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.

The effect of external fixator on bone metabolism in patients with open fracture of extremities.

This experiment aimed to explore the influence mechanism of external fixator on open fracture. A total of 128 patients with open tibiofibular fractures were included in this study. The patients were randomly divided into external fixator group (n=64) and control group (n=64) according to the order of admission. Double-blind controlled observation was used. The levels of osteocalcin (BGP), ß-CTX, P1 NP, BALP, including haptoglobin (Hp), ceruloplasmin (CER), serum adrenocorticotropic hormone (ACTH), cortisol (COR), C-reactive protein (CRP), white blood cell (WBC) and interleukin-6 (IL-6) were recorded in different groups. The postoperative VAS score and quality of life were recorded. Log-rank was used to analyze the difference in postoperative adverse reaction rates among different groups. External fixation stent treatment increased BGP, PINP, and BALP expression and decreased ß-CTX, Hp, CER, ACTH, COR, CRP, WBC, and IL-6 levels. Patients in the external fixation stent group had significantly lower VAS score quality of life scores and incidence of adverse events than the control group. External fixation stents protect open fracture patients by promoting bone metabolism.

The miRNAs let-7b and miR-141 Coordinately Regulate Vitellogenesis by Modulating Methyl Farnesoate Degradation in the Swimming Crab Portunus trituberculatus.

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.

Integrated physiological, transcriptome and metabolome analyses of the hepatopancreas of the female swimming crab Portunus trituberculatus under ammonia exposure.

Ammonia is a common environmental pollutant in aquatic ecosystem and is also a significant concern in closed aquaculture systems. The threat of ammonia has been increasing with rising anthropogenic activities including intensified aquaculture. In this study, we aimed to investigate ammonia toxicity and metabolism mechanisms in the hepatopancreas, a major organ for Vitellogenin (Vtg) synthesis and defending against ammonia stress, of female swimming crab Portunus trituberculatus which is an important fishery and aquaculture species, by integrating physiological, transcriptome and metabolome analyses. The results revealed that ammonia exposure (10 mg/L, an environmentally relevant concentration) resulted in a remarkable reduction in vtg expression and depression of multiple signaling pathways for reproductive regulators including methyl farnesoate, ecdysone and neuroparsin, demonstrating for the first time that ammonia impairs swimming crab female reproduction. In addition, a number of important genes and metabolites in glycolysis, the Krebs cycle, fatty acid ß-oxidation and synthesis were significantly downregulated, indicating that changes in ammonia levels lead to a general depression of energy metabolism in hepatopancreas. After ammonia exposure, an increased level of urea and a reduction of amino acid catabolism were observed in hepatopancreas, suggesting that urea cycle was utilized to biotransform ammonia, and amino acid catabolism was decreased to reduce endogenous ammonia generation. Furthermore, antioxidant systems were altered following ammonia exposure, which was accompanied by proteins and lipid oxidations, as well as cellular apoptosis. These results indicate that ammonia leads to metabolic suppression, oxidative stress and apoptosis in P. trituberculatus hepatopancreas. The findings improve the understanding for the mechanisms of ammonia toxicity and metabolism in P. trituberculatus, and provide valuable information for assessing potential ecological risk of environmental ammonia and improving aquaculture management.

Immune response and antioxidant status of Portunus trituberculatus inoculated with pathogens.

The white spot syndrome virus (WSSV), Vibrio parahaemolyticus and V. alginolyticus are serious epidemic pathogen affecting the cultured Portunus trituberculatus and resulted in severe economic losses to local farmers. The immune and antioxidant systems are believed to be closely involved in host responses to pathogens in aerobic animals, including crustaceans. In order to explore such host-pathogen interactions in the early stage of infection in P. trituberculatus, the mRNA transcript levels of six key immune-related genes (proPO, α2M, crustin, lysozyme, NOS, and NOX) and three key antioxidant-related genes (CuZnSOD, CAT, and GPx) and their corresponding enzymatic activity were investigated in response to challenge with the three pathogens. A decrease in the expression of the proPO, crustin, and lysozyme was observed, which may reflect the immunosuppressive mechanism of the pathogens against the host. Moreover, an increase was observed in the α2M expression with time, which indicated that the pathogens could affect proteinase cascade reactions associated with the proPO system by disturbing the balance between serine proteinases and their inhibitors. Moreover, WSSV, V. parahaemolyticus and V. alginolyticus induced to increase the transcription and enzyme activities of NOS and NOX. Additionally, significant variations in the expression of the anitioxidant-related genes CuZnSOD, CAT, and GPx and their enzyme activities implied that these enzymes played a critical role in the immune response against the pathogens. The present findings indicate that the immune parameters analyzed here may be markers of the physiological status of this species after bacterial or viral infections.

Characterization of three caspases and their pathogen-induced expression pattern in Portunus trituberculatus.

Caspases are a family of proteases involved in many important biological processes including apoptosis and inflammation. In this study, we analyzed the expression patterns and effects on immune response in various tissues of the edible crab Portunus trituberculatus. PtCas 2, PtCas 3 and PtCas 4 share overall sequence identities of 55.88%-74.86%, 8.47%-46.54% and 20.11%-50.87%, respectively, with their other crustacean species. PtCas 2, PtCas 3 and PtCas 4 have the same caspase domain and catalytic site found in known caspases. The expression levels of the three caspases differed between tissues. Following bacterial and viral infection, the expression levels of the three caspases reached a maximum level at 24 h post-infection (hpi) in case of bacteria, whereas it was 48 hpi in virus. Moreover, the WSSV, Vibrio alginolyticus or V. parahaemolyticus induced the activities of PtCas 2-4 in a time-dependent manner. These results indicate an involvement of caspases in bacterial and viral induced immune response and demonstrate for the first time that PtCas 2, PtCas 3 and PtCas 4 are essential for optimal response to bacterial and virus infection in crabs.

Effects of florfenicol on the antioxidant status, detoxification system and biomolecule damage in the swimming crab (Portunus trituberculatus).

Florfenicol (FLR) is the most commonly used antibacterial agent in aquaculture because of its wide spectrum of activity and few side-effects. We characterized the toxicokinetics of FLR in the swimming crab (Portunus trituberculatus) after intravenous (IV) dosing (20, 40 and 80mg/kg). The results showed that FLR significantly suppressed the antioxidant system of the hepatopancreas. FLR induced transcriptional expression of phase I and phase II detoxification genes (CYP3 and GST, respectively) in a dose- and clearance time-dependent manner and altered the expression of their corresponding enzymes (erythromycin N-demethylase and glutathione S-transferase, respectively). Moreover, FLR induced the transcription of ATP-binding cassette (ABC) transporter subfamily B (ABCB) and subfamily G (ABCG), although ABCG transcription was not induced by FLR at 20mg/kg. Additionally, higher FLR doses caused significant biomolecule damage during the first 48h after delivery. This study will provide an improved understanding of the exact mechanism underlying toxicity in aquatic organisms.

The immune responses and antioxidant status of Portunus trituberculatus individuals with different body weights.

Vibrio alginolyticus is a virulent pathogen that affects crab aquacultures. In the present study, the immune responses and antioxidant status of big and small (based on body weight and size) 80-, 100- and 120-day-old specimens of Portunus trituberculatus, challenged for 72 h with Vibrio alginolyticus, were studied. The total hemocyte count (THC), and phagocytic, prophenoloxidase and phenoloxidase activities, of the big individuals (BIs) were higher than those of the small individuals (SIs) (P < 0.05). The antioxidant status of the organisms showed a similar pattern: superoxide dismutase (SOD) activity and glutathione/oxidized glutathione (GSH/GSSG) in the cell-free hemolymph and hepatopancreases of the BIs were higher than in the SIs (P < 0.05). There were no significant differences in α2-macroglobulin (α2-M), antibacterial and bacteriolytic activities in the cell-free hemolymph, or glutathione peroxidase activity in the cell-free hemolymph or hepatopancreas between the BIs and SIs. The α2-M and crustin gene expression levels in the hemocytes, and SOD expression in the hemocytes and hepatopancreas, were also significantly higher in the BIs. The results suggest that, compared with the SIs, the BIs possessed a higher resistance to V. alginolyticus infection.

Chromosome-level genome of the long-tailed marine-living ornate spiny lobster, Panulirus ornatus.

Recent conservation efforts to protect rare and endangered aquatic species have intensified. Nevertheless, the ornate spiny lobster (Panulirus ornatus), which is prevalent in the Indo-Pacific waters, has been largely ignored. In the absence of a detailed genomic reference, the conservation and population genetics of this crustacean are poorly understood. Here, We assembled a comprehensive chromosome-level genome for P. ornatus. This genome-among the most detailed for lobsters-spans 2.65 Gb with a contig N50 of 51.05 Mb, and 99.11% of the sequences with incorporated to 73 chromosomes. The ornate spiny lobster genome comprises 65.67% repeat sequences and 22,752 protein-coding genes with 99.20% of the genes functionally annotated. The assembly of the P. ornatus genome provides valuable insights into comparative crustacean genomics and endangered species conservation, and lays the groundwork for future research on the speciation, ecology, and evolution of the ornate spiny lobster.

cDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda.

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5' untranslated region (UTR) of 14 bp, a 3' UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.

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.

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.

cDNA cloning, characterization and expression analysis of catalase in swimming crab Portunus trituberculatus: cDNA cloning and expression analysis of catalase gene of Portunus trituberculatus.

Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. In the present study, a full-length cDNA sequence of catalase was cloned from the haemocytes of swimming crab Portunus trituberculatus by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end method. The catalase cDNA sequence contained 1,851 bp with an open reading frame of 1,551 bp encoding 516 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of P. trituberculatus catalase. The deduced catalase protein had a calculated molecular mass of 58.5 kDa with an estimated isoelectric point of 6.90. Multiple alignment analysis revealed that the deduced amino acid sequence of catalase shared high identity of 68-95 % with those of other species. Quantitative real-time RT-PCR analysis revealed that P. trituberculatus catalase transcript was strongly detected in haemocytes, hepatopancreas, heart, stomach, intestine, gill, ovary and muscle. The expression level of catalase transcripts both in haemocytes and hepatopancreas changed rapidly and dynamically after Vibrio alginolyticus challenging. These facts indicate that catalase was perhaps involved in the acute response against invading bacteria and was an inducible protein involved in the host innate immune response through elimination of H(2)O(2) in crab.

Application of proteomics and metabolomics to assess ammonia stress response and tolerance mechanisms of juvenile ornate rock lobster Panulirus ornatus.

Ammonia is a common pollutant in the aquatic ecosystem and closed aquaculture systems. It may pose a threat to the lobster growth, reproduction and survival. However, there is lack of research of the mechanisms on the toxic effects ammonia at molecular levels. In this work, proteomics and metabolomics were integrated to analyze the proteome and metabolome responses in the ornate spiny lobster Panulirus ornatus treated with ammonia (20 mg L-1) for 48 h. A total of 199 proteins and 176 metabolites were significantly altered in P. ornatus following ammonia treatment. The responsive proteins and metabolites were predominantly involved in immune response, phase I and phase II biotransformation, carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Furthermore, an increase in urea levels was observed, and amino acid metabolism was induced, indicating that the urea cycle was utilized to biotransform ammonia so as to reduce endogenous ammonia content. Ammonia exposure also affected the antioxidant system and induced cellular apoptosis. Overall, our results provide comprehensive insights into the molecular mechanisms underlying the response of P. ornatus to ammonia stress. We believe that the data reported herein should contribute to the development of novel, efficient methods for P. ornatus aquaculture.

Screening and Verification of Molecular Markers and Genes Related to Salt-Alkali Tolerance in Portunus trituberculatus.

Salt-alkali tolerance is one of the important breeding traits of Portunus trituberculatus. Identification of molecular markers linked to salt-alkali tolerance is prerequisite to develop such molecular marker-assisted breeding. In this study, Bulked Segregant Analysis (BSA) was used to screen molecular markers associated with salt-alkali tolerance trait in P. trituberculatus. Two DNA mixing pools with significant difference in salt-alkali tolerance were prepared and 94.83G of high-quality sequencing data was obtained. 855 SNPs and 1051 Indels were firstly selected as candidate markers by BSA analysis, out of which, 20 markers were further selected via △index value (close to 0 or 1) and eight of those were successfully verified. In addition, based on the located information of the markers in genome, eight candidate genes related to salt-alkali tolerance were anchored including ubiquitin-conjugating enzyme, aspartate-tRNA ligase, vesicle-trafficking protein, and so on. qPCR results showed that the expression patterns of all these genes changed significantly after salt-alkali stress, suggesting that they play certain roles in salt-alkali adaptation. Our results will provide applicable markers for molecular marker-assisted breeding and help to clarify the mechanisms of salt-alkali adaptation of P. trituberculatus.

A chromosome-level genome of Portunus trituberculatus provides insights into its evolution, salinity adaptation and sex determination.

Portunus trituberculatus (Crustacea: Decapoda: Brachyura), commonly known as the swimming crab, is of major ecological importance, as well as being important to the fisheries industry. P. trituberculatus is also an important farmed species in China due to its rapid growth rate and high economic value. Here, we report the genome sequence of the swimming crab, which was assembled at the chromosome scale, covering ~1.2 Gb, with 79.99% of the scaffold sequences assembled into 53 chromosomes. The contig and scaffold N50 values were 108.7 kb and 15.6 Mb, respectively, with 19,981 protein-coding genes. Based on comparative genomic analyses of crabs and shrimps, the C2H2 zinc finger protein family was found to be the only gene family expanded in crab genomes, suggesting it was closely related to the evolution of crabs. The combination of transcriptome and bulked segregant analysis provided insights into the genetic basis of salinity adaptation and rapid growth in P. trituberculatus. In addition, the specific region of the Y chromosome was located for the first time in the genome of P. trituberculatus, and three genes were preliminarily identified as candidate genes for sex determination in this region. Decoding the swimming crab genome not only provides a valuable genomic resource for further biological and evolutionary studies, but is also useful for molecular breeding of swimming crabs.

Molecular characterization and expression analysis of extracellular copper-zinc superoxide dismutase gene from swimming crab Portunus trituberculatus.

An extracellular CuZnSOD cDNA was cloned from the haemocytes of swimming crab Portunus trituberculatus by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the ecCuZnSOD full-length cDNA consisted of 965 bp with an open reading frame of 579 bp. It encoded a polypeptide of 192 amino acids which had a predicted molecular weight of 20.0 kDa and with an estimated pI of 6.23. The deduced amino acid sequence contained a putative signal peptide of 31 amino acids. It is predicted to possess all the expected features of CuZnSOD members, including amino acids responsible for binding Cu and Zn, two putative CuZnSOD signatures, two cysteines and one N-linked glycosylation site. Sequence comparison showed that the CuZnSOD deduced amino acid sequence of P. trituberculatus has similarity of 80%, 76%, 55% and 50% to that of blue crab Callinectes sapidus, mud crab Scylla serrata, crayfish Pacifastacus leniusculus and freshwater prawn Macrobrachium rosenbergii, respectively. The ecCuZnSOD transcripts expressed in all examined tissues, including haemocytes, hepatopancreas, heart, stomach, intestine, gill, ovary and muscle. RT-PCR analysis indicated that ecCuZnSOD transcripts both in haemocytes and hepatopancreas increased in the first 3 h after Vibrio alginolyticus challenging, as the bacterial infection progressed, the challenged crabs decreased to levels significantly lower than control by 96 h post-infection. These facts indicated that ecCuZnSOD was potentially involved in the acute response against invading bacteria in P. trituberculatus.

Molecular cloning and characterization of prophenoloxidase gene in swimming crab Portunus trituberculatus.

Phenoloxidase (PO), a melanin-synthesizing enzyme, found as a zymogen (proPO) in hemolymp, plays an important role in arthropod defence. In this study, a prophenoloxidase (proPO) cDNA was cloned from the haemocytes of swimming crab Portunus trituberculatus by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone had a full-length of 3040 bp, with an open reading frame of 2019 bp, a 5'-untranslated region of 138 bp, and a long 3'-untranslated region of 1707 bp. It encoded a protein of 672 amino acids which has a predicted molecular weight of 77.4 kDa and with an estimated pI of 6.19. It is predicted to possess all the expected features of proPO members, including two putative tyrosinase copper-binding motifs with six histidine residues and a proteolytic activation site. Comparison of amino acid sequences showed that the proPO-deduced amino acid of P. trituberculatus has an overall similarity of 56%-87% to that of other crustaceans. Northern blot analysis revealed that the presence of proPO was expressed in haemocytes, hepatopancreas and ovary, but not in eyestalk, gill and muscle. RT-PCR analysis indicated that proPO showed different expression profiles in crab haemocytes and hepatopancreas after Vibrio alginolyticus challenging. These facts indicated that proPO was potentially involved in the acute response against invading bacteria in P. trituberculatus.

Integrative Proteomic and MicroRNA Analysis: Insights Into Mechanisms of Eyestalk Ablation-Induced Ovarian Maturation in the Swimming Crab Portunus trituberculatus.

Eyestalk ablation is the most common method to induce ovarian maturation in decapod crustacean aquaculture, but it jeopardizes broodstock survival and larvae production. It is important to understand the molecular basis underlying the maturation triggered by ablation and thereby develop an alternative measure for maturation manipulation. In this study, we investigate alterations of ovarian proteome and miRNA profile after ablation in a commercially important marine crab Portunus trituberculatus. Quantitative proteomic analysis using iTRAQ reveals that 163 proteins are differentially expressed following ablation, and modulation of methyl farnesoate metabolism and activation of calcium signaling may play important roles in the ovarian maturation induced by ablation. miRNA expression profiling identifies 31 miRNAs that show statistically significant changes. Integration of miRNA and proteome expression data with miRNA target prediction algorithms generates a potential regulatory network consisting of 26 miRNAs and 30 proteins linked by 71 possible functional associations. The miRNA-protein network analysis suggests that miRNAs are involved in promoting ovarian maturation by controlling expression of proteins related to methyl farnesoate synthesis, calcium signals, and energy metabolism. Experimental validation and temporal expression analysis indicate multiple miRNAs can act synergistically to regulate expression of Farnesoic acid O-methyltransferase and Calmodulin. Our findings provide new insights for elucidating the mechanisms underlying eyestalk ablation-induced ovarian maturation and could be useful for devising an alternative technique for manipulating reproduction in P. trituberculatus and other decapods.

Characterization of a chitinase-1 gene (PtCht-1) from a marine crab Portunus trituberculatus and its response to immune stress.

Chitinases play an important role in many biological processes in crustaceans, including molting, digestion, and immunity. In order to further explore the immune defense mechanism of chitinase in Portunus trituberculatus, the PtCht-1 gene was cloned by RACE (rapid-amplification of cDNA ends). This cDNA with a full length of 1910 bp, and an ORF (open reading frame) 1749 bp, coded for 582 amino acid residues and was classified into P. trituberculatus chitinase GH18-group4. It had the typical structural characteristics of GH18 chitinase family. Real-time PCR was used to analyze the expression of PtCht-1 in different tissues, molting stages, after pathogen infection, and low salinity (11‰). PtCht-1 was expressed in all tissues, with the highest expression in the hepatopancreas. In the hepatopancreas of different molting stages, the expression level decreased successively during post-molt stages (A/B), pre-molt stage (D) and inter-molt stage (C). Under normal circumstances, after artificial infection with WSSV and Vibrio parahaemolyticus, the expression of PtCht-1 in hepatopancreas reached the maximum at 48 h, and in hemolymph at 72 h and 24 h, respectively. Overall PtCht-1 expression was up-regulated compared with the control group. Low salinity stress significantly inhibited the expression of PtCht-1, up to 42 folds. Under low salinity stress, the time when WSSV infection reached the peak was markedly delayed by at least 24 h. The results of this study indicate that PtCht-1, as an immune factor, is likely involved in pathogen defense of P. trituberculatus, the immune function of which may be inhibited to some extent after low salinity stress.