Temporal and tissue-specific regulation of energy metabolism in the swimming crab Portunus trituberculatus during nitrite stress and recovery.

Nitrite is a common pollutant in aquaculture systems that poses a significant threat to aquatic animals. Energy metabolism is critical in ensuring survival of animals under environmental stressors. However, regulation of energy metabolism in crustaceans under nitrite stress has not been well understood. Here we investigated energy metabolism regulation during nitrite stress and recovery in different tissues of the swimming crab Portunus trituberculatus, an important aquaculture species in China. Our results revealed that nitrite can cause tissue hypoxia and impair energy homeostasis, and energy balance cannot be restored even after a 96-hour recovery. Following exposure, mobilization of glycogen and lipids exhibited different temporal patterns. In response to energy imbalance, AMPK signaling was activated to counter energy imbalance. However, prolonged nitrite stress impaired AMPK signaling, leading to a further decline in energy supply. The findings improve our understanding for nitrite toxicity in P. trituberculatus, and provide valuable information for aquaculture management.

Corazonin Stimulates Ecdysteroid Synthesis during the Molting Process of the Swimming Crab, Portunus trituberculatus.

The neuropeptide corazonin (Crz) exerts diverse physiological effects in insects, yet its role in crustaceans remains elusive. The abundant expression of Crz receptor (CrzR) in the Y-organs of several crustaceans suggests a potential involvement of Crz in regulating ecdysteroid synthesis. In this study, we examined the effects of PtCrz on ecdysteroid synthesis during the molting period of Portunus trituberculatus through PtCrz treatments and PtCrzR silencing. Our results showed that PtCrz peptide stimulates ecdysteroid levels and the gene expression involved in ecdysteroidogenesis both in vitro and in vivo, whereas dsPtCrzR treatments had opposite effects on ecdysteroid levels and associated gene expression. Thus, our study suggests that PtCrz may modulate ecdysteroid synthesis via Y-organ-expressed PtCrzR. Furthermore, we also discovered the involvement of PtCrz/PtCrzR signaling in regulating PtETH expression. Notably, the inhibitory effect of dsPtCrzR on ecdysteroid synthesis or PtETH expression can be reversed by PtCrz treatment, suggesting the potential existence of multiple receptors for PtCrz. This study provides new insights into the function of crustacean Crz and, for the first time, elucidates the presence of a neuropeptide that can stimulate ecdysteroid synthesis in crustaceans.

Dietary lauric acid promoted antioxidant and immune capacity by improving intestinal structure and microbial population of swimming crab (Portunus trituberculatus).

Lauric acid (LA), a saturated fatty acid with 12 carbon atoms, is widely regarded as a healthy fatty acid that plays an important role in disease resistance and improving immune physiological function. The objective of this study was to determine the effects of dietary lauric acid on the growth performance, antioxidant capacity, non-specific immunity and intestinal microbiology, and evaluate the potential of lauric acids an environmentally friendly additive in swimming crab (Portunus trituberculatus) culture. A total of 192 swimming crabs with an initial body weight of 11.68 ± 0.02 g were fed six different dietary lauric acid levels, the analytical values of lauric acid were 0.09, 0.44, 0.80, 1.00, 1.53, 2.91 mg/g, respectively. There were four replicates per treatment and 8 juvenile swimming crabs per replicate. The results indicated that final weight, percent weight gain, specific growth rate, survival and feed intake were not significantly affected by dietary lauric acid levels; however, crabs fed diets with 0.80 and 1.00 mg/g lauric acid showed the lowest feed efficiency among all treatments. Proximate composition in hepatopancreas and muscle were not significantly affected by dietary lauric acid levels. The highest activities of amylase and lipase in hepatopancreas and intestine were found at crabs fed diet with 0.80 mg/g lauric acid (P < 0.05), the activity of carnitine palmityl transferase (CPT) in hepatopancreas and intestine significantly decreased with dietary lauric acid levels increasing from 0.09 to 2.91 mg/g (P < 0.05). The lowest concentration of glucose and total protein and the activity of alkaline phosphatase in hemolymph were observed at crabs fed diets with 0.80 and 1.00 mg/g lauric acid among all treatments. The activity of GSH-Px in hepatopancreas significantly increased with dietary lauric acid increasing from 0.09 to 1.53 mg/g, MDA in hepatopancreas and hemolymph was not significantly influenced by dietary lauric acid levels. The highest expression of cat and gpx in hepatopancreas were exhibited in crabs fed diet with 1.00 mg/g lauric acid, however, the expression of genes related to the inflammatory signaling pathway (relish, myd88, traf6, nf-κB) were up-regulated in the hepatopancreas with dietary lauric acid levels increasing from 0.09 to 1.00 mg/g, moreover, the expression of genes related to intestinal inflammatory, immune and antioxidant were significantly affected by dietary lauric acid levels (P < 0.05). Crabs fed diet without lauric acid supplementation exhibited higher lipid drop area in hepatopancreas than those fed the other diets (P < 0.05). The expression of genes related to lipid catabolism was up-regulated, however, and the expression of genes related to lipid synthesis was down-regulated in the hepatopancreas of crabs fed with 0.80 mg/g lauric acid. Lauric acid improved hepatic tubular integrity, and enhanced intestinal barrier function by increasing peritrophic membrane (PM) thickness and upregulating the expression of structural factors (per44, zo-1) and intestinal immunity-related genes. In addition, dietary 1.00 mg/g lauric acid significantly improved the microbiota composition of the intestinal, increased the abundance of Actinobacteria and Rhodobacteraceae, and decreased the abundance of Vibrio, thus maintaining the microbiota balance of the intestine. The correlation analysis showed that there was a relationship between intestinal microbiota and immune-antioxidant function. In conclusion, the dietary 1.00 mg/g lauric acid is beneficial to improve the antioxidant capacity and intestinal health of swimming crab.

The function of the cytoplasmic dynein light chain PTKM23 in the transport of PTSMAD2 during spermatogenesis in Portunus trituberculatus.

Cytoplasmic dynein participates in transport functions and is essential in spermatogenesis. KM23 belongs to the dynein light chain family. The TGFß signaling pathway is indispensable in spermatogenesis, and Smad2 is an important member of this pathway. We cloned PTKM23 and PTSMAD2 from Portunus trituberculatus and measured their expression during spermatogenesis. PTKM23 may be related to cell division, acrosome formation and nuclear remodeling, and PTSMAD2 may participate in regulating the expression of genes related to spermatogenesis. We assessed the localization of PTKM23 with PTDHC and α-Tubulin, and the results suggested that PTKM23 functions in intracellular transport during spermatogenesis. We knocked down PTKM23 in vivo, and the expression of p53, B-CATAENIN and CYCLIN B decreased significantly, further suggesting a role of PTKM23 in transport and cell division. The localization of PTDIC with α-Tubulin and that of PTSMAD2 with PTDHC changed after PTKM23 knockdown. We transfected PTKM23 and PTSMAD2 into HEK-293 T cells and verified their colocalization. These results indicate that PTKM23 is involved in the assembly of cytoplasmic dynein and microtubules during spermatogenesis and that PTKM23 mediates the participation of cytoplasmic dynein in the transport of PTSMAD2 during spermatogenesis. This study provides a theoretical molecular biological basis for the breeding of P. trituberculatus.

Dietary Astaxanthin Can Promote the Growth and Motivate Lipid Metabolism by Improving Antioxidant Properties for Swimming Crab, Portunus trituberculatus.

This study aimed to assess the influence of varying dietary levels of astaxanthin (AST) on the growth, antioxidant capacity and lipid metabolism of juvenile swimming crabs. Six diets were formulated to contain different AST levels, and the analyzed concentration of AST in experimental diets were 0, 24.2, 45.8, 72.4, 94.2 and 195.0 mg kg-1, respectively. Juvenile swimming crabs (initial weight 8.20 ± 0.01 g) were fed these experimental diets for 56 days. The findings indicated that the color of the live crab shells and the cooked crab shells gradually became red with the increase of dietary AST levels. Dietary 24.2 mg kg-1 astaxanthin significantly improved the growth performance of swimming crab. the lowest activities of glutathione (GSH), total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and peroxidase (POD) were found in crabs fed without AST supplementation diet. Crabs fed diet without AST supplementation showed lower lipid content and the activity of fatty acid synthetase (FAS) in hepatopancreas than those fed diets with AST supplementation, however, lipid content in muscle and the activity of carnitine palmitoyl transferase (CPT) in hepatopancreas were not significantly affected by dietary AST levels. And it can be found in oil red O staining that dietary 24.2 and 45.8 mg kg-1 astaxanthin significantly promoted the lipid accumulation of hepatopancreas. Crabs fed diet with 195.0 mg kg-1 AST exhibited lower expression of ampk, foxo, pi3k, akt and nadph in hepatopancreas than those fed the other diets, however, the expression of genes related to antioxidant such as cMn-sod, gsh-px, cat, trx and gst in hepatopancreas significantly down-regulated with the increase of dietary AST levels. In conclusion, dietary 24.2 and 45.8 mg kg-1 astaxanthin significantly promoted the lipid accumulation of hepatopancreas and im-proved the antioxidant and immune capacity of hemolymph.

Combined effects of pentachlorophenol and nano-TiO2 with different sizes on antioxidant, digestive, and immune responses of the swimming crab Portunus trituberculatus.

Marine nano-titanium dioxide (nano-TiO2) and pentachlorophenol (PCP) pollution are escalating concerns in coastal areas. This study investigated the combined effects of continuous exposure to nano-TiO2 (25 nm, 100 nm) and PCP (0, 1, 10 µg/L) for 28 days on the antioxidant, digestive, and immune abilities of the swimming crab Portunus trituberculatus. Compared with the control group, the interaction between nano-TiO2 and PCP was significantly higher than exposure to a single stressor, with a pronounced decrease in amylase activity observed due to the reducing nano-TiO2 particle sizes. Resulting in increased MDA and SOD activity. The expression levels of Toll4, CSP3, and SER genes in crab hemolymph showed perturbations following exposure to nano-TiO2 and PCP. In summary, according to the results of CAT, GPX, PES and AMS enzyme activities, it was concluded that compared to the larger particle size (100 nm), the single stress of nano-TiO2 at a smaller particle size (25 nm) and co-stress with PCP have more significant impacts on P. trituberculatus. However, the potential physiological regulation mechanism of the interaction between these pollutants remains elusive and requires further study.

Characterization of a pseudohemocyanin gene (PtPhc1) and its immunity function in response to Vibrio parahaemolyticus infection in the swimming crab Portunus trituberculatus.

Pseudohemocyanin is a member of the hemocyanin superfamily, but little research is available on its function in immunology. In this study, a Portunus trituberculatus pseudohemocyanin gene, named PtPhc1, was obtained by gene cloning. The PtPhc1 cDNA was 2312 bp in length, encoding 684 amino acids while exhibiting a characteristic hemocyanin structural domain. Tissue expression analysis revealed ubiquitous expression of PtPhc1 across all tissues, with the highest level of expression observed in the hepatopancreas. The expression pattern of PtPhc1 in response to Vibrio parahaemolyticus infection was clarified using RT-qPCR in swimming crabs. Notably, the expression peaked at 24 h, and increased 1435-fold compared to the control group in the hepatopancreas. While the expression level reached the maximum value at 72 h, which was 3.24 times higher than that of the control group in hemocytes. Remarkably, the reduction in PtPhc1 expression led to a noteworthy 30% increase in the mortality rate of P. trituberculatus when exposed to V. parahaemolyticus. In addition, in vitro bacterial inhibition assays exhibited a dose-dependent suppression of bacterial proliferation by recombinant PtPhc1 protein, with a notable inhibition rate of 48.33% against V. parahaemolyticus at a concentration of 0.03 mg/mL. To the best of our knowledge, the results establish the function of pseudohaemocyanin in immunity for the first time, contributing to a deeper comprehension of innate immune regulatory mechanisms in aquatic organisms and advancing strategies for disease-resistant breeding.

Identification of transient receptor potential channel genes from the swimming crab, Portunus Trituberculatus, and their expression profiles under acute temperature stress.

BACKGROUND: Temperature is an important environment factor that is critical to the survival and growth of crustaceans. However, the mechanisms by which crustaceans detect changes in temperature are still unclear. The transient receptor potential (TRP) channels are non-selective cation channels well known for properties in temperature sensation. However, comprehensive understandings on TRP channels as well as their temperature sensing functions are still lacking in crustaceans. RESULTS: In this study, a total of 26 TRP genes were identified in the swimming crab, Portunus trituberculatus, which can be classified into TRPA, TRPC, TRPP, TRPM, TRPML, TRPN and TRPV. Tissue expression analysis revealed a wide distribution of these TRP genes in P. trituberculatus, and antennules, neural tissues, and ovaries were the most commonly expressed tissues. To investigate the responsiveness of TRP genes to the temperature change, 18 TRPs were selected to detect their expression after high and low temperature stress. The results showed that 12 TRPs showed induced gene expression in both high and low temperature groups, while 3 were down-regulated in the low temperature group, and 3 showed no change in expression in either group. CONCLUSIONS: This study characterized the TRP family genes in P. trituberculatus, and explored their involvement in response to temperature stress. Our results will enhance overall understanding of crustacean TRP channels and their possible functions.

E93 gene in the swimming crab, Portunus trituberculatus: Responsiveness to 20-hydroxyecdysone and methyl farnesoate and role on regulating ecdysteroid synthesis.

Ecdysone-induced protein 93 (E93) is a metamorphic determinant involved in crosstalk between 20-hydroxyecdysone (20E) and juvenile hormone (JH) during the insect molting process. The present study identified the E93 gene from the swimming crab, P. trituberculatus, and found it was widely distributed in adult tissues. PtE93 mRNA levels in Y-organ and epidermis fluctuated during the molt cycle, suggesting its involvement in juvenile molting. In vitro and in vivo treatments with 20E led to an induction of PtE93 expression in Y-organ and epidermis, while we found the opposite effect for methyl farnesoate (MF) treatments, a crustacean equivalent of insect JH. We also observed that two genes for ecdysteroid biosynthesis, Spook (Spo) and Shadow (Sad), were suppressed by 20E and induced by MF, showing a negative correlation between PtE93 and ecdysteroid biosynthesis. PtE93 RNA interference (RNAi) induced Spo and Sad expression levels, elevated ecdysteroid content in culture medium, and relieved the 20E inhibitory effect on ecdysteroid synthesis, indicating an inhibitory role of PtE93 on ecdysteroid synthesis. Overall, our results suggest that E93 may be involved in the crosstalk between 20E and MF during crustacean molting, and its presence in Y-organ is closely related to ecdysteroid synthesis.

Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus.

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

Comparison of freezing and heating treatment sequence on biochemical properties and flavor of swimming crabs (Portunus Trituberculatus) meat during freeze-thaw cycles.

The objective of this study was to compare the effect of freezing and heating treatment sequences on the biochemical properties and flavor of crab (Portunus trituberculatus) meat during freeze-thaw cycles. The results showed that pH, color, K and microstructure changes in the H-F group were not significant with increasing number of freeze-thaw cycles, but TVB-N values increased and WHC values decreased. However, with the increase in the number of freeze-thaw cycles, pH and WHC significantly decreased and TVB-N, L* and K values significantly increased in the C and F-H groups. Proteins were degraded in all groups, but the lower degree of degradation occurred in the H-F group. Although the total free amino acid content decreased with increasing number of freeze-thaw cycles in each group, the high content of AMP and IMP in the H-F group suggested that it still had a better flavor.

Differential molecular responses of hemolymph and hepatopancreas of swimming crab, Portunus trituberculatus, infected with Ameson portunus (Microsporidia).

Ameson portunus (Microsporidia) has caused serious economic losses to the aquaculture industry of swimming crab, Portunus trituberculatus. The hemolymph and hepatopancreas are the main immune organs of P. trituberculatus, and the main sites of A. portunus infection. Elucidating the response characteristics of hemolymph and hepatopancreas to microsporidian infection facilitates the development of microsporidiosis prevention and control strategy. This study performed comparative transcriptomic analysis of hemolymph (PTX/PTXA) and hepatopancreas (PTG/PTGA) of P. trituberculatus uninfected and infected with A. portunus. The results showed that there were 223 and 1309 differentially expressed genes (DEGs) in PTX/PTXA and PTG/PTGA, respectively. The lysosome pathway was significantly enriched after the invasion of the hemolymph by A. portunus. Also, immune-related genes were all significantly up-regulated in the hemolymph and hepatopancreas, suggesting that the invasion by A. portunus may activate host immune responses. Unlike hemolymph, antioxidant and detoxification-related genes were also significantly up-regulated in the hepatopancreas. Moreover, metabolism-related genes were significantly down-regulated in the hepatopancreas, suggesting that energy synthesis, resistance to pathogens, and regulation of oxidative stress were suppressed in the hepatopancreas. Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. The differential genes and pathways identified in this study can provide references for the prevention and control of microsporidiosis.

Host-parasite interactions: a study on the pathogenicity of different Mesanophrys sp. densities and hemocytes-mediated parasitic resistance of swimming crabs (Portunus trituberculatus).

Mesanophrys sp. is a parasitic ciliate that invades and destroys the hemocytes of the swimming crab (Portunus trituberculatus). In the present study, we employed an in vitro model to elucidate how Mesanophrys sp. destroys crab hemocytes. We also evaluated the relationship between the parasite's density, the destruction rate of the hemocytes, and the rapid proliferation pattern of parasites in host crabs. We found that the survival rate and cell integrity of crab hemocytes decreased with an increase in Mesanophrys sp. density, depicting a negative correlation between hemocyte viability and parasite density. Further analyses revealed that crab hemocytes could resist destruction by a low density (10 ind/mL) of Mesanophrys sp. for a long time (60 h). Mesanophrys sp. and its culture medium (containing the ciliate secretions) destroy the host hemocytes. The natural population growth rate of Mesanophrys sp. decreased with an increase in the parasite density, but the Mesanophrys sp. density did not affect the generation time of the parasites. In summary, Mesanophrys sp. can destroy crab hemocytes, and the degree of destruction is directly proportional to the parasite density. The resistance of crab hemocytes to Mesanophrys sp. decreased gradually with an increase in the parasite density.

Comparative Transcriptome Analysis of the Response to Vibrio parahaemolyticus and Low-Salinity Stress in the Swimming Crab Portunus trituberculatus.

Vibrio parahaemolyticus is one of the main pathogenic bacteria of Portunus trituberculatus and causes mass mortality of P. trituberculatus in aquaculture. In addition, low-salinity stimulation makes P. trituberculatus more susceptible to V. parahaemolyticus infections. In order to elucidate the molecular mechanism of resistance to V. parahaemolyticus in P. trituberculatus, comparative transcriptomic analysis of blood cells stimulated by low salinity and V. parahaemolyticus was carried out in this study. Transcriptome sequencing of low-salinity stress and pathogen infection at different time points was completed using Illumina sequencing technology. A total of 5827, 6432, 5362 and 1784 differentially expressed genes (DEGs) involved in pathways related to ion transport and immunoregulation were found under low-salinity stress at 12, 24, 48 and 72 h compared with the control at 0 h. In contrast, 4854, 4814, 5535 and 6051 DEGs, which were significantly enriched in Toll and IMD signaling pathways, were found at 12, 24, 48 and 72 h compared with the control at 0 h under V. parahaemolyticus infection. Among them, 952 DEGs were shared in the two treatment groups, which were mainly involved in apoptosis and Hippo signaling pathway. Cluster analysis screened 103 genes that were differentially expressed in two factors that were negatively correlated, including immunoglobulin, leukocyte receptor cluster family, scavenger receptor, macroglobulin and other innate-immune-related genes. These results provide data support for the analysis of the mechanisms of immunity to V. parahaemolyticus under low-salinity stress in P. trituberculatus and help to elucidate the molecular mechanisms by which environmental factors affect immunity.

Nutrient Composition of Ovary, Hepatopancreas and Muscle Tissues in Relation to Ovarian Development Stage of Female Swimming Crab, Portunus trituberculatus.

The swimming crab Portunus trituberculatus is one of the most important economic species in China and its mature ovary often determines its commercial value and production. Although the ovary maturation of crustaceans is generally affected by exogenous nutrition, the specific nutritional needs of ovary maturation of P. trituberculatus are poorly understood. To this end, we collected the P. trituberculatus samples with five ovarian maturation stages and measured their biochemical composition of the ovary, hepatopancreas, and muscle at each ovarian developmental stage. We further analyzed their relation to the ovarian developmental stage of P. trituberculatus by principal components analysis (PCA). We found the levels of branched-chain amino acids, long-chain polyunsaturated fatty acids (LC-PUFA), and monounsaturated fatty acids (MUFAs) in the ovary and hepatopancreas increased during the ovary maturation process, and also passively correlated with ovarian developmental stage, which highlights the necessity of these specific nutrients for oogenesis and for improving the nutrient quality of crabs. In addition, we found an increasing tendency of carotenoid content and phosphatidylcholine in phospholipid in the ovary from the pre-developmental stage to the proliferative stage, but not in the hepatopancreas and muscle, which highlights the possible involvement of carotenoids during the rapid oocyte development process. Our study may provide valuable information for developing a suitable broodstock diet that promotes the ovarian maturation of adult P. trituberculatus and ensures high-quality larval production.

Purification, Expression, and Characterization of Sarcoplasmic Calcium Binding Protein: A Novel Allergen of Portunus trituberculatus.

Swimming crab (Portunus trituberculatus), a crucial valuable crustacean, is a common factor causing food allergy. However, studies on allergens of P. trituberculatus are scarce. In this study, the sarcoplasmic calcium binding protein (SCP) of P. trituberculatus was expressed in Escherichia coli, purified with affinity chromatography, and the IgE-binding activity was evaluated through serological analyses. Further, the structure, physicochemical properties, and cross-reactivity were assessed via bioinformatics, immunologic, and spectroscopy techniques. The results indicated that P. trituberculatus SCP was an allergen displaying strong IgE-binding capacity, composed of 60% α-helix. It presented good immunologic and structural stability at 4-70 °C and pH 3-10, and only exhibited high IgG cross-reactivity among crustaceans, without cross-reactivity with other species tested. These results establish the foundations for further studies on SCP and are promising to promote the development of specific crustacean allergen detection and precise allergy diagnosis.

Nano-TiO2 aggravates the adverse effect of pentachlorophenol on antioxidant and immune response in anti-predatory mussels.

Nano-TiO2 can act as a vector to organic compounds, such as pentachlorophenol (PCP) posing a potential threat to the marine ecosystems. Studies showed that nano pollutant toxicity can be modulated by abiotic factors, but little is known about the potential influence of biotic stressors (such as predators) on the physiological responses to pollutants in marine organisms. We explored the effects of n-TiO2 and PCP on the mussel Mytilus coruscus in the presence of its natural predator, the swimming crab Portunus trituberculatus. Exposure to n-TiO2, PCP, and predation risk showed interactive effects on antioxidant and immune parameters of the mussels. Elevated activities of catalase (CAT), glutathione peroxidase (GPX), acid phosphatase (ACP) and alkaline phosphatase (AKP), suppressed activity of superoxide dismutase (SOD), lower levels of glutathione (GSH) and increased malondialdehyde (MDA) levels indicated dysregulation of the antioxidant system and immune stress induced by single PCP or n-TiO2 exposure. Integrated biomarker (IBR) response values showed the effect of PCP was concentration dependent. Of the two used n-TiO2 sizes (25 and 100 nm), larger particles induced higher antioxidant and immune disturbances indicating higher toxicity possibly due to higher bioavailability. Compared to single PCP exposure, the combination of n-TiO2 and PCP enhanced the imbalance of SOD/CAT and GSH/GPX and led to elevated oxidative lesions and activation of immune-related enzymes. Overall, the combined impacts of pollutants and biotic stress exhibited a greater magnitude of adverse effects on antioxidant defense and immune parameters in mussels. The toxicological effects of PCP were exacerbated in the presence of n-TiO2, and the deleterious impact of these stressors was further amplified under predator-induced risk after prolonged (28 days) exposure. However, the underlying physiological regulatory mechanisms governing the interplay of these stressors and predatory cues on mussels remain elusive, warranting further investigation.

Each Personality Performs Its Own Function: Boldness and Exploration Lead to Differences in the Territoriality of Swimming Crabs (Portunus trituberculatus).

The boldness and exploration of animals are closely related to their territoriality, with relevant studies having important applications in wildlife conservation. The present study establishes a behavior observation system measuring the boldness and exploration of swimming crabs (Portunus trituberculatus) to clarify the relationship between boldness, exploration, and territoriality, as well as to provide a behavioral basis for the construction of marine ranching. The behavioral tests of crabs in a safe environment (predator absence), a dangerous environment (predator presence), and habitat selection (complex and simple habitat) are analyzed. A territorial behavior score is calculated as an evaluation index of territoriality. The correlation between the swimming crabs' boldness, exploration, and territoriality is analyzed. The results show that there is no boldness-exploratory behavioral syndrome. In predator absence or presence environments, boldness is dominant in territorial behavior and positively correlates with territoriality. Exploration plays a vital role in habitat selection tests but has no significant correlation with territoriality. The experimental results preliminarily show that boldness and exploration jointly develop the difference in the space utilization ability of crabs with different personalities, improving the adaptability of swimming crabs in different conditions. The results of this study supplement the behavior rules of the dominant species of typical fishery resources in marine ranches, providing a basis for achieving animal behavior management function in marine ranches.

Optimization of Sperm Cryopreservation Formulation in Portunus trituberculatus.

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.

Putative Role of CFSH in the Eyestalk-AG-Testicular Endocrine Axis of the Swimming Crab Portunus trituberculatus.

It has been shown in recent studies that the crustacean female sex hormone (CFSH) plays a crucial role in the development of secondary sexual characteristics in Decapoda crustaceans. However, research on the function of CFSH in the eyestalk-AG-testicular endocrine axis has been inadequate. We cloned and identified a homolog of CFSH, PtCFSH, in this study. RT-PCR showed that PtCFSH was mainly expressed in the eyestalk. A long-term injection of dsPtCFSH and recombinant PtCFSH (rPtCFSH) in vivo showed opposite effects on spermatogenesis-related gene expression and histological features in the testis of P. trituberculatus, and was accompanied by changes in AG morphological characteristics and PtIAG expression. In addition, the phosphorylated-MAPK levels and the expression of several IIS pathway genes in the testis was changed accordingly in two treatments, suggesting that PtCFSH may regulate the testicular development via IAG. The hypothesis was further validated by a mixed injection of both dsPtCFSH and dsPtIAG in vivo. The following in vitro studies confirmed the negatively effects of PtCFSH on AG, and revealed that the PtCFSH can also act directly on the testis. Treatment with rPtCFSH reduced the cAMP and cGMP levels as well as the nitric oxide synthetase activity. These findings provide vital clues to the mechanisms of CFSH action in both the eyestalk-AG-testis endocrinal axis and its direct effects on the testis.