuran species, and its accumulation may lead to abortive moulting and large-scale deaths of the early juveniles. To date, the underlying mechanism for ammonia-induced alteration of the moulting process is still unknown. In this study, we aimed to investigate the effects of ammonia on the moulting as well as the potential mechanisms in early juveniles of the swimming crab Portunus trituberculatus, an important aquaculture species in China. We evaluated the survival rate and moulting rate of the juvenile crabs (C2) and analyzed the expression pattern of the genes in key components of molt signaling during a complete moulting cycle under different concentrations of ammonia nitrogen (the control group: <0.1 mg/L; the LA group: 5 mg/L; and the HA group: 20 mg/L). The results showed that: (1) the survival rate in the LA and HA groups was lower than that in the control group at the end of the experiment, and moulting death syndrome (MDS) was only observed in the HA group; (2) the moulting rate was higher in the LA group and lower in the HA group compared to the control group; (3) consistent with the results of the moulting experiment, MIH showed decreased expression, and genes related to ecdysteroid synthesis, ecdysteroid receptors, and responsive effectors exhibited increased expression in the LA group compared to the control group; and (4) although MIH expression was upregulated, increased expression of the genes associated with ecdysteroid synthesis, ecdysteroid receptors and downstream effectors still observed in the HA group. Our results indicated that low levels of ammonia can promote moulting in juvenile swimming crabs by inhibiting the expression of MIH and activating moult signaling, whereas high levels of ammonia inhibit moulting and lead to MDS through impairing moult signaling.

Methyl farnesoate (MF), a crucial sesquiterpenoid hormone, plays a pivotal role in the reproduction of female crustaceans, particularly in the vitellogenesis process. Despite extensive research on its functions, the molecular mechanisms that regulate MF levels during the vitellogenic phase remain largely elusive. This study investigates the roles of microRNAs (miRNAs), significant post-transcriptional regulators of gene expression, in controlling MF levels in the swimming crab Portunus trituberculatus. Through bioinformatic analysis, four miRNAs were identified as potential regulators targeting two genes encoding Carboxylesterases (CXEs), which are key enzymes in MF degradation. Dual luciferase reporter assays revealed that let-7b and miR-141 suppress CXE1 and CXE2 expression by directly binding to their 3′ UTRs. In vivo overexpression of let-7b and miR-141 significantly diminished CXE1 and CXE2 levels, consequently elevating hemolymph MF and enhancing vitellogenin expression. Spatiotemporal expression profile analysis showed that these two miRNAs and their targets exhibited generally opposite patterns during ovarian development. These findings demonstrate that let-7b and miR-141 collaboratively modulate MF levels by targeting CXEs, thus influencing vitellogenesis in P. trituberculatus. Additionally, we found that the expression of let-7b and miR-141 were suppressed by MF, constituting a regulatory loop for the regulation of MF levels. The findings contribute novel insights into miRNA-mediated ovarian development regulation in crustaceans and offer valuable information for developing innovative reproduction manipulation techniques for P. trituberculatus.