Search

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. • Lauric acid can improve the antioxidant capacity and up-regulate the expression of anti-inflammatory factors. • Lauric acid increased the thickness of peritrophic membrane in the gut of swimming crab. • Lauric acid reduced the abundance of vibrio in the intestine. • Excessive amounts of lauric acid can lead to tissue damage and metabolic disorders. [ABSTRACT FROM AUTHOR]

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. • Four C-type lectins and four crustins were identified in Portunus trituberculatus. • The eight genes were predominantly expressed in hepatopancreas or gills. • PtCTL-7 responded quickly to Vibro alginolyticus and exhibited much more strongly up-regulation in hemocytes. • Ptcrustin-1 rapidly responded to V. alginolyticus within 3 h in hemocytes, hepatopancreas and gills. • PtCTL-5 and PtCTL-8 displayed broad spectrum of binding activity and specific antimicrobial activity to a certain extent. [ABSTRACT FROM AUTHOR]

The immune deficiency (IMD) pathway is involved in both antiviral and antibacterial immune responses in Drosophila. IMD protein is the key adaptor to link the extracellular signal and the intracellular reaction to initiate the signal transduction in IMD pathway. In present study, the cDNA of the IMD (Pt -IMD) was identified from a marine crab, Portunus trituberculatus. The Pt -IMD is predicted to encode 170 amino acids with a death domain. Real-Time quantitative PCR analysis showed that Pt -IMD was constitutively expressed in hemocytes, intestine, gill, heart, muscle and hepatopancreas in normal crab. Moreover, the transcript of Pt -IMD in large-granule hemocytes is approximately 6-fold higher than semi-granular cells and agranular cells. Intracellular localization showed Pt -IMD was distributed mainly in the cytoplasm when it was over-expressed in Drosophila Schneider 2 (S2) cell. Functionally, over-expression of Pt -IMD could activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. Furthermore, Pt -IMD expression was also knock-down by RNAi to determine the function of Pt -IMD on regulation of the expression of different antimicrobial peptides (AMPs) in crab. In the primary cultured hemocytes challenged with or without Vibrio alginolyticus , after Pt -IMD was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), ALF3, crustin1, crustin3, arasin2, hyastatin1and hyastatin3 have been significantly inhibited in normal cell or bacterial infected cell, while the expression of lysozyme was normal in non-infected cells and was significantly induced in bacterial infected cells, which compared to the non-specific dsRNA treated cells. • Full-length cDNAs of Pt -IMD was cloned from Portunus trituberculatus. • Pt -IMD is constitutively expressed in tissues, and has highest abundances in hemocytes, especially in large granule cells. • Over-expressed Pt -IMD were mainly in the cytoplasm and could activate the promoters of Drosophila AMPs in S2 cell. • The expression of several crab AMPs have been significantly inhibited when Pt -IMD transcription was suppressed by RNAi. [ABSTRACT FROM AUTHOR]

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. • Immunologic activity of pseudohemocyanin PtPhc1 reported for the first time in crustaceans. • Knockdown of PtPhc1 led to increased mortality in pathogen-infected swimming crabs. • Pseudoanthocyanidin PtPhc1 exhibited bacteriostatic activity in vitro. [ABSTRACT FROM AUTHOR]

In recent years, the community structure of fishery resources in offshore waters has undergone significant changes, and the resources of traditional commercial fish have gradually declined; as a result, crustaceans have become the main catch. To prevent the loss of resources, it is necessary to carry out ecological stock enhancement and scientifically prevent and control the ecological risks of released species. In this study, based on the Ecopath with Ecosim model, the areas of marine ranching and adjacent waters in Haizhou Bay were used as a case study to construct a temporal dynamic model of Portunus trituberculatus , one of the typical crustacea released in offshore water of China. The ecological carrying capacity (ECC) of P. trituberculatus was assessed, and its impact on other functional groups and ecosystems was predicted. The results showed that the ECC of P. trituberculatus is 42.458 t km−2, and the entire ecosystem would become imbalanced if this value is exceeded. Under the four simulation scenarios, each ecosystem indicator showed positive responses to different degrees, with total system respiration (R) and total system biomass (B) increasing by 244.66% and 236.90%, respectively. We believe that the maturity of the ecosystem in Haizhou Bay is not high, and the marine organisms are mostly in trophic levels Ⅱ to Ⅲ. Therefore, there is still room for the stock enhancement of P. trituberculatus. The model framework provides a method for assessing the ECC of P. trituberculatus in offshore water and offers a scientific reference and practical basis for stock enhancement activities, resource management, and fishing strategies for P. trituberculatus in the offshore waters of China. • To assess the ecological capacity of Portunus trituberculatus. • To predict the variation of releasing capacity before and after stock enhancement. • To provide scientific recommendations for the stock enhancement sustainability. [ABSTRACT FROM AUTHOR]

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 r Pt -SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, r Pt -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. •A novel SRB homolog (Pt- SRB2) showed distributed primarily in the cell membrane and cytoplasm •The immunofluorescence staining showed Pt- SRB2 were mainly located in the inner lumen membrane of the hepatopancreatic tubules. •R Pt- SRB2-L showed binding affinity with some bacterial structures as well as some fatty acids, especially the eicosapentaenoic acid. [ABSTRACT FROM AUTHOR]

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. • Hemolymph and hepatopancreas transcriptomes of Portunus trituberculatus infected with Ameson portunus were analyzed. • Upregulation of immune-related genes suggests that microsporidian infection activates host immune responses. • Downregulation of metabolism-related genes affects host energy synthesis, resistance to pathogens, oxidative stress regulation. • Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. [ABSTRACT FROM AUTHOR]

Myeloid differentiation factor 88 (MyD88) is a universal and essential adaptor protein required for the Toll-like receptors (TLRs) pathway activation in invertebrates as well as in vertebrates. Herein, we characterized a MyD88 (Pt -MyD88) cDNA sequence in the swimming crab (Portunus trituberculatus). The Pt -MyD88 ORF is predicted to encode 469 peptides with an N-terminal death domain and a typical C-terminal TIR domain. Real-Time quantitative PCR analysis showed that the Pt -MyD88 transcriptions were constitutively expressed in hemocytes, gill, intestine, heart and muscle in normal crab. The expressions of Pt -MyD88 would be down-regulated by V. alginolyticus or LPS challenge, and be up-regulated by WSSV infection in hemocytes. Intracellular localization showed Pt -MyD88 was distributed mainly in the cytoplasm when it was over-expressed in human cell HEK293T or in Drosophila Schneider 2 (S2). Functionally, over-expression of Pt -MyD88 could either activate the NF-κB in HEK293T cells or activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. In primary cultured hemocytes of swimming crab, after Pt -MyD88 was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), hyastatin3, crustin1 and crustin3 have been significantly inhibited, while the expression of other AMPs is normal compared to non-specific dsRNA treated cells. • Full-length cDNAs of Pt -MyD88 was cloned from Portunus trituberculatus. • Pt -MyD88 transcriptions were reduced by V. alginolyticus or LPS challenge but induced upon WSSV infection. • Over-expressed Pt -MyD88 were distributed mainly in the cytoplasm in HEK293T cell or in S2 cell. • Pt -MyD88 could either activate the NF-κB in HEK293 cells or activate the promoters of Drosophila AMPs in S2 cell. • In Pt -MyD88 knock-down cells, the expression of ALF1, hyastatin3, crustin1 and crustin3 have been significantly inhibited. [ABSTRACT FROM AUTHOR]

• The study investigated the effect of exogenous estradiol on the lipid metabolism in hepatopancreas of Portunus trituberculatus. • Estradiol reduced the expression of genes related to lipid metabolism in hepatopancreas. • Estradiol treatments had higher percentages of 20:4n6, 20:5n3 and 22:6n3, but lower percentages of ∑MUFA and polar lipids than the control. • Estradiol improved the size of B cell compared to the control. Estradiol is an important sex steroid hormone that involved in regulation of animal lipid metabolism. However, the effect of estradiol on lipid metabolism in swimming crab (Portunus trituberculatus) is unclear. The present study investigated the effect of four concentrations of exogenous estradiol (0, 0.01, 0.1 and 1 μg g−1 crab weight) on the expression levels of lipid metabolism-related genes, lipid composition and histology of hepatopancreas in the P. trituberculatus. The results showed that the mRNA levels of carnitine palmitoyltransferase I and II (CPT-I and CPT-II) increased significantly at the low concentrations (0.01 μg g−1 and 0.1 μg g−1), while decreased significantly in the highest concentration (1 μg g−1). The mRNA levels of acyl-CoA oxidase (ACOX), fatty acid transport protein (FATP), fatty acid-binding protein (FABP), d iacylglycerol acyltransferase 1 (DGAT1) and acetyl-CoA carboxylase (ACC) were significantly down-regulated. The transcripts of fatty acid synthase (FAS) and fatty acyl desaturase (FAD) decreased significantly only in 1 μg g−1 treatment. All estradiol treatments (0.01, 0.1 and 1 μg g−1) had significantly higher percentages of 20:4n6, 20:5n3 and 22:6n3, but lower percentages of total monounsaturated fatty acids and polar lipids than the control treatment (0 μg g−1). Histological observations indicated the size of B cell became larger under estradiol treatment. The results indicated that estradiol promoted lipid catabolism in the hepatopancreas of P. trituberculatus. [ABSTRACT FROM AUTHOR]

Alpha-2 macroglobulin (A2M) is a ubiquitous protease inhibitor involved in the innate host defense system. Herein, two distinct A2M genes (designated as PtA2M-1 and PtA2M-2 , respectively) were isolated from the swimming crab Portunus trituberculatus. PtA2M-1 and PtA2M-2 encoded proteins with 1541 or 1516 amino acids, respectively, containing the typically functional domains of A2M. Unlike highly expressed in hemocytes of most arthropods, PtA2M-1 and PtA2M-2 were predominantly detected in gill, eyestalk and digestive tracks. During the embryonic stages, PtA2Ms were found to be expressed most highly in fertilized eggs, suggesting their maternal origin. After challenged with Vibrio alginolyticus , the transcripts of PtA2Ms showed similar time-dependent response expression pattern, while PtA2M-1 was more sensitive to Micrococcus luteus and Pichia pastoris infection than PtA2M-2. Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), however, PtLSZ and the phagocytosis-related genes (PtMyosin and PtRab5) were effectively inhibited. These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1- or PtA2M-2-silenced crabs. In addition, PtA2M-1 and PtA2M-2 could regulate the expression of antimicrobial peptide (AMP) genes (PtALF1-3 , PtCrustin1 and PtCrustin3) through the Toll and NF-κB pathways. Our findings together suggest that PtA2Ms might function in crab host defense via regulating the proPO system, phagocytosis and the expression of AMP genes. • Two alpha-2 macroglobulin genes were isolated from Portunus trituberculatus. • Maternal mRNAs of PtA2Ms were highly expressed in fertilized eggs. • The expression of PtA2Ms was induced by bacteria or fungi. • PtA2Ms were involved in the proPO-activating system and phagocytosis. • PtA2Ms regulated the expression of AMP genes through the Toll and NF-κB pathways. [ABSTRACT FROM AUTHOR]

Mannose-binding lectin (MBL) is a pattern recognition receptor (PRR) that plays an important role in the innate immune response. In this study, a novel mannose-binding lectin was cloned from the swimmimg crab Portunus trituberculatus (designated as PtMBL). The complete cDNA of PtMBL gene was 1208 bp in length with an open reading frame (ORF) of 732 bp that encoded 244 amino acid proteins. PtMBL shared lower amino acid similarity with other MBLs, yet it contained the conserved carbohydrate-recognition domain (CRD) with QPD motif and was clearly member of the collectin family. PtMBL transcripts were mainly detected in eyestalk and gill with sexually dimorphic expression. The temporal expression of PtMBL in hemocytes showed different activation times after challenged with Vibrio alginolyticus , Micrococcus luteus and Pichia pastoris. The recombinant PtMBL protein revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria. It could also bind and agglutinate (Ca2+-dependent) both bacteria and yeast. Furthermore, the agglutinating activity could be inhibited by both d -galactose and d -mannose, suggesting the broader pathogen-associated molecular patterns (PAMPs) recognition spectrum of PtMBL. These results together indicate that PtMBL could serve as not only a PRR in immune recognition but also a potential antibacterial protein in the innate immune response of crab. • PtMBL contained the conserved carbohydrate-recognition domain with QPD motif. • PtMBL transcripts could be quickly induced by bacterial challenge. • The recombinant PtMBL exhibited antimicrobial activity against the tested bacteria. • The rPtMBL could bind and agglutinate (Ca2+-dependent) both bacteria and yeast. • The rPtMBL could not only bind d -galactose but also d -mannose. [ABSTRACT FROM AUTHOR]

Clip domain serine proteases (cSPs), a family of multifunctional proteins, play a crucial role in innate immune system. Here, we report the functional characterization of two clip domain serine proteases (PtcSP1 and PtcSP3) from the swimming crab Portunus trituberculatus. The recombinant N-terminal clip domains and the C-terminal SP-like domains of PtcSP1 and PtcSP3 were expressed in Escherichia coli system, and assayed for various biological functions: protease activity, antimicrobial activity, bacterial clearance and microbial-binding activity. The recombinant SP-like domains of PtcSP1 and PtcSP3 exhibited trypsin-like protease activity, while their recombinant clip domains showed strong antibacterial activity and could bind to bacteria and yeast, suggesting the potential roles of PtcSP1 and PtcSP3 in immune defense and pattern recognition. Unlike PtcSP3, PtcSP1 revealed the opsonic activity as shown by a higher bacterial clearance rate of Vibrio alginolyticus coated with the combination of the recombinant clip domain and SP-like domain of PtcSP1 as compared with V. alginolyticus only. Knockdown of PtcSP1 or PtcSP3 by RNA interference resulted in a significant decrease of total phenoloxidase (PO) activity in crab, suggesting that PtcSP1 and PtcSP3 are involved in the proPO system. In addition, suppression of PtcSP1 or PtcSP3 changed the expression of PtALFs and complement-like components. All these findings suggest that PtcSP1 and PtcSP3 are multifunctional immune molecules and perform different protective functions in crab defense. • PtcSP1 and PtcSP3 are two trypsin-like serine proteases. • The clip domain of PtcSP1 and PtcSP3 had strong antibacterial and microbial-binding activity. • PtcSP1 functioned as an opsonin. • Suppression of PtcSP1 or PtcSP3 transcript reduce the total PO activity in crab. • Knockdown of PtcSP1 or PtcSP3 could significantly change the expression levels of complement-like factors and AMPs. [ABSTRACT FROM AUTHOR]

Abstract The receptor for the globular head of complement component C1q, gC1qR, is a multifunctional and multiligand binding protein with a crucial role in host defense. In the present study, a full-length cDNA sequence of a gC1qR homolog (PtgC1qR) in Portunus trituberculatus was identified. PtgC1qR was a 268-amino-acid polypeptide with a conserved MAM33 domain and a mitochondrial targeting sequence in the first 56 amino acids. The transcripts of PtgC1qR were detected in all examined tissues with the highest level detected in the hepatopancreas. Compared with other early embryonic stages, PtgC1qR was highly expressed in the fertilized eggs and embryos at the cleavage stage, which suggest PtgC1qR may be a maternal gene. The transcripts of PtgC1qR in hemocytes exhibited time-dependent response expression pattern after challenged with bacteria (Vibrio alginolyticus , Micrococcus luteus) and fungi (Pichia pastoris). Moreover, the recombinant PtgC1qR (rPtgC1qR) exhibited strong antibacterial activity and microbial-binding activity, suggesting its crucial role in immune defense and recognition. Further phenoloxidase (PO) assay showed that rPtgC1qR could suppress the crab PO activity in vitro in a dose-dependent manner, and it could result in nearly 100% inhibition of PO activity under the concentration of 11.65 μM. Knockdown of PtgC1qR could significantly enhance the expression of serine protease related genes (PtSP1 - 3 and PtSPH), proPO-associated genes (PtproPO and PtPPAF) and C3-like genes (Ptα2M1 and PtTEP). However, the phagocytosis related genes (PtMyosin , PtRab5 and PtArp) and Ptα2M2 were significantly down-regulated in the PtgC1qR silenced crabs. These findings together demonstrate that PtgC1qR might function in crab immune response via its antibacterial activity, immune recognition or regulating the proPO system, complement pathway and phagocytosis. Highlights • A gC1qR homolog was isolated from Portunus trituberculatus. • PtgC1qR expression was induced by bacteria or fungi. • PtgC1qR exhibited antibacterial activity and microbial binding activity. • PtgC1qR suppressed the crab PO activity in vitro. • PtgC1qR regulates the expression of phagocytosis related and C3-like genes. [ABSTRACT FROM AUTHOR]

The housekeeping gene encoding β-actin appears to be the most widely-used internal reference for gene expression studies in experimental animals or their cell lines. However, the effectiveness of β-actin to normalize mRNA levels expression in many crustacean species is still object of debate. To date, it is still unclear if β-actin is suitable to be utilized as the internal reference in qualitative real-time gene expression study in crab species. To address this concern, we evaluated 5 candidate reference genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, elongation factor 1-α (EF1-α), and 18 S ribosomal RNA (18 S rRNA) in the swimming crabs ( Portunus trituberculatu s) models. Our data showed that the β-actin gene expression varied significantly across individual swimming crab individuals in gills or hemocytes and the expression of 18 S rRNA, EF1-α, cyclophilin or GAPDH gene were relatively stable compared to that of β-actin. Moreover, the expression stability of the reference genes among different tissues in normal crabs or after WSSV challenge was also tested by geNorm and NormFinder software. Among tissues, 18 S rRNA was most stably expressed in different tissues, followed by cyclophilin A and EF1-α, compared to β-actin and GAPDH. Upon to viral simulation, GAPDH was found to be the most stable internal control gene in gills and cyclophilin A was ranked as the most stable gene in hemocytes. [ABSTRACT FROM AUTHOR]

C-type lectin plays an important role in the innate immune response of crustaceans including Portunus trituberculatus which is an important marine species. In the present study, we cloned the full length of a C-type lectin (designated as PtCTL4) from P. trituberculatus via 3′RACE. The full length of the nucleic acid sequence has a length of 654 bp including an open reading frame (ORF) of 480 bp. PtCTL4 possesses conserved CTL features, while containing a CRD domain with Ca 2+ binding site 2 and six conserved cysteine residues. Quantitative RT-PCR analysis showed that PtCTL4 expression level was highest in the hepatopancreas, while it was relatively low in other tissues such as hemocytes, eyestalk, muscle, and gonad. The expression level of PtCTL4 reached a maximum at 3 h after challenge with Vibrio alginolyticus , then decreased to the lowest level at 12 h, and returned to normal level at 48 h. Hemagglutination analysis showed that the recombinant PtCTL4 (rPtCTL4) can agglutinate rabbit erythrocyte. The rPtCTL4 can agglutinate Gram-positive bacteria ( Bacillus aquimaris , Micrococcus lysodeik , and Staphylococcus aureus ) and Gram-negative bacteria ( Aeromonas hydrophila , V. alginolyticus , and Chryseobacterium indologenes ) in the presence of Ca 2+ . This study indicated that PtCTL4 acts as a pattern recognition receptor in the innate immune response of P. trituberculatus. [ABSTRACT FROM AUTHOR]

The swimming crab Portunus trituberculatus is one of the most popular seafoods in South Korea. Despite yearly reports of foodborne illness caused by swimming crab consumption, the microbiota in the whole body of the crab has not been fully characterized. A total of 60 crabs were collected from different sites in the Yellow Sea in the spring and autumn, and the crab microbiota was analyzed by 16S rRNA pyrosequencing. Crab microbial diversity was higher in the autumn than in the spring. Psychrobacter, Vagococcus, Carnobacterium, Lactococcus, and Streptococcus were dominant genera in the spring, whereas Roseovarius and various other genera were dominant in the autumn. The composition of the microbiota differed significantly between spring and autumn (p < 0.01). The relative abundances of potential pathogens, including Lactococcus garvieae, in crabs were higher in spring than in autumn. Thus, the potential risk of foodborne illness by crab intake could be higher in spring than autumn. This study extends our knowledge of potential foodborne illness according to microbiota and may improve the management of crab products. [ABSTRACT FROM AUTHOR]

C-type lectins are a superfamily of Ca 2+ -dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including nonself-recognition and pathogen elimination. In the present study, two single-CRD containing C-type lectins were identified from swimming crab Portunus trituberculatus (designated as PtCTL-2 and PtCTL-3). The open reading frame (ORF) of PtCTL-2 encoded polypeptides of 485 amino acids with a signal peptide and a single carbohydrate-recognition domain (CRD), while PtCTL-3's ORF encoded polypeptides of 241 amino acids with a coiled-coil region and a single-CRD. The key motifs determining carbohydrate binding specificity in PtCTL-2 and PtCTL-3 were EPR (Glu-Pro-Arg) and QPD (Gln-Pro-Asp). EPR is a motif being identified for the first time, whereas QPD is a typical motif in C-type lectins. Different PAMPs binding features of the two recombinant proteins - PtCTL-2 (rPtCTL-2) and PtCTL-3 (rPtCTL-3) have been observed in our experiments. rPtCTL-2 could bind three pathogen-associated molecular patterns (PAMPs) with relatively high affinity, including glucan, lipopolysaccharide (LPS) and peptidoglycan (PGN), while rPtCTL-3 could barely bind any of them. However, rPtCTL-2 could bind seven kinds of microbes and rPtCTL-3 could bind six kinds in microbe binding assay. Moreover, rPtCTL-2 and rPtCTL-3 exhibited similar agglutination activity against Gram-positive bacteria, Gram-negative bacteria and fungi in agglutination assay. All these results illustrated that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) with broad nonself-recognition spectrum involved in immune defense against invaders. In addition, the results of carbohydrate binding specificity showed that PtCTL-2 with novel key motif had broad carbohydrate binding specificity, while PtCTL-3 with typical key motif possessed different carbohydrate binding specificity from the classical binding rule. Furthermore, PtCTL-2 and PtCTL-3 could also function as opsonin to enhance encapsulation of hemocytes against Ni-NTA beads. [ABSTRACT FROM AUTHOR]

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 80 mg/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 20 mg/kg. Additionally, higher FLR doses caused significant biomolecule damage during the first 48 h after delivery. This study will provide an improved understanding of the exact mechanism underlying toxicity in aquatic organisms. [ABSTRACT FROM AUTHOR]

Clip domain serine proteases (clip-SPs) play critical roles in various immune responses in arthropods, such as hemolymph coagulation, antimicrobial peptide (AMP) synthesis, cell adhesion and melanization. In the present study, we report the molecular and functional characterization of a clip domain serine protease (PtcSP2) from the swimming crab Portunus trituberculatus . The N-terminal clip domain and the C-terminal SP-like domain of PtcSP2 were expressed in Escherichia coli system, and assayed for their activities. Sequence similarity and phylogenetic analysis revealed that PtcSP2 may belong to the chymotrypsin family, which was confirmed by protease activity assay of the recombinant SP-like domain. The clip domain of PtcSP2 exhibited strong antibacterial activity and microbial-binding activity, suggesting the potential role in immune defense and recognition. Knockdown of PtcSP2 by RNA interference could significantly reduce PtcSP2 transcript levels, but neither decrease the total phenoloxidase (PO) activity in crab nor significantly alter the expression levels of serine protease inhibitors PtPLC and PtSerpin. These results indicate that PtcSP2 is not involved in the proPO system. However, suppression of PtcSP2 led to a significant change in the expression of AMP genes PtALFs and PtCrustin but not PtALF5. All these findings suggest that PtcSP2 is a multifunctional chymotrypsin-like serine protease and may participate in crab innate immunity by its antibacterial activity, immune recognition or regulation of AMP expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a novel C-type lectin (designated as PtCTL1) was identified and characterized from Portunus trituberculatus . The full-length cDNA of PtCTL1 was of 702 bp, containing a 5′ untranslated region (UTR) of 91 bp, a 3′ UTR of 110 bp with a poly (A) tail, and an open reading frame (ORF) of 501 bp encoding a polypeptide of 166 amino acids with a putative signaling peptide of 21 amino acids. A C-type lectin carbohydrate-recognition domain (CRD) containing four conserved cysteines was identified in the amino acid sequence of PtCTL1. The cDNA fragment encoding the mature peptide of PtCTL1 was recombined into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame and expressed in Escherichia coli Origami (DE3). The recombinant PtCTL1 (rPtCTL1) can agglutinate all the tested bacteria, including three Gram-positive bacterial strains and three Gram-negative bacterial strains. In addition, erythrocyte agglutination and LPS-binding activity were observed in a Ca 2+ -dependent manner. The erythrocyte agglutination was inhibited by EDTA, indicating that PtCTL1 was Ca 2+ -dependent. The mRNA transcripts of PtCTL1 were detected mainly in the tissues of hepatopancreas and hemocytes and its levels were significantly up-regulated in hemocytes following Vibrio alginolyticus challenge. These results indicate that PtCTL1 may function as a pattern recognition receptor (PRR) for protecting P. trituberculatus from bacterial infection. Moreover, such findings also provide evidence for further understanding the innate immunology of invertebrate. [ABSTRACT FROM AUTHOR]

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 , α 2 M , 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 α 2 M 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. [ABSTRACT FROM AUTHOR]

Heat shock protein 40 (HSP40) is a kind of molecular chaperone involved in various immune responses. However, the exact roles of HSP40 in immune defense against bacteria remain largely unclear. In this study, the activation function of a type Ⅰ HSP40 from Portunus trituberculatus (PtHSP40-Ⅰ) in the TLR pathway was investigated. The results showed that PtHSP40-Ⅰ can bind to lipopolysaccharide (LPS) and peptidoglycan (PGN). The PtHSP40-Ⅰ also exhibited binding activity toward the extracellular leucine-rich repeat (LRR) domain of Toll-like receptor (TLR). Moreover, the PtHSP40-Ⅰ could promote the transcription factor Dorsal translocated from cytoplasm into the nucleus in hemocytes and participated in regulating the expression of anti-lipopolysaccharide factor (ALF) and crustin. These findings provided insights into the activation mechanisms of TLR pathway mediated by HSP40 and offered basic theory of disease control in P. trituberculatus aquaculture. • PtHSP40-Ⅰ could bind to lipopolysaccharide and peptidoglycan. • PtHSP40-Ⅰ could bind to the leucine-rich repeat domain of the Toll-like receptor. • PtHSP40-Ⅰ promoted PtDorsal translocated from cytoplasm into nucleus. • PtHSP40-Ⅰ regulated the expression of anti-lipopolysaccharide factor and crustin. [ABSTRACT FROM AUTHOR]

Class B scavenger receptors (SRBs), which are present in mammals and insects, have been implicated in a wide range of functions. Herein, a novel SRB homologue, Pt SRB, was cloned from the swimming crab, Portunus trituberculatus . Pt SRB has 538 amino acid residues, and it consists of two transmembrane regions, a large extracellular loop, and two intracellular tails. A phylogenetic analysis showed that Pt SRB distinctly clustered with Marsupenaeus japonicas SRB-1 and most Drosophila SRB homologues, including Croquemort, Peste, NinaD, and Santa Maria, but was separate from the Drosophila sensory neuron membrane protein, Mj SRB-2, and all vertebrate SRBs. Real-time quantitative PCR analyses showed that the Pt SRB gene was constitutively expressed in all tissues tested. When Pt SRB was overexpressed in human embryonic kidney 293T cells, it was distributed in the membrane and cytoplasm. Moreover, in vitro assays showed that r Pt SRB bound microbial lipopolysaccharide with low affinity, and lipoteichoic acid and peptidoglycan with high affinity. Pt SRB transcripts were down-regulated after challenge with Vibrio alginolyticus or white spot syndrome virus, but not after a Candida lusitaniae challenge. This study provides valuable data for understanding the role of SRBs in the host defense against microbial pathogens, which will facilitate future studies of host–pathogen interactions in crabs. [ABSTRACT FROM AUTHOR]

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 α 2 -macroglobulin (α 2 -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 α 2 -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. [ABSTRACT FROM AUTHOR]

The Toll signaling pathway is one of the most important regulators of the immune response in both vertebrates and invertebrates. Herein, three novel Toll (PtToll1–3) cDNA sequences were cloned from the swimming crab, Portunus trituberculatus . PtToll1 has 1003 amino acid residues and consists of an extracellular domain containing 15 leucine-rich repeats (LRRs) and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain of 139 residues. PtToll2 encodes 1196 peptides, with an extracellular domain containing 28 LRRs and a cytoplasmic TIR domain. PtToll3 is 1229 residues long and contains 26 LRRs and a cytoplasmic TIR domain. Based on sequence and phylogenetic analyses, PtToll1 distinctly clustered with almost all crustacean Tolls, except Litopenaeus vannamei Toll3. However, PtToll2 and PtToll3 were separated from most reported crustacean Tolls, which mostly clustered with Drosophila melanogaster (Dm) Toll8, L. vannamei Toll3, and DmToll6. Reverse transcription PCR and real-time quantitative PCR analyses showed that PtToll1 and PtToll3 were constitutively expressed in all tissues tested, but PtToll2 mRNA was only highly enriched in gills. Upon challenges with Vibrio alginolyticus , Candida lusitaniae , or white spot syndrome virus (WSSV), the three Tolls exhibited different responses: the PtToll1 transcript was up-regulated in response to C. lusitaniae or V. alginolyticus challenge, but did not respond to WSSV challenge; both PtToll2 and PtToll3 mRNAs were down-regulated 12 h after C. lusitaniae or V. alginolyticus infection. However, WSSV elicited the expression of PtToll2 at 6 h post-infection, but suppressed transcription of PtToll3 at 24 h post-infection. The study provides valuable data for understanding the role of Toll pathways in the host defense against microbial pathogens, which will facilitate future studies on host–pathogen interactions in crabs. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a cytoplasmic adapter protein that mediates signals induced by the tumor necrosis factor receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R). In the present study, the full-length cDNA of TRAF6 (Pt-TRAF6) was identified in a marine crab, Portunus trituberculatus . Pt-TRAF6 ORF is predicted to encode a 599-amino acid protein, including a RING type zinc finger, two TRAF-type zinc fingers, and a meprin and TRAF homology (MATH) domain. The overall amino acid sequence identity between Pt-TRAF6 and other TRAF6s ranged from 50.9 to 51.3% for shrimp and from 16.1 to 19.4% for insects. The Pt-TRAF6 gene contains six exons and five introns, which is different from the organization of the insect TRAF6 gene. Pt-TRAF6 transcripts were broadly expressed in all tissues tested, and their expression was higher in hemocytes, gills, the intestine, and heart than in muscle. Interestingly, the level of Pt-TRAF6 transcript differed between male and female crabs. After Vibrio alginolyticus or lipopolysaccharide (LPS) challenge, the Pt-TRAF6 transcript was down-regulated in hemocytes and up-regulated in gills. Moreover, Pt-TRAF6 expression was altered sooner in the LPS challenge group than in the V. alginolyticus challenge group. These results indicate that Pt-TRAF6 may respond to Gram-negative bacterial infections. [ABSTRACT FROM AUTHOR]

Pacifastin-related inhibitor is a new family of serine protease inhibitors that regulate the proteolytic cascade in multiple biological processes. Contrary to the knowledge on the structure and inhibitory mechanism of pacifastin-like members in locust, very little is known about their functions. Here, we report the inhibitory activities in relation to the structural characteristics of pacifastin light chain (PtPLC) gene identified from the swimming crab Portunus trituberculatus . The mature PtPLC and five PLD-related domains with critical residues were expressed in Escherichia coli , and assayed for their activities. The recombinant PtPLC (rPtPLC) displayed inhibitory activities against trypsin and chymotrypsin in a dose dependent manner, with a preference for trypsin. Except for rPtPLC-D4, the other four rPtPLC-related domains could inhibit at least one of serine proteases. The enzyme specificity of PtPLC domains generally corresponded to the nature of the P1 residue at the reactive site. rPtPLC was able to inhibit the growth of Gram-negative bacteria Vibrio alginolyticus and Pseudomonas aeruginosa , but not the Gram-positive bacterium and fungus tested. Further phenoloxidase (PO) assay showed the rPtPLC could depress the crab proPO system activation in vitro , and lead to 72.8% inhibition of PO activity at the concentration of 9.11 μM. It also suppressed proPO activation induced by rPtcSP and rPtSPH1. As the first functional study of the recombinant PLC protein in crustaceans, the present results together indicate that PtPLC functions in the crab immune response possibly via inhibiting bacterial growth and regulating the proPO system. [ABSTRACT FROM AUTHOR]

In order to determine the effects of different short - term low salinity stress on non - volatile flavor substances of Portunus trituberculatus , four salinity levels (24‰, 16‰, 12‰ and 8‰) were selected. The contents of free amino acids (FAA), nucleotides and lactic acid in both muscle and hepatopancreas of P. trituberculatus were determined by HPLC and spectrophotometry respectively. Results showed that muscle contained a relatively higher level of sweetish free amino acids (SFA), however short - term low salinity stress greatly increased the level of bitter free amino acid (BFA); hepatopancreas contained a higher level of umami free amino acids (UFA) and a higher equivalent umami concentration (EUC) value, which increased significantly with the decrease of salinity level and the degree of umami flavor reached the highest when salinity dropped to 8‰. The lactic acid content in muscle increased at 12‰ and then decreased significantly at 8‰ while no significant differences were observed in hepatopancreas. Results above revealed the flavor change in two edible parts of P.trituberculatus under short - term low salinity stress which provided a guidance for more systematic nutritional flavor evaluation in breeding process and consumption demand. • Short-term low salinity stress affects the contents of non-volatile flavor substances. • 2. Umami feature index in hepatopancreas reached the highest when salinity 8‰. • Short-term low salinity stress stimulated bitter feature in muscle tissue. [ABSTRACT FROM AUTHOR]

Serpin or serine protease inhibitor is the largest family of protease inhibitors involved in many innate immune pathways, particularly the prophenoloxidase (proPO) activating system in arthropod. Here, we report the molecular and functional characterization of PtSerpin identified from the swimming crab Portunus trituberculatus. The genomic sequence encoding mature peptide of PtSerpin gene contained two exons of 84 and 1098 bp separated by one intron of 111 bp. The recombinant PtSerpin (rPtSerpin) with a predicted size of 44 kDa was expressed in Escherichia coli system, purified and assayed for its activities. The rPtSerpin exhibited inhibitory activity against trypsin in a dose-dependent manner, but did not affect chymotrypsin, which could define a role for PtSerpin as a trypsin inhibitor. The rPtSerpin could inhibit the growth of Gram-negative bacterium Vibrio alginolyticus, but not the tested Gram-positive bacterium and fungus. Further phenoloxidase (PO) assay showed PO activity was dramatically increased in hemocyte lysate supernatant of P. trituberculatus upon bacterial challenge. The rPtSerpin could depress the crab proPO system activation in vitro, and it could lead to 100% inhibition of PO activity under the concentration of 8.62 µM. Moreover, the rPtSerpin was able to inhibit the PO activity induced by rPtcSP and rPtSPH1. These results together indicate that PtSerpin is a potential trypsin inhibitor and may participate in crab innate immunity by the inhibition of bacterial growth and the regulation of proPO system. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is one of the typical metal pollutants in the Bohai Sea. To evaluate the acute toxicological effects of Cd on marine crustaceans, juvenile Fenneropenaeus chinensis and Portunus trituberculatus were exposed to Cd at environmentally relevant concentrations (5 and 50 μg/L) for 96 h. Cd accumulation, antioxidants and metabolite profiles were characterized to elucidate the responses of juvenile crustaceans to Cd stress. Significant Cd accumulation was observed in both juvenile crustaceans in 50 μg/L Cd-treated group. Results showed that Cd exposure induced hormesis based on the alterations of GSH, SOD and CAT activities (i.e. increased levels in the low concentration of Cd treatment and recovered levels in the high concentration of Cd treatment) in juvenile P. trituberculatus. Similarly, the responses of GSH contents presented hormesis pattern in Cd-treated juvenile F. chinensis. Na+-K+-ATPase contents were significantly elevated in 50 μg/L Cd-treated group. In addition, untargeted NMR-based metabolomics indicated Cd caused the disturbance in osmotic regulation and energy consumption in both juvenile F. chinensis and P. trituberculatus via different pathways. The immunotoxicity and movement disorder were uniquely demonstrated in juvenile P. trituberculatus after Cd exposure. [Display omitted] ● GSH, SOD and CAT presented typical hormetic curves in juvenile P. trituberculatus. ● GSH also presented hormesis pattern in juvenile F. chinensis. ● Cd disturbed osmotic regulation and energy consumption in juvenile crustaceans. ● Cd potentially induced immunotoxicity and movement disorder in juvenile crabs. [ABSTRACT FROM AUTHOR]

Abstract: Serine proteases (SPs), with their homologues (SPHs), a family of multifunctional proteins, play a crucial role in innate immune system. In our present study, we made an appropriate correction: serine protease homologue PtcSPH (Li et al., [1]) obtained from the swimming crab Portunus trituberculatus was actually a serine protease and re-designated as PtcSP. Sequence analysis revealed PtcSP and PtSP (Li et al., [2]) might be encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Eight exons were identified in genomic DNA sequence of PtcSP. A comprehensive phylogenetic analysis was made combined with our previous reports (Cui et al., [3]; Li et al., [1,2]). The result showed SPs and SPHs of P. trituberculatus had different origins in gene evolution. To further characterize the function(s) of proteins, the recombinant serine proteases or homologues were assayed for various biological functions: proteinase activity, antimicrobial activity and microorganisms binding activity. The recombinant protein PtcSP exhibited trypsin-like protease activity and antibacterial activity. PtSPH1 (Li et al., [2]) lacked proteolytic activity but displayed binding activity to yeast and the crab pathogenic bacterium, Vibrio alginolyticus. Further, the N-terminal clip domain of PtcSP had antibacterial activity and the C-terminal SP-like domain had trypsin-like protease activity. [Copyright &y& Elsevier]

Abstract: Anti-lipopolysaccharide factor (ALF) is an important antimicrobial peptide (AMP) that can bind and neutralize major component of Gram-negative bacteria cell wall, lipopolysaccharide (LPS). Seven isoforms of anti-lipopolysaccharide factors (PtALF1-7) were previously identified from the swimming crab Portunus trituberculatus in our laboratory. Here, polymorphisms of PtALF1-7 were detected and their association with resistance/susceptibility to Vibrio alginolyticus (a main Gram-negative bacteria causing high mortality in P. trituberculatus) were investigated. We identified 127, 96, 103, 53 and 158 single nucleotide polymorphisms (SNPs) in genomic fragments of PtALF1-3, PtALF4, PtALF5, PtALF6 and PtALF7, respectively. Among them, totally sixteen SNPs were significantly associated with resistance/susceptibility to V. alginolyticus (P < 0.05). Of these sixteen SNPs, most were located in introns and noncoding exons, while two synonymous SNPs and one nonsynonymous SNP were in coding exons. Additionally, simple sequence repeats (SSRs) were only identified in introns and noncoding exons of PtALF4, PtALF5 and PtALF7. Although no significant difference of allele frequencies was found, these SSRs had different polymorphic alleles according to the repeat number between susceptible and resistant stocks. After further confirmation, polymorphisms investigated here might be applied as potential molecular markers for future selection of resistant strains to diseases caused by Gram-negative bacteria. [Copyright &y& Elsevier]

Abstract: Anti-lipopolysaccharide factors (ALFs), exhibiting binding and neutralizing activities to lipopolysaccharide (LPS), are the potent antimicrobial peptides of innate immunity in crustaceans. In this study, a unique isoform of ALF (PtALF6) was identified from eyestalk cDNA library of the swimming crab Portunus trituberculatus. The full-length cDNA of PtALF6 was 669 bp encoding 115 amino acids, relatively short to other known ALFs. The deduced peptide of PtALF6 was conserved; it contained the signal peptide and LPS-binding domain, especially the two conserved cysteine residues at both ends of the domain. Predicted tertiary structures of PtALF6 containing four β-strands and three α-helices were similar to that described in Limulus polyphemus. The genomic fragment of PtALF6 contained three exons separated by two introns. Unlike most ALFs expressed in hemocytes, PtALF6 transcript was predominantly detected in gill with 14.05-fold higher than that in hemocytes. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF6 transcript in hemocytes showed a clear time-dependent response expression pattern with two significant peaks at 12 h and 32 h post-injection. The recombinant PtALF6 protein revealed antimicrobial activity against the test Gram-negative and Gram-positive bacteria, but did not inhibit the growth of fungus Pichia pastoris. These results together indicate that PtALF6 is a potential antimicrobial protein against Gram-negative and Gram-positive bacteria infection, and may play an important role in innate immune response of P. trituberculatus. [Copyright &y& Elsevier]

Abstract: Anti-lipopolysaccharide factors (ALFs), as the potent antimicrobial peptides, are becoming predominant candidates for potential therapeutic agents of bacterial and viral diseases. In this study, a unique isoform of ALF (PtALF7) was identified from hemocytes cDNA library of the swimming crab Portunus trituberculatus. The PtALF7 cDNA contained an open reading frame (ORF) of 372 bp encoding 123 amino acids. The deduced peptide of PtALF7 shared high similarity with our previously reported PtALF1–3 but low with PtALF4–6. The PtALF7 gene consisted of three exons interrupted by two introns, and was clearly transcribed from different genomic loci compared with other PtALF isoforms. Totally 128 SNPs including 12 in coding region and 116 in noncoding region were detected in PtALF7 gene by direct sequencing of 20 samples. The mRNA expression of PtALF7 transcript was primarily observed in hemocytes followed by gill and eyestalk, but barely detectable in hepatopancreas. After challenge with Vibrio alginolyticus, a main pathogen causing high mortality in P. trituberculatus, the PtALF7 transcript in hemocytes showed a clear time-dependent response expression pattern with obvious decrease at 6 h and significant increase at 24 h. The recombinant PtALF7 protein exhibited antimicrobial activity against the test Gram-negative and Gram-positive bacteria, but did not inhibit the growth of fungus Pichia pastoris. These results together indicate a potential involvement for PtALF7 in the innate immune response of P. trituberculatus. [Copyright &y& Elsevier]

Abstract: Crustins are cationic, cysteine-rich antimicrobial proteins, containing a single whey acidic protein (WAP) domain in the C-terminal end. Different from the reported Ptcrustin in the hemocytes, two novel crustin genes (PtCrustin2 and PtCrustin3) were cloned completely from the eyestalk cDNA library of Portunus trituberculatus in this study. All PtCrustins share the consensus cysteine motif and are considered as Type I crustins. Four exons and three introns are identified in genomic DNA sequence of PtCrustin3 while three exons and two introns in PtCrustin2. The mRNA transcripts of PtCrustin2 and PtCrustin3 are mainly detected in eyestalk and gills, but not in hemocytes. Although both PtCrutins are up-regulated after challenge of three microorganisms, PtCrustin3 seems to respond more quickly to microbial challenge than Ptcrustin2. Unlike most crustins, both recombinant PtCrustin2 and PtCrustin3 exhibit antibacterial activity against Gram-positive bacteria Micrococcus luteus and Staphyloccocus aureus and Gram-negative bacterium Pseudomonas aeruginosa. In addition, rPtCrustin2 is moderately active against yeast Pichia pastoris and rPtCrustin3 show significant activity against Gram-negative bacterium Vibrio alginolyticus. These results indicate that PtCrustin2 and PtCrustin3 are two novel crustins and play different roles in immune response of P. trituberculatus against microbial challenge. [Copyright &y& Elsevier]

Abstract: A new isoform of anti-lipopolysaccharide factor (PtALF5) was cloned from eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA of PtALF5 was 1045 bp encoding 120 amino acids. PtALF5 shared lower amino acid similarity with other ALFs, yet it contained the conserved LPS-binding domain and was clearly member of the ALF family. The genomic fragment of PtALF5 contained two exons separated by one intron. Several tandem repeats were found in intron. The mRNA transcript of PtALF5 was predominantly expressed in the hemocytes but barely detectable in muscle. After challenge with Vibrio alginolyticus, a main pathogen causing high mortality in P. trituberculatus, the PtALF5 transcript in hemocytes showed a clear time-dependent response expression pattern with obvious decrease at 6 h and significant increase at 24 h. The recombinant PtALF5 protein revealed antimicrobial activity against Gram-negative bacteria V. alginolyticus and Pseudomonas aeruginosa, but did not inhibit the growth of the tested Gram-positive bacteria and fungus. These results together suggest that PtALF5 is a potent antibacterial protein against Gram-negative bacteria infection, and might function as a promising therapeutic agent in disease control of crab aquaculture. [Copyright &y& Elsevier]

Abstract: Previously, we had reported two homologues of the thioredoxin (Trx) super-family (PtTrx1 and PtTrx2) identified from eyestalk and haemocytes cDNA library of swimming crab Portunus trituberculatus, respectively. It was the first report of two thioredoxin homologues from the same crustacean species. Here, we focused on the molecular characterization, genomic organization and expression pattern of PtTrx1 and PtTrx2. The full-length cDNA sequences of PtTrx1 and PtTrx2 were 739 and 1300 bp, encoding 105 and 133 amino acids, respectively. They both had a conserved CGPC active site and highly similar tertiary structures, which containing four β-sheets and four α-helixes. Specifically, PtTrx2 was encoded by a nuclear gene and its cellular localization was targeted to mitochondria by an N-terminal mitochondrial pre-sequence. Sequence analysis revealed PtTrx1 and PtTrx2 were encoded by different genomic locus. As the first analyzed genomic structure of PtTrxs in crustaceans, two introns with microsatellites were found in the open reading frame region of these genes. Quantitative real-time PCR analysis revealed the mRNA expression of PtTrx1 transcripts were mainly detected in gill, while, PtTrx2 in eyestalk and gill. The temporal expression levels of PtTrxs transcripts in haemocytes showed different expression patterns after challenge with Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. These results together indicate that PtTrxs should be involved in the responses to pathogen challenge of P. trituberculatus. [Copyright &y& Elsevier]

Abstract: Anti-lipopolysaccharide factors (ALFs) are the potent antimicrobial peptides that can bind and neutralize lipopolysaccharide (LPS). In this study, a new isoform of the ALF homologs (PtALF4) was cloned from eyestalk cDNA library of swimming crab Portunus trituberculatus. PtALF4 shared lower amino acid similarity with other ALFs, yet it contained the conserved LPS-binding domain and was clearly member of the ALF family. The genomic sequence of PtALF4 consisted of three exons interrupted by two introns. Several tandem repeats were found in both introns. Unlike most ALFs expressed in hemocytes, PtALF4 transcript was predominantly detected in eyestalk. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF4 transcript in hemocytes showed a clear time-dependent response expression pattern with two significant peaks. The recombinant proteins of PtALF1, PtALF3 and PtALF4 revealed different antimicrobial activities against bacteria or fungus. These results together suggest that PtALF isoforms might be potent immune effectors to provide multiple protective functions against invading bacteria or fungus in P. trituberculatus. [Copyright &y& Elsevier]

Abstract: In our previous studies, five serine proteases containing clip domain were characterized from the swimming crab Portunus trituberculatus. To further investigate the characterization and function of serine proteases, one serine protease (PtSP) and one serine protease homolog (PtSPH1) without clip domain were identified from haemocytes cDNA library in this paper. They both possessed an SP or SP-like domain at the C-terminal. In contrast to PtSP, absence of Ser catalytic residue resulted in the loss of serine protease activity of PtSPH1. Phylogenetic analysis suggested either SPs or SPHs might not have a single origin in gene evolution. Six introns presented in PtSP genomic DNA with one uncommon splice site (GG) was discovered at exon 1/intron 1 boundary region. Four introns with common splice sites were found in PtSPH1 genomic DNA. RT-PCR results showed that PtSP mRNA was mainly distributed in haemocytes, gill and eyestalk, whereas PtSPH1 transcript was mainly expressed in stomach. PtSP showed slight increase during the first 48 h compared to control groups except 8 h point after Micrococcus luteus challenge. However, significant up-regulation was observed in the expression level of PtSPH1 challenged by Gram-negative bacteria Vibrio alginolyticus, Gram-positive bacteria M. luteus and fungi Pichia pastoris during the first 48 h. It indicates that PtSPH1 might be more sensitive to microorganism challenges compared with PtSP. [Copyright &y& Elsevier]

Abstract: Four genes including three clip domain serine proteases (PtcSP1, PtcSP2 and PtcSP3) and one clip domain serine protease homologue (PtcSPH) of the swimming crab Portunus trituberculatus (Decapoda: Brachyura: Portunidae) were characterized based on analysis of expressed sequence tags from haemocytes and eyestalk cDNA libraries. The relative four peptidases, which share high structural similarity to the clip-SPs of other arthropod species, appeared to possess a clip domain at the N-terminus and an enzymatically active serine protease domain at the C-terminus except PtcSPH for its second catalytic residue Asp. (D) replaced by Ala (A). Alignment among the four full-sequences showed that PtcSP2 and PtcSP3 had the highest identical score (58%) while the similarity of other sequences was lower than 24%. The mRNA transcripts of PtcSPs and PtcSPH could be detected widely in all the examined tissues with remarkable different expression levels. The temporal expressions of PtcSPs and PtcSPH demonstrated different time-dependent expression pattern post Vibrio alginolyticus, Micrococcus luteus, and Pichia pastoris challenge. Especially, the expression of PtcSPH transcripts showed greater change against V. alginolyticus compared with the other two microorganisms. These findings suggest that PtcSPs and PtcSPH play different roles in the antibacterial defence mechanism of P. trituberculatus crab. [Copyright &y& Elsevier]

Abstract: Serine protease inhibitors (Serpins) play a key role in diverse immune biological processes. A serine protease inhibitor (Serpin), namely PtSerpin, was identified from the haemocyte cDNA library of swimming crab Portunus trituberculatus. The full-length PtSerpin cDNA was 1593 bp, including an open reading frame (ORF) of 1227 bp encoding a polypeptide of 408 amino acids with estimated molecular mass of 45.048 kDa and theoretical isoelectric point of 7.23. Predicted tertiary structure of PtSerpin contained three β-sheets and nine α-helices. Multiple sequence alignment revealed that deduced amino acid sequence of PtSerpin shared the highest similarity with serpin SPI from green mud crab Scylla paramamosain (SpSerpin). Phylogenetic analysis supported PtSerpin and SpSerpin were closely related to serpins from Penaeus monodon and Daphnia pulex while other decapods formed a separate group. Although the mRNA transcripts of PtSerpin could be detected in all the examined tissues, the higher levels were present in haemocytes and gills which are the major organs respond to pathogenic microorganism. After challenged by Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris, the temporal expression of PtSerpin gene in haemocytes showed different activation times against bacteria and fungi within the experimental period of 72 h. These findings suggest that PtSerpin is involved in the antibacterial defense mechanism of P. trituberculatus crab. [Copyright &y& Elsevier]

Abstract: Among all the serine proteinase inhibitor families (SPIs), the pacifastin-related inhibitor is seldom isolated. A pacifastin-related SPI named as PtPLC was identified from the swimming crab Portunus trituberculatus in this study. The full-length of PtPLC was cloned from haemocytes cDNA library by the combination of homology cloning and rapid amplification of cDNA ends (RACE). The PtPLC contained an open reading frame (ORF) of 1098 bp encoding a putative pacifastin-related precursor of 365 amino acids, a 5′-untranslated region (UTR) of 33 bp, and a 3′-UTR of 524 bp. The estimated molecular weight of mature PtPLC was 40.51 kDa and its isoelectric point was 5.04. Eight PLD domains in PtPLC shared a common characteristic of conserved array of six cysteine residues (Cys–Xaa9–12–Cys–Asn–Xaa–Cys–Xaa– Cys–Xaa2–3–Gly–Xaa3–4–Cys–Thr–Xaa3–Cys). The mRNA expression of PtPLC transcripts was highly detected in haemocytes, gill, hepatopancreas and stomach. The temporal expression levels of PtPLC transcripts in haemocytes showed different expression patterns after challenged by Gram-negative bacteria Vibrio alginolyticus, Gram-positive bacteria Micrococcus luteus and fungus Pichia pastoris. There were two peaks in the mRNA expression profile after M. luteus stimulation. And after V. alginolyticus and P. pastoris stimulation, there were three peaks in the mRNA expression profiles. These findings suggest that PtPLC is involved in the antibacterial defense mechanism of P. tritubercualtus. [Copyright &y& Elsevier]

Abstract: Expressed sequence tags (ESTs) analysis has been shown to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. Two full-length enriched cDNA libraries were constructed from hemocytes and eyestalk of Portunus trituberculatus, respectively, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. A total of 99 unigenes including 64 unigenes (6.00% of 1066 unigenes) in hemocytes library and 35 unigens (6.86% of 510 unigenes) in eyestalk library are identified to be immune genes. These genes are categorized into six classes, viz. antimicrobial peptides, redox proteins, melanization related proteins, chaperone proteins, clottable proteins and other immune factors. The content and category of immune genes in eyestalk library indicate eyestalk might have unrecognized role in crab immunity. Five immune genes containing multiple protein isoforms are identified and characterized, including anti-lipopolysaccharide factor (PtALF1-7), crustin (PtCrustin1-3), thioredoxin (PtTrx1-2), clip domain serine proteinase (PtcSP1-5) and kazal-type proteinase inhibitor (PtKPI1-4). Sequence alignment and phylogenetic analysis reveal PtALF1-7 contain two conserved cysteine residues and might be encoded by multiple genomic loci. PtCrustin1-3 share the consensus cysteine motif and are considered as Type I crustins. PtTrx1 possesses the critical structural cysteine residue C73 of Trx-1, while PtTrx2 has the N-terminal mitochondrial translocation signal of Trx-2. Sequence analysis shows PtcSP1-5 contain one clip domain and one partial SP catalytic triad domain. PtKPI1-4 present one typical Kazal domain consisting of six conserved cysteine residues. Some protein isoforms are tissue-specific, which might suggest they have different origins and perform diverse functions. Except PtALF1-3 and PtCrustin1, the other isoformes in this study are firstly identified from P. trituberculatus. Especially, PtTrx2 are firstly identified from crustaceans. Our research will provide useful genomic information of P. trituberculatus and be helpful in understanding the molecular mechanisms of crab immunity. [Copyright &y& Elsevier]

Abstract: Anti-lipopolysaccharide factors (ALFs), as the potent antimicrobial peptides, can bind and neutralize lipopolysaccharide (LPS) and exhibit broad spectrum antimicrobial activities. In this study, three isoforms of the ALF homologues (PtesALF1–3) were identified from eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA sequences of PtesALF1, 2 and 3 were 1138, 1052 and 1057 bp encoding 92, 108 and 123 amino acids, respectively. PtesALF1–3 contained two conserved cysteine residues and shared high similarity with other reported ALFs. Predicted tertiary structures of PtesALF2 and 3 containing four β-strands and three α-helix were similar to that described in Limulus polyphemus, while PtesALF1 had only one α-helix in its spatial structure. Sequence analysis revealed PtesALF1–3 were encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Totally 89 SNPs including 18 in coding region and 71 in noncoding region were detected by direct sequencing of 30 genomic samples. The mRNA expression of PtesALF1 and PtesALF1–3 transcripts was mainly detected in haemocytes but showed different expression pattern in other tissues including hepatopancreas, gill, eyestalk and muscle. After challenge with Vibrio alginolyticus, the temporal expression level of PtesALF1–3 transcripts in haemocytes showed a clear time-dependent response expression pattern with two peaks within the experimental period of 32 h, while PtesALF1 was up-regulated only once with obvious decrease at 6 h and significant increase at 24 h. These results suggest that the PtesALF isoforms have different tissue specificity and might provide multiple protective functions against invading bacteria in P. trituberculatus. [Copyright &y& Elsevier]

Abstract: Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. [ABSTRACT FROM AUTHOR]

Abstract: Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. In this study, the full-length cDNA of a novel c-type lysozyme was cloned from the haemocytes of swimming crab Portunus trituberculatus using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of lysozyme was of 984 bp, containing a 5′ untranslated region (UTR) of 90 bp, a 3′ UTR of 222 bp with a poly (A) tail, and an open reading frame (ORF) of 672 bp encoding an 18 amino acids signal peptide and a mature polypeptide of 205 amino acids with the predicted molecular weight of 23.76 kDa. The conserved catalytic sites Glu51 and Asp69, and the eight conserved cysteine residues identified in lysozyme suggesting it belonged to c-type lysozyme. Also, the swimming crab c-type lysozyme may belong to calcium-binding c-type lysozymes because of containing the three Asp residues (Asp98, Asp103 and Asp104), which are conserved in calcium-binding c-type lysozymes. Quantitative real-time RT-PCR analysis revealed that the mRNA transcripts of lysozyme were expressed at the highest level in haemocytes, with higher level in gill, moderately in hepatopancreas and muscle while low in stomach and heart. The mRNA expression of lysozyme in haemocytes was down-regulated significantly after challenge with Vibrio alginolyticus at 3 h, and then kept at the low expression level until 24 h. As time progressed, the mRNA expression of c-type lysozyme was recovered to the control level after 24 h injection. The results indicate swimming crab c-type lysozyme may be an inducible acute-phase protein and bacterial challenge could decline its mRNA expression. [Copyright &y& Elsevier]

Abstract: ESTs have been generated for many crustacean species, providing an invaluable resource for peptide discovery in members of this arthropod subphylum. Here, these data were mined for novel peptide-encoding transcripts, with the mature peptides encoded by them predicted using a combination of online peptide prediction programs and homology to known arthropod sequences. In total, 70 mature full-length/partial peptides representing members of 16 families/subfamilies were predicted, the vast majority being novel; the species from which the peptides were identified included members of the Branchiopoda (Daphnia carinata and Triops cancriformis), Maxillopoda (Caligus clemensi, Caligus rogercresseyi, Lepeophtheirus salmonis and Lernaeocera branchialis) and Malacostraca (Euphausia superba, Marsupenaeus japonicus, Penaeus monodon, Homarus americanus, Petrolisthes cinctipes, Callinectes sapidus and Portunus trituberculatus). Of particular note were the identifications of an intermediate between the insect adipokinetic hormones and crustacean red pigment concentrating hormone and a modified crustacean cardioactive peptide from the daphnid D. carinata; Arg7-corazonin was also deduced from this species, the first identification of a corazonin from a non-decapod crustacean. Our data also include the first reports of members of the calcitonin-like diuretic hormone, FMRFamide-related peptide (neuropeptide F subfamily) and orcokinin families from members of the Copepoda. Moreover, the prediction of a bursicon α from the euphausid E. superba represents the first peptide identified from any member of the basal eucaridean order Euphausiacea. In addition, large collections of insect eclosion hormone- and neuroparsin-like peptides were identified from a variety of species, greatly expanding the number of known members of these families in crustaceans. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Hsp70 can stimulate cells of the innate immune system directly by acting as “danger”-signaling molecules. To understand the immune defense mechanisms of swimming crab Portunus trituberculatus (Decapoda: Brachyura: Portunidae), the cDNA of Hsp70 (designated PtHsp70) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE). The full-length PtHsp70 cDNA was 2195 bp, including an open reading frame (ORF) of 1950 bp encoding a polypeptide of 650 amino acids with estimated molecular mass of 71.1514 kDa and theoretical isoelectric point of 5.38. FASTA and BLAST analysis indicated that PtHsp70 should be an inducible cytosolic member of the Hsp70 family. The coding region of PtHsp70 was uninterrupted and four SNPs with 1133C/T, 1311C/T, 1551C/T and 1809 A/G were detected by direct sequencing of 20 genomic samples. Using fluorescent real-time quantitative PCR, the transcriptional expression of PtHsp70 showed a clear time-dependent response after challenge by Vibrio alginolyticus, the main causative agent of emulsification disease causing large mortality in P. trituberculatus. This is the first report on the expression of Hsp70 induced by pathogen stimulation in Brachyura. Phylogenetic analysis revealed that the inducible Hsp70s were divided into two groups in crab and PtHsp70 was clustered into the Hsp/Hsc group (Clade I) by maximum-likelihood (ML) and Bayesian inference (BI) methods. GAP repeat and GGMP motif of inducible Hsp70 gene in the crab species were only found in Clade I. [Copyright &y& Elsevier]