Swimming Crab Portunus Trituberculatus Research Articles

Survival, Energy Status, and Cellular Stress Responses of the Juvenile Swimming Crab Portunus trituberculatus under Acute Nitrite Stress

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.

A swimming crab portunus trituberculatus re-identification method based on RNN encoding of striped key regions

A swimming crab portunus trituberculatus re-identification method based on RNN encoding of striped key regions

Molecular identification of an insulin-like peptide from the swimming crab Portunus trituberculatus and evidence for its glucoregulation function

Insulin-like peptides (ILPs) are essential to the animal kingdom for regulating growth and development, reproduction, behavior, metabolism, and lifespan. In crustaceans, the most well-known ILP is insulin-like androgenic gland hormone, a key hormone in regulating sex differentiation and reproduction. Identification of other ILPs and their functions are still limited. In this study, an insulin-like peptide gene of the swimming crab Portunus trituberculatus was cloned and characterized. Its transcripts were mainly found in nerve tissues and expression could be induced by glucose, implying a putative role in glucoregulation. After depletion of endogenous ILP, injection of ILP dsRNA (dsILP) significantly elevated blood glucose levels and recombinant ILP (rILP) decreased hemolymph glucose levels, further clarifying the involvement of acquired ILP in hemolymph glucose regulation. Injection of dsILP decreased PtAkt, PtGS, PtPFK and increased PtGSK and PtPEPCK gene expression. The opposite profile was observed after glucose and rILP injection, indicating that PtILP might negatively regulate hemolymph glucose levels via the IIS (insulin/IGF-1 signaling) pathway by inhibiting gluconeogenesis and promoting glycogen synthesis and glycolysis. This study has refined the mechanism of glucose regulation in crustaceans and laid the foundation for further studies on ILP function.

Ammonia Stress Disturbs Moult Signaling in Juvenile Swimming Crab Portunus trituberculatus

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.

Adaptive changes of swimming crab (Portunus trituberculatus) associated bacteria helping host against dibutyl phthalate toxification

The pollution of dibutyl phthalate (DBP) in aquatic environments is becoming an extensive environmental problem and detrimental to aquatic animals. Here, we quantified the response pattern of the bacterial community and metabolites of swimming crab (Portunus trituberculatus) juveniles exposed to 0.2, 2, and 10 mg/L DBP using 16 S rRNA gene amplicon sequencing coupled with metabolomic technique. The results showed that DBP changed the bacterial community compositions in a concentration-dependent pattern and decreased the Shannon index at the second developmental stage of the swimming crabs. The Rhodobacteraceae taxa were specifically enriched by crabs when challenged by 2 and 10 mg/L DBP, with an increased in Shannon index and enhanced drift in its assembly. Moreover, DBP changed the metabolic profiling of the swimming crab, highlighted by increased levels of lactate, valine, methionine, lysine, and phenylalanine in the 10 mg/L DBP-exposed crabs. Rhodobacteraceae presented the most considerable contribution to the metabolic potentials in phthalate and benzoate degradation, lactate production, and amino acid biosynthesis. Overall, our results indicated an adaptive change of crab-associated bacteria helped the host resist DBP stress. The findings extend our insights into the relationship between the microbiota and its host metabolism under DBP stress and reveal the potential microbiota modalities for DBP detoxification.

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.

Characterization and split-split-plot analysis of steamed factors affecting the swimming crab (Portunus trituberculatus) flavor quality

Swimming crab (Portunus trituberculatus) is a kind of popular crab for being rich in nutrition and intense in flavor. The time and heat exposure cause differences in flavor during steaming, dramatically affecting first impression and acceptability. The quality of P. trituberculatus with different steaming methods (steam with Room Temperature Water, RTW and steam with Boiling Water, BW) was evaluated using consumer acceptability, sensory characterization, and instrumental analysis. The split-split plot analysis was employed to compared the effects of the edible parts, steaming methods and steaming time. The results demonstrated that the hedonic score of crab meat was 1.3 times higher than that of hepatopancreas. Edible parts were the most important factor followed by the steaming method without interaction. The female crab meat (F-M) and male crab meat (M-M) were correlated with positive emotions, while opposite for hepatopancreas. RTW was more likely to evoke positive emotions, while BW followed the opposite trend. Instrumental analysis revealed that the types of volatiles produced with BW were similar to those by RTW, but their contents are generally 1.5–2.5 times higher. In sum, P. trituberculatus which were steamed for 25 min using RTW may be more acceptable.

Transcriptome analysis reveals the potential mechanism of carotenoids change in hepatopancreas under low-temperature storage from swimming crab (Portunus trituberculatus)

The hepatopancreas of swimming crab (Portunus trituberculatus) rich in carotenoids would undergo serious color deterioration during cold storage, and then made portunid lose its commodity value. In this study, we firstly elucidated the change mechanism of its carotenoids during storage at the molecular level using transcriptome technology. We concluded that low-temperature would inhibit aerobic respiration of portunid, leading to a lower pH and inducing the degradation of carotenoids. After that, longer cold storage time would increase the oxidative stress in portunid, resulting in a further decrease in carotenoids content. Finally, the strong autolysis of portunid could release carotenoids stored in other parts such as ovary to the external environment, resulting in the increase of carotenoids detection content. This research could provide a basis for further developing the fresh-keeping technology of portunid during low-temperature storage.

Application of light-emitting diodes (LEDs) fishing lights to improve catch rates of small-scale trammel net fishery in the Yellow Sea, China

The small-scale trammel net fisheries are essential to the economy and well-being of coastal communities in the Yellow Sea, China. However, these fisheries are low efficient and urgently demand technical innovations to improve catch rates. In this study, we conducted field experiments in three traditional fishing grounds (Rizhao, Yantai, and Weihai) to test whether the addition of different colored light-emitting diodes (LEDs) fishing lights in the trammel nets could improve the catch rates of seven commercial species. Results showed that the effects of LEDs on catch rates were species-specific. Green LEDs could significantly increase the catch per unit effort (CPUE) of black rockfish (Sebastes schlegelii), fat greenling (Hexagrammos otakii), marbled flounder (Pseudopleuronectes yokohamae), stone flounder (Platichthys bicoloratus), Asian paddle crab (Charybdis japonica), and mantis shrimp (Oratosquilla oratoria) by 40%-72% compared with unilluminated nets, while had no significant effect on the catch rates of swimming crab (Portunus trituberculatus). Red LEDs harvested 58%-86% higher CPUE of fat greenling, marbled flounder, stone flounder, mantis shrimp, and swimming crab while catching fewer black rockfish and Asian paddle crab. Blue and white LEDs exhibited similar performance, which could significantly capture more black rockfish, fat greenling, and mantis shrimp and did not affect the catch rates of other species. The effects of LEDs were also size-dependent for certain species, and the catch rates generally increased with the length classes. Taking economic benefits into consideration, the green and red LEDs provided the highest profitability for Rizhao, while green LEDs performed best in Weihai and Yantai. This study is the first investigation on how artificial lights affected the catch rates of trammel nets. The findings of our study illustrated that trammel nets equipped with LEDs could serve as a viable fishing strategy in small-scale trammel net fisheries.

Evaluation of Krill Meal in Commercial Diets for Juvenile Swimming Crab (Portunus trituberculatus).

An 8-week feeding trial was carried out to assess the effect of dietary krill meal on growth performance and expression of genes related to TOR pathway and antioxidation of swimming crab (Portunus trituberculatus). Four experimental diets (45% crude protein and 9% crude lipid) were formulated to obtain different replacements of fish meal (FM) with krill meal (KM); FM was replaced with KM at 0% (KM0), 10% (KM10), 20% (KM20), and 30% (KM30); fluorine concentration in diets were analyzed to be 27.16, 94.06, 153.81, and 265.30 mg kg-1, respectively. Each diet was randomly divided into 3 replicates; ten swimming crabs were stocked in each replicate (initial weight, 5.62 ± 0.19 g). The results indicated that crabs fed with the KM10 diet had the highest final weight, percent weight gain (PWG), and specific growth rate (SGR) among all treatments (P < 0.05). Crabs fed with the KM0 diet had the lowest activities of total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), glutathione (GSH), and hydroxyl radical scavenging activity and had the highest concentration of malondialdehyde (MDA) in the hemolymph and the hepatopancreas (P < 0.05). In the hepatopancreas, the highest content of 20:5n-3 (EPA) and the lowest content of 22:6n-3 (DHA) were shown in crabs fed with the KM30 diet among all treatments (P < 0.05). With the substitution level of FM with KM gradually increasing from 0% to 30%, the color of the hepatopancreas changed from pale white to red. Expression of tor, akt, s6k1, and s6 in the hepatopancreas was significantly upregulated, while 4e-bp1, eif4e1a, eif4e2, and eif4e3 were downregulated with dietary replacement of FM with KM increasing from 0% to 30% (P < 0.05). Crabs fed with the KM20 diet had notably higher expression of cat, gpx, cMnsod, and prx than those fed with the KM0 diet (P < 0.05). Results demonstrated that 10% replacement of FM with KM can promote growth performance and antioxidant capacity and notably upregulate the mRNA levels of genes related to TOR pathway and antioxidant of swimming crab.

Light spectrum impacts on development respiratory metabolism and antioxidant capacity of larval swimming crab Portunus trituberculatus

The effects of the spectrum on the development, respiratory metabolism, and antioxidant capacity of the larval swimming crab Portunus trituberculatus were studied. Seven light spectra, i.e., purple (400 nm), blue (425 nm), cyan (510 nm), green (525 nm), yellow (598 nm), red (638 nm), and white (full spectrum), were estimated. The larvae had the optimum survival rate and development under cyan light. On the contrary, larvae in red and yellow lights had poor growth performance. The oxygen consumption rate (OCR) dropped while the ammonia excretion rate (AER) rose as the larvae developed. Early larvae’s oxygen-nitrogen ratio (O: N) fell when exposed to red light, suggesting more protein was utilized in the respiratory process. Regarding the antioxidant system, crab had the lowest malondialdehyde (MDA) under green, cyan and yellow light, and the highest total antioxidant capacity (T-AOC) in cyan light. Taken together, the current results suggest that cyan was the optimum spectrum for the development of P. trituberculatus larvae.

Changes in Protein Degradation and Non-Volatile Flavor Substances of Swimming Crab (Portunus trituberculatus) during Steaming.

To investigate the effect of steaming time (0, 5, 10, 15, 20, and 25 min) on the protein degradation and non-volatile flavor substances of swimming crab (Portunus trituberculatus), the moisture content, total nitrogen (TN), non-protein nitrogen (NPN), free amino acids (FAAs), flavor nucleotides, electronic tongue analysis, and sensory evaluation were determined. The results showed that the contents of NPN and total FAAs were the highest after crabs steamed for 10 min. Meanwhile, the AMP (adenosine monophosphate) content reached the maximum value (332.83 mg/100 g) and the taste active value (TAV) reached 6.67, which indicated that AMP contributes the most to the taste of steamed crab at 10 min. The electronic tongue distinguished the taste difference well, and the sensory score was the highest at 15 min. Combined with equivalent umami concentration (EUC) and TAV value, swimming crab (weight = 200 ± 20 g) steamed for 10-15 min tasted best.

Dietary phenylalanine level could improve growth performance, glucose metabolism and insulin and mTOR signaling pathways of juvenile swimming crabs, Portunus trituberculatus

An 8-week feeding trial was conducted to determine the optimal dietary phenylalanine requirement of juvenile swimming crab (Portunus trituberculatus). Six experimental diets (45.0% crude protein and 8.0% crude lipid) were formulated to contain 0.89%, 1.15%, 1.41%, 1.64%, 1.90% and 2.18% phenylalanine, respectively. Each diet was randomly divided into triplicate groups with 30 juvenile swimming crabs (initial weight 22.87 ± 0.03 g). The highest percent weight gain (PWG) and feed efficiency (FE) were presented in crabs fed with 1.64% phenylalanine diet, and the lowest PWG and FE were observed in crabs fed diet with 0.89% phenylalanine (P < 0.05), while no significant difference was presented in survival among all treatments (P > 0.05). Crabs fed the diet containing 0.89% phenylalanine had the lowest content of crude lipid in hepatopancreas among all treatments (P < 0.05). Hematological parameters related to glucose and lipids metabolism and enzyme activities involved in glycolysis and gluconeogenesis were significantly affected by dietary phenylalanine levels (P < 0.05). The contents of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), n-6 polyunsaturated fatty acids (n-6 PUFAs) and n-3 polyunsaturated fatty acids (n-3 PUFAs) in the hepatopancreas were notably affected by the dietary phenylalanine levels (P < 0.05). The enzyme activities related to glycolysis, gluconeogenesis and glycogen content in hepatopancreas were significantly influenced by dietary phenylalanine levels (P < 0.05). The mRNA levels of genes related to glycolysis and gluconeogenesis in the hepatopancreas were significantly affected by dietary phenylalanine levels (P < 0.05). Moreover, the insulin and mammalian target of rapamycin (mTOR) signaling pathway were notably activated by dietary phenylalanine levels (P < 0.05). Based on two slope broken-line regression analysis of PWG against the dietary phenylalanine levels, the optimal dietary phenylalanine requirement was estimated to be 1.60% dry matter (3.55% dietary protein) for juvenile swimming crab.

Comparative transcriptomics reveals the immune dynamics during the molting cycle of swimming crab Portunus trituberculatus.

Molting is one of the most important biological processes of crustacean species, and a number of molecular mechanisms facilitate this complex procedure. However, the understanding of the immune mechanisms underlying crustacean molting cycle remains very limited. This study performed transcriptome sequencing in hemolymph and hepatopancreas of the swimming crab (Portunus trituberculatus) during the four molting stages: post-molt (AB), inter-molt (C), pre-molt (D), and ecdysis (E). The results showed that there were 78,572 unigenes that were obtained in the hemolymph and hepatopancreas of P. trituberculatus. Further analysis showed that 98 DEGs were involved in immunity response of hemolymph and hepatopancreas, and most of the DEGs participated in the process of signal transduction, pattern recognition proteins/receptors, and antioxidative enzymes system. Specifically, the key genes and pathway involved in signal transduction including the GPCR126, beta-integrin, integrin, three genes in mitogen-activated protein kinase (MAPK) signaling cascade (MAPKKK10, MAPKK4, and p38 MAPK), and four genes in Toll pathway (Toll-like receptor, cactus, pelle-like kinase, and NFIL3). For the pattern recognition proteins/receptors, the lowest expression level of 11 genes was found in the E stage, including C-type lectin receptor, C-type lectin domain family 6 member A and SRB3/C in the hemolymph, and hepatopancreatic lectin 4, C-type lectin, SRB, Down syndrome cell adhesion molecule homolog, Down syndrome cell adhesion molecule isoform, and A2M. Moreover, the expression level of copper/zinc superoxide dismutase isoform 4, glutathione peroxidase, glutathione S-transferase, peroxiredoxin, peroxiredoxin 6, and dual oxidase 2 in stage C or stage D significantly higher than that of stage E or stage AB. These results fill in the gap of the continuous transcriptional changes that are evident during the molting cycle of crab and further provided valuable information for elucidating the molecular mechanisms of immune regulation during the molting cycle of crab.

Feeding strategy trade-off and selection of marine nocturnal gastropods under predation risk

The circadian rhythm is one of the most crucial and universal biological rhythms in living organisms. As a typical nocturnal creature, the Pacific abalone (Haliotis discus hannai) exhibits rhythmic behaviors in terms of passively selecting whether to avoid predators or to forage, and active adaptation to light cycle changes is regulated by the biological clock. However, no basic data are available to help us to understand these rhythmic behaviors in the abalone species. In the present study, quantification of behavioral data for the abalone and its predator swimming crab Portunus trituberculatus in short-term (24 h) and long-term (40 days) polyculture scenarios suggests that the distance and duration of movement, percentage of feeding individuals, and cumulative duration of feeding of the abalone individuals were significantly lower under the short-term predation risk than the long-term predation risk. The concentrations of 5-hydroxytryptamine (5-HT), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and hexokinase (HK) in hemolymph, and expression levels of 5-HT1A receptor and 5-HT2 receptor in cerebral ganglion were significantly higher under the long-term predation risk than the short-term predation risk. The concentration of lactate dehydrogenase (LDH) and glycogen content in adductor muscle of the abalone was significantly higher under the short-term predation risk than the long-term predation risk, thereby implying their role in anaerobic metabolism and aerobic metabolism as primary energy sources under the short-term and long-term predation risk, respectively. The concentrations of 5-HT and cAMP, and the expression levels of Bmal1 and 5-HT2 receptor exhibited no significant signs of cosine rhythmicity under the short-term predation risk, but changes in the movement and feeding behaviors of the abalone still occurred at the night only. Correlation analysis shows that the expression levels of Bmal1 and Clock had significantly positive correlations with the circadian changes in the movement parameters of the abalone, thereby suggesting a dominant role in the rhythmic expression of endogenous circadian clock genes regulating the rhythmic behavior of the abalone. These findings provide new insights into the origin and evolution of biological rhythms in nocturnal marine animals and a reference for developing rational stock enhancement plans, and would improve protection for marine benthic biodiversity.

Comprehensive Genome-Wide Analysis of Wnt Gene Family and Expression Profiling during Limb Regeneration in Portunus trituberculatus

Wnt genes encode a family of secretory glycoproteins that are involved in various stages of organ development through regulation of cell proliferation, differentiation, and growth. Recently, Wnt genes have been shown to play an important role in regeneration processes. However, there have been no previous genome-wide analyses of the Wnt gene family members in crab species. In this study, a total of 13 Wnt family genes were identified from the genome of the swimming crab Portunus trituberculatus and classified into three main groups based on the conserved domain, protein sequence, and motifs. Chromosome location analysis showed that tandem duplication may have resulted in the expansion of the PtWnt gene family. RNA-seq results indicated that most PtWnt genes had a higher Fragments Per Kilobase of transcript sequences per Million base pairs (FPKM) value in the regenerating limb bud muscle than in the normal limb muscle, and the genes were enriched in a number of pathways that had biological functions underlying limb regeneration such as the cell surface receptor signaling pathway, the hippo signaling pathway, receptor binding, and basal cell carcinoma. Moreover, quantitative real-time PCR (RT-qPCR) analysis confirmed that the expression levels of nine PtWnts except for PtWnt1, PtWnt2, and PtWnt6 had a consistently increasing trend during limb regeneration. PtWnt1, PtWnt2, and PtWnt6 exhibited significantly up- or down-regulated expression at different limb regeneration stages. These results provide valuable information for further evolutionary and functional characterization of PtWnt genes and indicate the complexity and specialized mechanism of the Wnt signaling pathway regulating regeneration in crustaceans.

Differences in Agonistic Behavior and Energy Metabolism between Male and Female Swimming Crab Portunus trituberculatus Based on the Analysis of Boldness.

Simple SummaryBoldness is a widely studied personality trait that is positively related to an individual’s willingness to fight. To explore the agonistic behavior between female and male crabs with different degrees of boldness, we quantified the boldness of females and males using a behavior observation system and used the experimentally determined boldness and classification. The results showed that boldness affected the agonistic behavior between female and male swimming crabs, the fighting willingness and capacity of male crabs is higher than that of female individuals and is higher in bold crabs than in shy individuals. Energy reserves and metabolic rates may be one of the factors affecting the agonistic behavior in crabs and that the agonistic behavior resulted in significant changes in key energy metabolites.Individual differences in metabolism and agonistic behavior have been a key research area in evolution and ecology recently. In this study, we investigated the boldness of swimming crabs Portunus trituberculatus and explored the agonistic behavior between female and male crabs, specifically examining competitions between bold females vs. bold males (BF–BM), bold females vs. shy males (BF–SM), shy females vs. shy males (SF–SM), and shy females vs. bold males (SF_BM) and its relationship with energy metabolism. The main results revealed the following: There was no significant difference in boldness between females and males, while there were more bold individuals than shy in both females and males. Bold individuals initiated significantly more fights than shy individuals, and male initiators won significantly more fights than female initiators. The duration and intensity of fight between bold individuals was significantly higher than fights between shy individuals. For males, the concentration of glucose in the hemolymph was significantly higher in shy crabs than bold crabs, while there was no significant difference between shy and bold individuals in females. After fighting, the concentration of glycogen in claws was lower than that before fighting, and the concentrations of glucose and lactate in hemolymph were significantly higher after fighting than before. We found that the fighting willingness and ability were higher in male crabs than females and higher in bold crabs than shy. Fighting ability varied between sexes and was influenced by boldness and energy state.

A pattern recognition receptor ficolin from Portunus trituberculatus (Ptficolin) regulating immune defense and hemolymph coagulation

Ficolins, belonging to the fibrinogen-related protein superfamily, are important pattern recognition receptors in innate immunity. Here, a ficolin gene Ptficolin was characterized from the swimming crab Portunus trituberculatus. The completed cDNA sequence of Ptficolin encoded a signal peptide, a coiled-coil region and a fibrinogen-like domain but without the typical collagen region of vertebrate ficolins. Ptficolin showed higher expression in stomach and hepatopancreas, and presented a time-dependent response after pathogen challenge and injury stimulation. The recombinant Ptficolin (rPtficolin) could bind to various PAMPs and microorganisms, and agglutinate microorganisms and rabbit erythrocytes in a Ca2+-dependent manner, with strong binding ability to N-acetyl sugars. Meanwhile, rPtficolin promoted the hemocyte phagocytosis and clearance activity of Vibrio, while Ptficolin knockdown impaired the bacterial phagocytosis and clearance ability, suggesting the opsonin activity of Ptficolin. Knockdown of Ptficolin could downregulate the transcription of most complement-like genes and AMPs, but enhance the expression of most proPO system-related genes and key genes of Toll, IMD and JNK pathways. Moreover, knockdown of Ptficolin led to the increased hemolymph clotting time and the decreased expression of clotting-related genes. Our results indicate that Ptficolin could recognize and eliminate invading pathogens, and might be a prominent component in hemolymph coagulation of crab.

Identification and functional characterization of C-type lectins and crustins provide new insights into the immune response of Portunus trituberculatus

A meticulous understanding of the immune characteristics of aquaculture animals is the basis for developing precise disease prevention and control strategies. In this study, four novel C-type lectins (PtCTL-5, PtCTL-6, PtCTL-7 and PtCTL-8) including a single carbohydrate-recognition domain (CRD), and four novel crustins (Ptcrustin-1, Ptcrustin-2, Ptcrustin-3 and Ptcrustin-4) with a single whey acidic protein (WAP) domain were identified from the swimming crab Portunus trituberculatus. Tissue distribution analysis indicated that most of the target genes were predominantly expressed in the hepatopancreas in all examined tissues, except for Ptcrustin-1 which were mainly expressed in the gills. Our results showed that the eight genes displayed various transcriptional profiles across different tissues. In hemocytes, the PtCTL-7 responded quickly to Vibrio alginolyticus and exhibited much more strongly up-regulation than other three PtCTLs. The Ptcrustin-1 rapidly responded to V. alginolyticus within 3 h in all the three tested tissues. Furthermore, recombinant proteins of PtCTL-5 and PtCTL-8 were successfully obtained, and both of them displayed bacterial binding activities toward V. alginolyticus, V. harveyi and Staphylococcus aureus, and only showed antibacterial activity against V. harveyi. These findings provided new insights into the diverse immune response of P. trituberculatus and laid theoretical foundations for the development of precise disease prevention and control strategies in P. trituberculatus farming. Moreover, the specific anti-V. harveyi activities exhibited by rPtCTL-5 and rPtCTL-8 suggested their promising application prospects for controlling diseases caused by V. harveyi.

Putative role of corazonin in the ovarian development of the swimming crab Portunus trituberculatus

Corazonin (Crz) is a neuropeptide that widely distributed in insects and crustaceans. The Crz is proposed to have pleiotropic functions in insects, but its physiological roles in crustaceans are poorly understood. In the present study, Crz and its putative receptor (CrzR) were identified from the swimming crab, Portunus trituberculatus, and their interaction was validated using the Dual-Luciferase reporter assay system. Tissue distribution analysis showed the PtCrz was mainly derived from center nerve system, while its receptor was highly expressed in Y-organ, the main site for ecdysteroids synthesis. Exposure of YO to synthetic Crz and CrzR dsRNA respectively led to the transcriptional changes of two ecdysteroidogenesis genes, further indicating a putative role of Crz signaling on ecdysteroids synthesis. During the ovarian development, the mRNA levels of PtCrz and PtCrzR increased significantly in vitellogenic stages, suggesting a potential role of Crz signaling in vitellogenesis. The hypothesis was further strengthened by in vitro experiments that the expression of vitellogenin (Vg), Vg receptor (VgR), cyclinB, and Cdc2 in ovary explants could be induced by synthetic Crz, whereas reduced by CrzR dsRNA. In addition, since 20-hydroxyecdysone also showed a stimulating effect on Vg expression, an indirect regulation of Crz signaling on ovarian development via ecdysteroids might also exist.