Phascolosoma esculenta is a unique aquatic invertebrate native to China, whose habitat is highly susceptible to environmental pollution, making it an ideal model for studying aquatic toxicology. Mitochondrial thioredoxin (Trx2), a key component of the Trx system, plays an essential role in scavenging reactive oxygen species (ROS), regulating mitochondrial membrane potential, and preventing ROS-induced oxidative stress and apoptosis. This study investigated the toxicity of cadmium (Cd) on P. esculenta and the role of P. esculenta Trx2 (PeTrx2) in Cd detoxification. The results showed that Cd stress altered the activities of T-SOD and CAT, as well as the contents of GSH and MDA in the intestine. After 96 h of exposure, histological damages such as vacuolization, cell necrosis, and mitophagy were observed. Suggesting that Cd stress caused oxidative damage in P. esculenta. Furthermore, with the prolongation of stress time, the expression level of intestinal PeTrx2 mRNA initially increased and then decreased. The recombinant PeTrx2 (rPeTrx2) protein displayed dose-dependent redox activity and antioxidant capacity and enhanced Cd tolerance of Escherichia coli. After RNA interference (RNAi) with PeTrx2, significant changes in the expression of apoptosis-related genes (Caspase-3, Bax, Bcl-2, and Bcl-XL) were observed. Proving that PeTrx2 rapidly responded to Cd stress and played a vital role in mitigating Cd-induced oxidative stress and apoptosis. Our study demonstrated that PeTrx2 is a key factor for P. esculenta to endure the toxicity of Cd, providing foundational data for further exploration of the molecular mechanisms underlying heavy metal resistance in P. esculenta.

Metallothioneins (MTs) are non-enzymatic metal-binding proteins widely found in animals, plants, and microorganisms and are regulated by metal-responsive transcription factor 1 (MTF1). MT and MTF1 play crucial roles in detoxification, antioxidation, and anti-apoptosis. Therefore, they are key factors allowing organisms to endure the toxicity of heavy metal pollution. Phascolosoma esculenta is a marine invertebrate that inhabits intertidal zones and has a high tolerance to heavy metal stress. In this study, we cloned and identified MT and MTF1 genes from P. esculenta (designated as PeMT and PeMTF1). PeMT and PeMTF1 were widely expressed in all tissues and highly expressed in the intestine. When exposed to 16.8, 33.6, and 84 mg/L of zinc ions, the expression levels of PeMT and PeMTF1 in the intestine increased first and then decreased, peaking at 12 and 6 h, respectively, indicating that both PeMT and PeMTF1 rapidly responded to Zn stress. The recombinant pGEX-6p-1-MT protein enhanced the Zn tolerance of Escherichia coli and showed a dose-dependent ABTS free radical scavenging ability. After RNA interference (RNAi) with PeMT and 24 h of Zn stress, the oxidative stress indices (MDA content, SOD activity, and GSH content) and the apoptosis indices (Caspase 3, Caspase 8, and Caspase 9 activities) were significantly increased, implying that PeMT plays an important role in Zn detoxification, antioxidation, and anti-apoptosis. Moreover, the expression level of PeMT in the intestine was significantly decreased after RNAi with PeMTF1 and 24 h of Zn stress, which preliminarily proved that PeMTF1 has a regulatory effect on PeMT. Our data suggest that PeMT and PeMTF1 play important roles in the resistance of P. esculenta to Zn stress and are the key factors allowing P. esculenta to endure the toxicity of Zn.

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.

The spermatogenesis of crustaceans includes nuclear deformation and acrosome formation. The mechanism of acrosome formation is one focus of reproductive biology. In this study, Macrobrachium rosenbergii was selected as the research object to explore the mechanism of acrosome formation. The acrosome contains a large number of acrosomal enzymes for the hydrolysis of the egg envelope. How these acrosomal enzymes are transported to the acrosomal site after synthesis is the key scientific question of this study. The acroframosome (AFS) structure of caridean sperm has been reported. We hypothesized that acrosomal enzymes may be transported along the AFS framework to the acrosome by motor proteins. To study this hypothesis, we obtained the full-length cDNA sequences of Mr-kifc1 and Mr-Acrosin from the testis of M. rosenbergii. The Mr-kifc1 and Mr-Acrosin mRNA expression levels were highest in testis. We detected the distribution of Mr-KIFC1 and its colocalization with Mr-Acrosin during spermatogenesis by immunofluorescence. The colocalization of Mr-KIFC1 and microtubule indicated that Mr-KIFC1 may participate in sperm acrosome formation and nucleus maturation. The colocalization of Mr-KIFC1 and Mr-Acrosin indicated that Mr-KIFC1 may be involved in Acrosin transport during spermiogenesis of M. rosenbergii. These results suggest that Mr-KIFC1 may be involved in acrosomal enzymes transport during spermiogenesis of M. rosenbergii.

Portunus trituberculatus is a very important marine economic species, and its aquaculture industry has been developing rapidly. However, the phenomenon of marine wild capture of P. trituberculatus and germplasm degradation has become increasingly serious. It is necessary to develop the artificial farming industry and carry out germplasm resource protection, for which sperm cryopreservation technology is an effective method. This research compared three methods (mesh-rubbing, trypsin digestion, and mechanical grinding) for acquiring free sperm, and the best method was mesh-rubbing. Then, the optimal cryopreservation conditions were selected, and the optimal formulation was sterile calcium-free artificial seawater, the optimal cryoprotectant was 20% glycerol, and the best equilibrium time was 15 min at 4 °C. The optimal cooling program was suspending the straws at 3.5 cm on the liquid nitrogen surface for 5 min and then storing them in liquid nitrogen. Finally, the sperm were thawed at 42 °C. However, the expression of sperm-related genes and the total enzymatic activities of frozen sperm were significantly decreased (p < 0.05), which showed that sperm cryopreservation damaged the sperm. Our study improves the sperm cryopreservation technology and the yield of aquaculture in P. trituberculatus. Additionally, the study provides a certain technical basis for the establishment of a sperm cryopreservation library of crustaceans.