Identification of an essential role against shrimp pathogens of prophenoloxidase activating enzyme 1 (PPAE1) from Fenneropenaeus merguiensis hemocytes.

Prophenoloxidase (proPO) activating enzymes, known as PPAEs, are pivotal in activating the proPO system within invertebrate immunity. A cDNA encoding a PPAE derived from the hemocytes of banana shrimp, Fenneropenaeus merguiensis have cloned and analyzed, referred to as FmPPAE1. The open reading frame of FmPPAE1 encompasses 1392 base pairs, encoding a 464-amino acid peptide featuring a presumed 19-amino acid signal peptide. The projected molecular mass and isoelectric point of this protein stand at 50.5 kDa and 7.82, respectively. Structure of FmPPAE1 consists of an N-terminal clip domain and a C-terminal serine proteinase domain, housing a catalytic triad (His272, Asp321, Ser414) and a substrate binding site (Asp408, Ser435, Gly437). Expression of the FmPPAE1 transcript is specific to hemocytes and is heightened upon encountering pathogens like Vibrio parahaemolyticus, Vibrio harveyi, and white spot syndrome virus (WSSV). Using RNA interference to silence the FmPPAE1 gene resulted in reduced hemolymph phenoloxidase (PO) activity and decreased survival rates in shrimp co-injected with pathogenic agents. These findings strongly indicate that FmPPAE1 plays a vital role in regulating the proPO system in shrimp. Furthermore, upon successful production of recombinant FmPPAE1 protein (rFmPPAE1), it became evident that this protein exhibited remarkable abilities in both agglutinating and binding to a wide range of bacterial strains. These interactions were primarily facilitated through the recognition of bacterial lipopolysaccharides (LPS) or peptidoglycans (PGN) found in the cell wall. This agglutination process subsequently triggered melanization, a critical immune response. Furthermore, rFmPPAE1 exhibited the ability to actively impede the growth of pathogenic bacteria harmful to shrimp, including V. harveyi and V. parahaemolyticus. These findings strongly suggest that FmPPAE1 not only plays a pivotal role in activating the proPO system but also possesses inherent antibacterial properties, actively contributing to the suppression of bacterial proliferation. In summary, these results underscore the substantial involvement of FmPPAE1 in activating the proPO system in F. merguiensis and emphasize its crucial role in the shrimp's immune defense against invading pathogens.

Determination of the efficacy of using a serine protease gene as a DNA vaccine to protect against Vibrio parahaemolyticus infection in Litopenaeus vannamei.

Serine proteases are proteolytic enzymes that exhibit biological roles in many biological systems. Previously, a Vibrio parahaemolyticus serine protease was reported to be a virulence factor. Here, the serine protease gene of V. parahaemolyticus was investigated as a DNA vaccine against V. parahaemolyticus in Litopenaeus vannamei. The serine protease gene was mutated to replace the conserved residues His82, Asp131 and Ser231 with Gly, Asp and Pro, respectively. Then, a pcDNA3.1 vector to express mutVpSP (mutant serine protease) was constructed for in vitro and in vivo DNA vaccine investigation. In vivo mutVpSP transcriptional analysis revealed expression in various immunized white shrimp tissues, such as hemocytes, hepatopancreas, stomach, intestine, gills, and muscle. The efficiency of prevention of V. parahaemolyticus infection was investigated in vaccinated shrimp, and the lowest cumulative mortality percentage was 30%, while the control shrimp had a 60% cumulative mortality rate. The immune system was stimulated in shrimp vaccinated with the DNA vaccine. The mRNA expression of the shrimp immune-responsive genes phenoloxidase, peroxinectin and C-type lectin was significantly upregulated. Additionally, the humoral and cellular immune responses, including the PO, phagocytic, and encapsulation activities and nodule formation, were elevated. These results suggested that the serine protease could be a V. parahaemolyticus virulence determinant and that this DNA vaccine could be applied as an effective vaccine candidate for control of acute hepatopancreatic necrosis disease syndrome (AHPND) in shrimp.

Galectin, another lectin from Fenneropenaeus merguiensis, contributed in shrimp immune defense.

Numerous lectins act as pattern recognition receptors (PRRs) in the innate immune system of invertebrates. Here, a galectin (FmGal) was isolated from hemocytes of Fenneropenaeus merguiensis. FmGal contained one open reading frame encoding a peptide of 338 amino acids. The primary sequence of FmGal comprised a carbohydrate recognition domain with a specific galactose binding site. The FmGal transcripts were found mostly in hemocytes of healthy shrimp. The expression of FmGal was up-regulated upon challenge with Vibrio parahaemolyticus and white spot syndrome virus (WSSV). Gene-silencing with FmGal double-stranded RNA resulted in extreme down-regulation of FmGal. Knockdown with a co-injection of pathogens reduced the survival rate of shrimp. The recombinantr protein of FmGal (rFmGal) required Ca2+ to agglutinate pathogenic bacteria and exhibited sugar-specificity to galactose, lactose, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The ELISA-validated binding of rFmGal revealed higher affinity to LTA than LPS. rFmGal did not exhibit antibacterial activity but could enhance the phagocytosis and encapsulation of pathogenic invaders by hemocytes. Encapsulation was suppressed by galactose and lactose. Moreover, rFmGal also promoted the in vivo clearance of V. parahaemolyticus. FmGal, a galectin in F. merguiensis, participated in shrimp immunity, functioning as a PRR which might be involved in certain cellular responses.

Effects of the interaction between a clip domain serine protease and a white spot syndrome virus protein on phenoloxidase activity.

Clip domain serine proteinases participate in invertebrate innate immunity by acting as crucial enzymes in the signaling cascade involved in shrimp immunity. To functionally characterize its role in Fenneropenaeus merguiensis, FmclipSP cDNA was cloned and characterized. The FmclipSP gene comprised 1353 bp with an open reading frame of 1110 bp and encoded 369 amino acids. The protein contained clip and serine protease domains. FmClipSP mRNA is highly expressed in hemocytes, and its expression was significantly upregulated by bacterial or viral pathogen challenge. Furthermore, FmClipSP recombinant protein (rFmClipSP) was produced and possessed protease activity, stimulating prophenoloxidase activity. Additionally, rFmClipSP exhibited antibacterial activity against pathogens and nonpathogens. ELISA results demonstrated the binding ability of rFmClipSP to a recombinant protein of VP28 (rVP28). Interestingly, the binding significantly inhibited prophenoloxidase activity. Altogether, we partially characterized the function of FmclipSP and demonstrated its association with VP28. This study indicates the importance of clipSP as a component of F. merguiensis innate immunity. However, the role of clipSP in crustaceans remains unclear and requires further investigation.

A unique lectin composing of fibrinogen-like domain from Fenneropenaeus merguiensis contributed in shrimp immune defense and firstly found to mediate encapsulation.

In invertebrates, both fibrinogen-related proteins (FREPs) and C-type lectins are acknowledged to act as pattern recognition receptors (PRRs) to participate particularly in an innate immunity. Hereby, a unique C-type lectin designated as FmLFd was isolated from the hemocytes of Fenneropenaeus merguiensis. FmLFd contained one open reading frame which encoding a peptide of 312 amino acid residues and a signal peptide of 18 amino acids. The primary sequence of FmLFd was composed of a fibrinogen-like domain (Fd) with a Ca2+-binding site and possessing specificity to bind N-acetyl glucosamine (GlcNAc). The FmLFd transcripts were detected mainly in hemocytes of healthy shrimp. The expression of FmLFd was significantly up-regulated upon challenge shrimp with Vibrio parahaemolyticus and Vibrio harveyi which more potent than by white spot syndrome virus (WSSV). The knocking down shrimp with FmLFd double-stranded RNA caused dramatical gene down-regulation. The gene silencing with co-injection of pathogens resulted in reduction of the shrimp survival rate. Recombinant protein of FmLFd (rFmLFd) could agglutinate and bind directly to both Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner and showed the sugar specificity to GlcNAc and bacterial saccharides; peptidoglycan (PGN), lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Recombinant protein of Fd domain (rFd) displayed the lower activity and specificity only to PGN. The binding between recombinant proteins of FmLFd and its domain confirming by ELISA demonstrated that both rFmLFd and rFd could bind to PGN, LPS and LTA with the highest affinity respected to PGN including a less extent of rFd. Besides, rFmLFd but not rFd could bind to WSSV proteins with the highest binding affinity to capsid VP15 and decreasing in order to envelope VP28 and tegument VP39A, respectively. It was presumed that entire molecule of FmLFd exhibited the antimicrobial ability by inhibiting the growth of pathogenic V. parahaemolyticus and this action was not affected by GlcNAc. Otherwise, FmLFd, a lectin containing fibrinogen-like domain, was firstly reported to be capable of promoting encapsulation by hemocytes. Altogether, we concluded that FmLFd belonged to a FREP family indentified by the existence of a conserved fibrinogen-like domain with possessing an ability to bind GlcNAc. It was a new C-type lectin existed in F. merguiensis and might presumably act as a kind of PRRs to participate in the shrimp immune defense towards bacterial and viral pathogens.

FmLC6: An ultimate dual-CRD C-type lectin from Fenneropenaeus merguiensis mediated its roles in shrimp defense immunity towards bacteria and virus.

C-type lectins are a member of pattern recognition receptors (PRRs) that can interact with pathogen-associated molecular patterns of invading microorganisms by using their conserved motifs in carbohydrate recognition domain (CRD). The binding can trigger various immune responses in both direct and indirect mechanisms. Hereby, an ultimate C-type lectin with dual CRDs each of which containing a different motif was identified from hepatopancreas of Fenneropenaeus merguiensis (mentioned as FmLC6). The full-length cDNA of FmLC6 consisted of 1148 bp comprising one 1005 bp open reading frame (ORF) encoding a signal peptide and a mature protein of 317 residues. FmLC6 was composed of two CRDs with a highly conserved QPD (Gln-Pro-Asp) motif and one variant EPQ (Glu-Pro-Gln) motif for illustrating the carbohydrate binding affinity. The transcription of FmLC6 was detected only in hepatopancreas of normal shrimp. After injection with pathogens or immunostimulants, the expression of FmLC6 was significantly up-regulated and reached the highest level at 12 h post-injection except with lipoteichoic acid challenge. The FmLC6 expression was severely suppressed by knockdown based-silencing. This gene silencing with co-injection by Vibrio parahaemolyticus caused increasing in cumulative mortality and reduction of the median lethal time. Purified recombinant proteins of an entire ORF and two individual CRDs of FmLC6 produced in Escherichia coli could induce a broad spectrum of microbial agglutination with calcium dependence. The agglutination induced by rFmLC6, rCRD1 and rCRD2 was suppressed by galactose plus mannose, galactose and mannose, respectively which this event was confirmed by the inhibition of hemagglutination. All three recombinant proteins possessed ability to inhibit the bacterial growth with a dose-response. Purified rFmLC6 could bind directly to white spot syndrome virus particles and also its recombinant proteins including VP15, VP39A and VP28 with different affinity. Altogether, these results indicate that FmLC6 acts as a PRR to recognize invading microorganisms and leads to mediating the immune response to cooperation in pathogenic elimination via the binding, agglutination and antimicrobial activity.

An alternative function of C-type lectin comprising low-density lipoprotein receptor domain from Fenneropenaeus merguiensis to act as a binding receptor for viral protein and vitellogenin.

A diversity of C-type lectins (CTLs) was coming reported and they are known to participate in invertebrate innate immunity by act as pattern recognition receptor (PRR). In the present study, a unique CTL containing low-density lipoprotein receptor (LDLR) domain from Fenneropenaeus merguiensis (designated as FmLdlr) was cloned. Its sequence contained a single LDLR domain and one carbohydrate recognition domain (CRD) with a QAP motif putative for galactose-specific binding. The expression of FmLdlr was detected only in hemocytes of healthy shrimp. Its expression was significantly up-regulated by Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenge. The knockdown by FmLdlr dsRNA resulted in severe gene down-regulation. The gene silencing with pathogenic co-inoculation led to reduction of the median lethal time and increasing in the cumulative mortality including the remained WSSV in WSSV co-challenge group. Recombinant proteins of FmLdlr and two domains could agglutinate various bacterial strains which LDLR domain revealed the lowest activity. Only FmLdlr and CRD could enhance phagocytosis and encapsulation by hemocytes. Both FmLdlr and CRD except LDLR domain exhibited the antibacterial activity by inhibiting the growth of pathogenic V. parahaemolyticus in cultured medium and disk diffusion assay. Only FmLdlr and CRD could bind to WSSV proteins, envelope VP28, tegument VP39A and also capsid VP15, which FmLdlr had the higher binding affinity than that of CRD. Altogether, we concluded that FmLdlr contributed in shrimp immune defense through the main action of CRD in capable of bacterial agglutination, enhancing the phagocytosis and encapsulation, antimicrobial activity and binding to viral proteins. Interestingly, ELISA approach revealed that LDLR domain displayed the highest binding affinity to vitellogenin than whole molecule and CRD. We signified a new function of FmLdlr that it might presumably act as a receptor for vitellogenin transportation in hemolymph during vitellogenesis of shrimp.

Lipopolysaccharide- and ß-1,3-glucan-binding protein from Litopenaeus vannamei: Purification, cloning and contribution in shrimp defense immunity via phenoloxidase activation.

Lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP) existed in diversity of invertebrates including shrimp plays a crucial role in an innate immunity via mediating the recognition of invading pathogens. In this study, LGBP was cloned and characterized from the hepatopancreas of Litopenaeus vannamei, named as LvLGBP. Its full-length cDNA of 1282 bp contained an open reading frame (1101 bp) encoding a peptide of 367 amino acids. The LGBP primary structure contained a glycosyl hydrolase domain, two integrin binding motifs, two kinase C phosphorylation sites, and two polysaccharide recognition motifs which were identified as a polysaccharide binding motif and a ß-1,3-glucan recognition motif. The LvLGBP transcripts were expressed mainly in the hepatopancreas. Upon challenge with Vibrio parahaemolyticus or white spot syndrome virus (WSSV), the LvLGBP mRNA expression was significantly up-regulated to reach a maximum at 48 h post injection. Its expression was also induced by lipopolysaccharide (LPS) or ß-1,3-glucan stimulation. RNAi-based silencing resulted in the critical suppression of LvLGBP expression. Knockdown of LvLGBP gene with co-inoculation by V. parahaemolyticus or WSSV led to increase in the cumulative mortality and reduce in the median lethal time. Native LGBP was detected only in the hepatopancreas as verified by Western blotting. Purified LGBP from the hepatopancreas exhibited the agglutinating and binding activity towards Gram-negative bacterium V. parahaemolyticus with calcium-dependence. Its agglutinating activity was dominantly inhibited by LPS with higher potential than ß-1,3-glucan. Purified LvLGBP could significantly activate the hemocyte phenoloxidase activity in the presence of LPS (12.9 folds), while slight activation was detected with ß-1,3-glucan (2.0 folds). It could enhance the encapsulation by hemocytes but did not have antibacterial activity. These results provided evidence that LvLGBP might act as a pathogenic recognition protein to activate shrimp immune defense against invading pathogens via the agglutination, binding and enhancing encapsulation and phenoloxidase activity of the hemocytes.

A mannose-specific C-type lectin from Fenneropenaeus merguiensis exhibited antimicrobial activity to mediate shrimp innate immunity.

Being one type of pattern recognition receptors (PRRs), lectins exhibit a crucial role in the defense mechanism of invertebrates which are deficient in an adaptive immune system. A new C-type lectin called FmLC3 was isolated from hepatopancreas of Fenneropenaeus merguiensis by cloning approaches, RT-PCR and 5' and 3' RACE (rapid amplification of cDNA ends). A full-length cDNA of FmLC3 contains 607 bp with one open reading frame of 480bp, encoding a 159-amino acids peptide. The predicted primary structure of FmLC3 is composed of a signal peptide, a carbohydrate recognition domain with an EPN motif and one Ca2+ binding site-2, including a double-loop region assisted by two conserved disulfide linkages. FmLC3 had a molecular mass of 17.96kDa and pI of 4.92. In normal or unchallenged shrimp, the mRNA expression of FmLC3 was detected only in hepatopancreas whilst its native proteins were found in hemolymph, heart, stomach and intestine but not in the expressed tissue, indicating that after being synthesized in hepatopancreas, FmLC3 would be secreted to other tissues. The significant up-regulation of FmLC3 was manifested in shrimp challenged with Vibrio harveyi or white spot syndrome virus. After knockdown with gene-specific double-stranded RNA and following by co-pathogenic inoculation, the FmLC3 expression was severely suppressed with coherence of increasing in cumulative mortality and reduction of the median lethal time. Recombinant FmLC3 (rFmLC3) had agglutinating activity towards diverse bacterial strains in a Ca2+-dependent manner. Its activity was inhibited by lipopolysaccharide and mannose, implying that FmLC3 was mannose-binding C-type lectin. Moreover, rFmLC3 could bind directly to various microbial strains with Ca2+-requirement. Otherwise, rFmLC3 exhibited the antimicrobial activity by inhibiting effectively the microbial growth in vitro. All these results signified that FmLC3 might act as PRR to recognize with a broad specificity for diverse pathogens, and contribute in shrimp immune response via the agglutination, binding and antimicrobial activity.

FmLC5, a putative galactose-binding C-type lectin with two QPD motifs from the hemocytes of Fenneropenaeus merguiensis participates in shrimp immune defense.

Crustaceans are deficient in adaptive immune system. They depend completely on an innate immunity to protect themselves from invading microorganisms. One kind of pattern recognition receptors that contribute roles in the innate immunity is lectin. A new C-type lectin gene designated as FmLC5 was isolated from Fenneropenaeus merguiensis. Its full-length cDNA is composed of 1526bp and one open reading frame of 852bp encoding a peptide of 284 amino acids. The deduced amino acid sequence of FmLC5 comprises a signal peptide of 20 contiguous amino acids with a molecular mass of 31.47kDa and an isoelectric point of 4.35. The primary structure of FmLC5 consists of two similar carbohydrate recognition domains (CRDs), each CRD contains a Ca2+ binding site-2 and a QPD motif specific for galactose-binding. The FmLC5 transcripts were detected only in the hemocytes analyzed by RT-PCR and in situ hybridization. The FmLC5 expression was significantly up-regulated after challenge with Vibrio harveyi, white spot syndrome virus (WSSV) or lipopolysaccharide. RNAi-based silencing with co-injection by V. harveyi or WSSV resulted in critical suppression of the FmLC5 expression, increasing in mortality and reduction of the median lethal time. These results conclude that FmLC5 is unique putative galactose-binding C-type lectin in F. merguiensis that may contribute as receptor molecule in the immune response to defend the shrimp from pathogenic bacteria and viruses.

Lipopolysaccharide-specific binding C-type lectin with one CRD domain from Fenneropenaeus merguiensis (FmLC4) functions as a pattern recognition receptor in shrimp innate immunity.

In crustaceans, an innate immune system is solely required because they lack an adaptive immunity. One kind of pattern recognition receptors (PRRs) that plays a particular role in the innate immunity of aquatic shrimp is lectin. A new diverse C-type lectin (FmLC4) was cloned from the hepatopancreas of Fenneropenaeus merguiensis by using RT-PCR and 5' and 3' rapid amplification of cDNA ends approaches. A full-length FmLC4 cDNA comprises 706 bp with an open reading frame of 552 bp, encoding a peptide of 184 amino acids. The predicted primary sequence of FmLC4 consists of a signal peptide of 19 amino acids, a molecular mass of 20.4 kDa, an isoelectric point of 5.13, one carbohydrate recognition domain with a QPD motif and a Ca2+ binding site as well as a double-loop characteristic supported by two conserved disulfide bonds. The FmLC4 mRNA expression was found only in the hepatopancreas of normal shrimp and significantly up-regulated upon challenge the shrimp with Vibrio harveyi or white spot syndrome virus (WSSV). Recombinant FmLC4 (rFmLC4) could agglutinate various bacterial strains with Ca2+-dependence. Lipopolysaccharide (LPS) could specifically inhibit the agglutinating activity and potently bind to rFmLC4, indicating that FmLC4 was LPS-specific binding C-type lectin. Moreover, rFmLC4 itself displayed the in vivo effective clearance of the pathogenic bacterium V. harveyi. Altogether, FmLC4 may serve as LPS-specific PRR to recognize opportunistic bacterial and viral pathogens, and thus to play a role in the immune defense of aquatic shrimp via the binding and agglutination.

Lipopolysaccharide- and ß-1,3-glucan-binding protein from Fenneropenaeus merguiensis functions as a pattern recognition receptor with a broad specificity for diverse pathogens in the defense against microorganisms.

In crustaceans, lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP) plays an important role in innate immunity by mediating the recognition of pathogens to host cells. Hereby, LGBP was cloned from Fenneropenaeus merguiensis hepatopancreas. Its full-length cDNA (1280 bp) had an open reading frame of 1101 bp, encoding a peptide of 366 amino acids. The LGBP primary structure comprises a recognition motif for ß-1,3-linkage of polysaccharides, two integrin binding motifs, a kinase C phosphorylation site and a bacterial glucanase motif. The LGBP mRNA was strongly expressed in hepatopancreas and significantly up-regulated to get the maximum at 12 h upon Vibrio harveyi challenge. Recombinant LGBP (rLGBP) could agglutinate Gram-negative and Gram-positive bacteria including yeast with Ca2+-dependence. V. harveyi agglutination induced by rLGBP was intensively inhibited by lipoteichoic acid, less in order were lipopolysaccharide, ß-1,3-glucan and N-acetyl neuraminic acid. Western blotting revealed that rLGBP bound widely to Gram-negative and Gram-positive bacteria and also yeast. By ELISA quantification, rLGBP could bind to ß-1,3-glucan better than to lipopolysaccharide and lipoteichoic acid. These findings suggest that LGBP may function as a receptor which recognizes invading diverse pathogens and contribute in F. merguiensis immune response.

Sialic acid-specific lectin participates in an immune response and ovarian development of the banana shrimp Fenneropenaeus merguiensis.

A sialic acid-specific lectin was purified from the hemolymph of Fenneropenaeus merguiensis by repetitive affinity fetuin-agarose column chromatography. The purified F. merguiensis lectin (called FmL) consisted of two distinct 30.9 and 32kDa subunits with identical N-terminal amino acid sequences of ten residues. FmL was also composed of sugar moieties; glucosamine, glucose, mannose and N-acetyl neuraminic acid but not N-glycolyl neuraminic acid. It was postulated to be a glycoprotein as it was positively stained by glycoprotein staining kit and detected by some bionylated plant lectins. Deglycosylation by either peptide N-glycosidase F or trifluoromethanesulfonic acid turned both types of FmL subunits to 28kDa peptides. The internal peptide sequence of FmL was similar to a fibrinogen-related domain of human ficolin and the horseshoe crab lectin. Determination of the lectin concentrations in the hemolymph was performed by ELISA while its hemaglutinating activity (HA) was tested by hemagglutination. Both specific lectin concentrations and HA increased as shrimp developed ovarian maturation stages 2 to 4. Their constitutive levels were found in pre-vitellogenic females and higher than those of males. Both specific lectin concentrations and HA of FmL were inducible to the highest levels at 12h after F. merguiensis was challenged by pathogenic Vibrio harveyi. The FmL-induced agglutination of V. harveyi was specifically abolished by sialic acid, fetuin and bacterial cell wall components. These findings might indicate the implication in an immune response of FmL to protect the shrimp themselves or their spawning eggs towards pathogenic bacteria in surrounding environment.

Cloning and the mRNA expression of a C-type lectin with one carbohydrate recognition domain from Fenneropenaeus merguiensis in response to pathogenic inoculation.

Crustaceans are deficient in an adaptive immune system and depend solely on their innate immunity. One kind of pattern recognition proteins which plays an important role in the shrimp immunity is lectin. A new C-type lectin called FmLC2 was cloned from the stomach of the banana shrimp Fenneropenaeus merguiensis by means of RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE). Its full-length cDNA contains 1098 bp with a single open reading frame of 738 bp, encoding a peptide of 245 amino acids. The deduced amino acid sequence of FmLC2 consists of a signal peptide of 17 amino acids with a molecular mass of 28,115 Da and an isoelectric point of 6.94. The primary structure of FmLC2 comprises a single carbohydrate recognition domain (CRD) with a QPD (Gln-Pro-Asp) motif and one Ca(2+) binding site. Like other C-type lectins, its CRD structure contains a double-loop characteristic being stabilized by two conserved disulfide linkages. The mRNA expression of FmLC2 was detected specifically in the stomach and gills, less was found in the hepatopancreas. Upon inoculation of shrimp with Vibrio harveyi or white spot syndrome virus (WSSV), the FmLC2 expression either in stomach or gills was higher than in the hepatopancreas. Besides, its expression in these tissues was up-regulated to reach the highest levels at 12 or 18 h for V. harveyi or WSSV stimulation, respectively. RNAi-based silencing of FmLC2 resulted in suppression of its expression, increases in mortality when the shrimp were challenged with V. harveyi or WSSV, and the median lethal time was reduced compared with controls. These results suggest that FmLC2 may serve as receptor molecules which recognize invading bacterial and viral pathogens and thus contribute a role in the shrimp immune response.

Molecular cloning of a C-type lectin with one carbohydrate recognition domain from Fenneropenaeus merguiensis and its expression upon challenging by pathogenic bacterium or virus.

Lectins, one type of pattern recognition proteins, play important roles in an innate immunity of crustaceans including shrimp. A new C-type lectin designated FmLC1 was cloned from the hepatopancreas of banana shrimp Fenneropenaeus merguiensis by procedures of PCR and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length cDNA is composed of 706bp with a single open reading frame of 477bp, encoding a peptide of 158 amino acid residues. Its deduced amino acid sequence comprises a putative signal peptide of 17 amino acids and has an estimated molecular mass of 17,934Da with a theoretical pI of 4.46. The primary sequence of FmLC1 contains a single carbohydrate recognition domain (CRD) with an EPS (Glu-Pro-Ser) motif and one Ca(2+) binding site, stabilized by two disulfide bonds. FmLC1 mRNA was detected to express specifically in the hepatopancreas, a master organ in shrimp. Its expression in the hepatopancreas was up-regulated to reach the maximum at 12 or 48h following challenge of shrimp with Vibrio harveyi or white spot syndrome virus, respectively. These results suggest that FmLC1 may participate in recognition of invading pathogens such as bacteria and viruses, and play roles in the immune response of shrimp even at different stages of the clearance of pathogens.

Gene expression and activity of digestive enzymes during the larval development of Asian seabass (Lates calcarifer).

Knowledge of digestive enzyme development during larval stages provides a better understanding of the digestive and nutritional physiology of fish larvae. This study characterized the ontogeny of key digestive enzymes in Asian seabass larvae from hatching to juvenile stage (30 days post hatch, dph) using molecular and biochemical approaches. Gene expression and activity of pepsinogen (pg), trypsinogen (try), chymotrypsinogen (ctr), bile salt-activated lipase (bal), α-amylase (amy), leucine aminopeptidase (lap) and alkaline phosphatase (alp) were determined. Gene expression and enzyme activity of all digestive enzymes were detectable from hatching. Pepsinogen mRNA levels were close to detection limit during 0-15 dph, but were highly expressed from 18 dph and onwards. This coincided with a marked increase in specific and individual pepsin activity, indicating complete development of digestive function. Expression levels of try, ctr, amy and bal were high between 3 and 15 dph and thereafter a decreasing trend was observed. Intestinal enzymes, lap and alp, showed highest expression levels during the yolk sac stage, and thereafter levels decreased. Activity of all digestive enzymes increased from around 18 dph and onwards. In conclusion, the development of main digestive enzymes in Asian seabass larvae shows a similar pattern to that of other marine fish species.

Molecular cloning of a C-type lectin with two CRD domains from the banana shrimp Fenneropenaeus merguiensis: early gene up-regulation after Vibrio harveyi infection.

A diverse class of pattern-recognition proteins called lectins play important roles in shrimp innate immunity. A novel C-type lectin gene (FmLC) was cloned from the hepatopancreas of banana shrimp Fenneropenaeus merguiensis by means of PCR and 5' and 3' rapid amplification of cDNA ends (RACE). The full-length cDNA consists of 1118 bp with one 1002 bp open reading frame, encoding 333 amino acids. Its deduced amino acid sequence contains a putative signal peptide of 20 amino acids. FmLC contains two carbohydrate recognition domains, CRD1 and CRD2, that share only 30% identity with each other. The first CRD comprises a QPD motif with specificity for binding galactose and a single Ca(2+) binding site, while the second CRD consists of an EPN motif for a mannose-specific binding site. FmLC had a close evolutionary relationship to other dual-CRD lectins of penaeid shrimp. Expression results showed that transcripts of FmLC were detected only in the hepatopancreas, none was found in other tissues. After challenging either whole shrimp or hepatopancreas tissue fragments with Vibrioharveyi, the expression of FmLC was up-regulated. This indicates that FmLC is inducible and may be involved in a shrimp immune response to recognize potential bacterial pathogens.

Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR.

An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph.

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca(2+)-dependent. The hemagglutinating activity of FmL was stable up to 55 degrees C and at pH 7.5-8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression.

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.