Lipopolysaccharide And Β-1,3-glucan Binding Protein Research Articles

Spirulina elicits the activation of innate immunity and increases resistance against Vibrio alginolyticus in shrimp.

The effect of Spirulina dried powder (SDP) on the immune response of white shrimp Litopenaeus vannamei was studied invitro and invivo. Incubating shrimp haemocytes in 0.5mgml(-1) SDP caused the degranulation of haemocytes and a reduction in the percentage of large cells within 30min. Shrimp haemocytes incubated in 1mgml(-1) SDP significantly increased their phenoloxidase (PO) activity, serine proteinase activity, and respiratory burst activity (RB, release of superoxide anion). A recombinant protein of lipopolysaccharide and β-1,3-glucan binding protein (LGBP) of the white shrimp was produced, named rLvLGBP, and examined for its binding with SDP. An ELISA binding assay showed that rLvLGBP binds to SDP with a dissociation constant of 0.0507μM. In another experiment, shrimp fed diets containing SDP at 0 (control), 30, and 60gkg(-1) after four weeks were examined for LGBP transcript level and lysozyme activity, as well as phagocytic activity, clearance efficiency, and resistance to Vibrio alginolyticus. These parameters were significantly higher in shrimp receiving diets containing SDP at 60gkg(-1) or 30gkg(-1) than in controls. In conclusion, shrimp haemocytes receiving SDP provoked the activation of innate immunity as evidenced by the recognition and binding of LGBP, degranulation of haemocytes, reduction in the percentage of large cells, increases in PO activity, serine proteinase activity, superoxide anion levels, and up-regulated LGBP transcript levels. Shrimp receiving diets containing SDP had increased lysozyme activity and resistance against V. alginolyticus infection. This study showed the mechanism underlying the immunostimulatory action of Spirulina and its immune response in shrimp.

Isolation of prawn (Exopalaemon carinicauda) lipopolysaccharide and β-1, 3-glucan binding protein gene and its expression in responding to bacterial and viral infections

The pattern recognition proteins (PRPs) play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene in the ridge tail white prawn (Exopalaemon carinicauda) (EcLGBP) was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crustaceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at different levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus (WSSV). The expression of EcLGBP in response to V. parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

Enhancement of Vibriosis Resistance in Litopenaeus vannamei by Supplementation of Biomastered Silver Nanoparticles by Bacillus subtilis

virulent and most dreadful viral outbreaks in shrimp culture. The potential antibacterial effects of Bacillus subtilis silver nanoparticles (AgNPs) from the gut of Litopenaeus vannamei were studied. The AgNPs has ~ 420 nm in UV-visible spectrum, diameter 5-25 nm with smooth spherical shape (characterized by transmission electron microscopy) and 2θ values corresponding to the presence of silver crystal (X-ray diffraction spectrum). The AgNPs showed promising activity against Vibrio parahaemolyticus (21.25 ± 2.55 mm) and V. harveyi (19.27 ± 1.36 mm) as compared with B. subtilis, silver nitrate (AgNO3) and control. Four different experiments were conducted using different feeding behavior of L. vannamei. In comparison to untreated control group, final weight (14.89 ± 0.03 g), weight gain (9.36 ± 0.01), specific growth rate (SGR) 14.41 ± 0.09%, feed conversion ratios (FCR) 1.47 ± 0.12, higher survival and haemocyte counts were significantly greater in shrimp fed with AgNPs. The gill of entire experimental animal showed morphological alteration in histopathological investigation. The AgNPs were then tested for shrimp challenged with the V. parahaemolyticus. Statistical analysis revealed significant differences in shrimp survival between AgNPs, B. subtilis and control group. In the infective experimental study, cumulative survival of the control group (10 ± 0.321%) whereas the shrimp with AgNPs (90.66 ± 0.523%) and (71 ± 0.577%) with B. subtilis. Subsequently, real-time PCR was observed for immune related genes to determine the mRNA levels of prophenoloxidase (proPO), anti lipopolysaccharide factor (ALF2 and 4), peroxinectin (PE), superoxide dismutase (SOD), 18S, lipopolysaccharide and β -1,3-glucan-binding protein (LGBP ) and serine protein (SP). The expression of all immune related genes (mRNA levels) studied was significantly upregulated in the AgNPs diet shrimp in contrast to the B. subtilis and control. This study discovers that the biomastered AgNPs give a promising potential new tool for inhibiting vibriosis in shrimp culture.

Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) bind to seaweed polysaccharides and activate the prophenoloxidase system in white shrimp Litopenaeus vannamei

Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), important pattern recognition proteins (PRPs), recognize lipopolysaccharide (LPS) and β-1,3-glucan (βG), known as pathogen-associated molecular patterns (PAMPs), and subsequently trigger innate immunity. Several seaweed polysaccharides and seaweed extracts increase immune parameters and resistance to pathogens. Here, we constructed the expression vector pET28b-LvLGBP and transferred it into Escherichia coli BL21 (DE3) for protein expression and to produce the recombinant protein LGBP (rLvLGBP) in white shrimp Litopenaeus vannamei. We examined the binding of rLvLGBP with seaweed-derived polysaccharides including alginate, carrageenan, fucoidan, laminarin, Gracilaria tenuistipitata extract (GTE), and Sargassum duplicatum extract (SDE), and examined the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and each polysaccharide. We also examined the binding of rLvLGBP with LPS and βG, and the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS (rLvLGBP-LPS) or a mixture of rLvLGBP and βG (rLvLGBP-βG). An ELISA binding assay indicated that rLvLGBP binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE with dissociation constants of 0.1138–0.1770 μM. Furthermore, our results also indicated that the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE significantly increased by 328%, 172%, 200%, 213%, 197%, 194%, 191%, and 197%, respectively compared to controls (cacodylate buffer). We conclude that LvLGBP functions as a PRP, recognizes and binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE, and subsequently leads to activating innate immunity in shrimp.

Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and β-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway.

The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: Activity and gene transcription.

The non-specific immune parameters, disease resistance and immune genes expressions in Macrobrachium rosenbergii were evaluated at 120 days of post feeding the diets containing the extracts of banana, Musa acuminate, fruit's peel (banana peels extract, BPE) at 0, 1.0, 3.0 and 6.0 g kg(-1). Results showed that prawns fed with a diet containing BPE at the level of 1.0, 3.0 and 6.0 g kg(-1) for 120 days had a significantly higher survival rate (30.0%, 40.0% and 56.7%, respectively) than those fed with the control diet after challenge with Lactococcus garvieae for 144 h, and the respective relative survival percentages were 22.2%, 33.3%, and 51.9%, respectively. Dietary BPE supplementation at 3.0 and/or 6.0 g kg(-1) for 120 days showed a significant increase total haemocyte count (THC), granular cell (GC), superoxide dismutase (SOD) activity, phenoloxidase (PO) activity, transglutaminase (TG) activity, and phagocytic activity and clearance efficiency to L. garvieae infection, and meanwhile, the significant decrease in haemolymph clotting times and respiratory bursts (RBs) per haemocyte of prawns were revealed. Furthermore, the mRNA expressions of prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PE), transglutaminase (TG), and crustin (CT) were significantly increased. We therefore recommend that BPE can be used as an immunomodulator for prawns through dietary administration at 6.0 g kg(-1) for a long term (over 120 days) to modify immune responses and genes expression following the enhanced resistance against pathogens.

White Shrimp Litopenaeus vannamei That Have Received Gracilaria tenuistipitata Extract Show Early Recovery of Immune Parameters after Ammonia Stressing.

White shrimp Litopenaeus vannamei immersed in seawater (35‰) containing Gracilaria tenuistipitata extract (GTE) at 0 (control), 400, and 600 mg/L for 3 h were exposed to 5 mg/L ammonia-N (ammonia as nitrogen), and immune parameters including hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, lysozyme activity, and hemolymph protein level were examined 24~120 h post-stress. The immune parameters of shrimp immersed in 600 mg/L GTE returned to original values earlier, at 96~120 h post-stress, whereas in control shrimp they did not. In another experiment, shrimp were immersed in seawater containing GTE at 0 and 600 mg/L for 3 h and examined for transcript levels of immune-related genes at 24 h post-stress. Transcript levels of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), cytMnSOD, mtMnSOD, and HSP70 were up-regulated at 24 h post-stress in GTE receiving shrimp. We concluded that white shrimp immersed in seawater containing GTE exhibited a capability for maintaining homeostasis by regulating cellular and humoral immunity against ammonia stress as evidenced by up-regulated gene expression and earlier recovery of immune parameters.

Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein from red claw crayfish, Cherax quadricarinatus

Abstract A pattern recognition protein (PRP), lipopolysaccharide and β-1,3-glucan binding protein (LGBP), cDNA was cloned from the hemocytes of red claw crayfish, Cherax quadricarinatus by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1456 bp has an open reading frame of 1095 bp encoding a protein of 364 amino acids including a 15 amino acid signal peptide. The calculated molecular mass of the mature protein (349 aa) was 39.92 kDa with an estimated pI of 4.50. Sequence comparison of the deduced amino acid sequence of C. quadricarinatus LGBP (named as CqLGBP) showed a high identity of 82%, 77%, 76% and 74% with Procambarus clarkii LGBP, Homarus gammanus BGBP, Penaeus monodon BGBP and Litopenaeus stylirostlis LGBP, respectively. The CqLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. The recombinant CqLGBP were expressed in Escherichia coli BL21 with pGEX-6P-2 expression vector. The titer of rabbit anti-CqLGBP serum was above 1:12,800. Microorganism binding assay showed CqLGBP binds two kinds of crustacean pathogens Spiroplasma eriocheiris and Aeromonas hydrophila. Quantitative RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with S. eriocheiris and A. hydrophila. A significant enhancement of CqLGBP transcriptions was apparent post-injection in response to bacterial infection compared to the control group. These data should be helpful to better understand the function of CqLGBP in the crayfish immune system.

Effects of dietary supplementation with β-glucan and synbiotics on immune gene expression and immune parameters under ammonia stress in Pacific white shrimp

Abstract The goal of this study was to investigate the effects of dietary supplementation with β-glucan and synbiotics (i.e., microencapsulated Bacillus subtilis and Pediococcus acidilactici ) on immune gene expression and immune parameters of Pacific white shrimp ( Litopenaeus vannamei ) as well as modulation of immune parameters when shrimp were exposed to ammonia stress. Shrimp were fed one of four treatment diets [basal diet (C), β-glucan-containing diet (β-glu), β-glucan plus B. subtilis -containing diet (β-glu + Bs ), and β-glucan plus P. acidilactici -containing diet (β-glu + Pa )] for 90 days. The microencapsulated bacteria colonized in the proximal and central parts of the shrimp intestine. The β-glu diet increased expression of lipopolysaccharide and β-1,3-glucan-binding protein ( LGBP ), and the β-glu + Bs diet induced expression of the serine protease and prophenoloxidase genes. In addition, β-glu + Pa increased the LGBP transcription. However, the mRNA level of peroxinectin did not differ significantly among the experimental groups. Shrimp fed the β-glu and β-glu + Pa diets had higher superoxide dismutase activity (SOD) compared to shrimp fed the basal diet. In addition, the β-glu + Bs and β-glu + Pa diets increased phenoloxidase activity (PO). However, there were no significant differences in total hemocyte count (THC) and lysozyme activity among the treatment diets. Ammonia stress led to increased THC and decreased SOD activity, but it seemed to have no adverse effects on PO and lysozyme activity. Thus, both β-glucan and synbiotic supplementation had beneficial effects on several immune parameters, and the effect of dietary synbiotics was superior to that of β-glucan only for PO activity.

Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and β-1,3 glucan-binding protein from Fenneropenaeus indicus

Lipopolysaccharide and β-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of β-1,3 glucan (β-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein from oriental river prawn, Macrobrachium nipponense

The lipopolysaccharide and β-1,3-glucan binding protein (LGBP), one of the pattern recognition proteins, plays an important role in the innate immune response of invertebrates. A 1,506 bp full-length cDNA of a LGBP gene was cloned and characterized from the oriental river prawn Macrobrachium nipponense (named as MnLGBP). Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,119 bp, encoding a protein of 372 amino acids including a 21-aa signal peptide. The calculated molecular mass of the mature protein (351 aa) was 39.9 kDa with an estimated pI of 4.63. The MnLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. Sequence comparison based on the deduced amino acid sequence of MnLGBP showed varied identity of 89, 76 and 74% with those of Macrobrachium rosenbergii LGBP, Marsupenaeus japonicus β-1,3-glucan binding proteins, and Fenneropenaeus chinensis LGBP, respectively. Quantitative RT-PCR results showed that MnLGBP was expressed in nerve, intestine, muscle, gill, heart, haemocytes and at the highest level in hepatopancreas. After challenge with the pathogen, Aeromonas hydrophila and Vibrio parahaemolyticus, the expression of MnLGBP mRNA was significantly upregulated in the hepatopancreas compared to the control group. At the same time, the mRNA level of MnproPO increased dramatically at 48 h after injection of bacteria. These data should be helpful to better understand the function of MnLGBP in the prawn immune system.

Lipopolysaccharide and β-1,3-glucan binding protein in the hard clam (Meretrix meretrix): molecular characterization and expression analysis.

Pattern recognition molecules play an important role in innate immunity by recognizing conserved molecular patterns that are present on the surface of invading microorganisms. In this study, a lipopolysaccharide and β-1,3-glucan binding protein (LGBP) gene was cloned from the hard clam Meretrix meretrix (designated as Mm-LGBP) by the expressed sequence tags and rapid amplification of cDNA ends method. The cDNA was 1827 bp in length, consisting of a 71-bp 5'-terminal untranslated region, a 62-bp 3'UTR, and a 1734-bp open reading frame encoding a 577-amino acid polypeptide with an estimated molecular mass of 60.7 kDa and a theoretical isoelectric point of 5.56. Characteristic potential polysaccharide binding, cell adhesion, and glucanase motifs were identified in the Mm-LGBP, indicating that Mm-LGBP should be a new member of the LGBP family. Quantitative real-time polymerase chain reaction was developed to detect the mRNA expression level of Mm-LGBP in 6 different tissues. Higher-level mRNA expression of Mm-LGBP was detected in the gill and digestive gland tissues. The upregulation of Mm-LGBP mRNA after Vibrio anguillarum challenge showed that Mm-LGBP play a pivotal role in antibacterial immunity.

Administration of Bacillus subtilis strains in the rearing water enhances the water quality, growth performance, immune response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Litopenaeus vannamei

In this study, vegetative cell suspensions of two Bacillus subtilis strains, L10 and G1 in equal proportions, was administered at two different doses 105 (BM5) and 108 (BM8) CFU ml−1 in the rearing water of shrimp (Litopenaeus vannamei) for eight weeks. Both probiotic groups showed a significant reduction of ammonia, nitrite and nitrate ions under in vitro and in vivo conditions. In comparison to untreated control group, final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR) and digestive enzymatic activity were significantly greater in the BM5 and BM8 groups. Significant differences for survival were recorded in the BM8 group as compared to the control. Eight weeks after the start of experiment, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 80%, whereas cumulative mortality of the shrimp that had been given probiotics was 36.7% with MB8 and 50% with MB5. Subsequently, real-time RT-PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan- binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was only significantly up-regulated in the BM5 group compared to the BM8 and control groups. These results suggest that administration of B. subtilis strains in the rearing water confers beneficial effects for shrimp aquaculture, considering water quality, growth performance, digestive enzymatic activity, immune response and disease resistance.

Molecular and immunological responses of the giant freshwater prawn, Macrobrachium rosenbergii, to the organophosphorus insecticide, trichlorfon

Trichlorfon is an organophosphorus (OP) insecticide that is used as an agriculture pesticide to destroy insects, a human medicine to combat internal parasites, and an ectoparasiticide in the livestock and aquaculture industries, but which has caused aquatic toxicity in the prawn industry. The aim of this study was to investigate the effects of trichlorfon on molecular and enzymatic processes of the immunological response of the giant freshwater prawn, Macrobrachium rosenbergii, at 0, 0.2, and 0.4mgL−1 with 0, 3, 6, 12, and 24h of exposure. The total hemocyte count (THC), respiratory bursts (RBs), phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity were examined to evaluate immunological responses and oxidative stress. Results showed that THCs of the prawn exposed to trichlorfon at both concentrations (0.2 and 0.4mgL−1) had increased after 12 and 24h; SOD and PO activities had significantly increased at 3h, whereas production of RBs had dramatically increased as oxidative stress at each sampling time after exposure to trichlorfon compared to the control. A potential biomarker of OPs, acetylcholinesterase (AChE) revealed a significant decrease after exposure for 6h, and showed a time-dependent tendency. Immune gene expressions, including prophenoloxidase (proPO), the lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglubulin (α2M), transglutaminase (TG), and copper, zinc (Cu,Zn)-SOD, of prawns exposed to trichlorfon at 0, 0.2, and 0.4mgL−1 for 0, 6, and 24h were further evaluated. Expressions of all of the immune genes significantly decreased when prawns were exposed to 0.4mgL−1 trichlorfon for 24h, and among them, an increase in SOD expression was seen after exposure to 0.4mgL−1 for 6h. Prawns exposed to trichlorfon within 24h exhibited the decrease of circulating hemocytes, and also the induction of oxidative stress, which caused subsequent damage to DNA formation of immune genes. From these results, we concluded that immunological responses and immune gene expressions of prawn exposed to trichlorfon at 0.4mgL−1 for 24h were perturbed, thus causing a deficiency in immunity and subsequent increased susceptibility to pathogen infections.

Immune responses and gene expression in hepatopancreas from Macrobrachium rosenbergii challenged by a novel pathogen spiroplasma MR-1008

Freshwater prawn Macrobrachium rosenbergii inoculated with 100 μl novel pathogen spiroplasma strain MR-1008 in logarithmic phase (108 spiroplasmas ml−1) were examined for alkaline phosphatase (AKP) activity, acid phosphatase (ACP) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, as well as expressions of 7 immune related genes in hepatopancreas after 1–28 d. Hematoxylin-eosin (HE) staining showed obvious pathological features in hepatopancreas connective and epithelial tissue. Enzyme activity analyze showed that hepatopancreas AKP and ACP activity increased markedly (P < 0.05) when inoculated with spiroplasma MR-1008 after 5 d and 10 d, respectively. SOD enzyme activity changed less obviously and slightly increased at 1 day post-inoculation, but CAT activity decreased significantly after 5 d inoculation. The expression levels of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglobulin (α2M), AKP, ACP, CAT, and copper/zinc SOD (Cu, Zn-SOD) genes in the hepatopancreas were examined by Real-Time PCR (qRT-PCR) and the results demonstrated that these immune related genes were induced by challenge with spiroplasma MR-1008. The results suggested that the prawn immune responses could be activated or inhibited by spiroplasma MR-1008, and that the hepatopancreas also plays key roles in innate immunity for defense against the pathogen.

Norepinephrine regulates prophenoloxidase system-related parameters and gene expressions via α- and β-adrenergic receptors in Litopenaeus vannamei

The total (THC) and differential haemocyte counts (DHC), phenoloxidase (PO) activity, and prophenoloxidase (proPO) system-related genes were investigated in haemocytes of Litopenaeus vannamei that received saline, norepinephrine (NE), and NE co-treated with various adrenergic receptor (AR) antagonists both in vivo and in vitro. Results showed that semi-granular and granular cells of shrimp which received NE, NE + phentolamine (Phe), NE + prazosin (Pra), NE + propanolol (Pro) and NE + metoprolol (Met) significantly decreased, while the PO activity of the shrimp received NE + Phe in vivo was significant higher than all the other treatments. PO activities of haemocytes exposed to saline, Pra + NE, and Met + NE were significantly higher than those of haemocytes exposed to NE, Phe + NE, and Pro + NE in vitro. Similar phenomena in lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP), proPO-I, proPO-II, serine proteinases (SP), and peroxinectin (PE) messenger (m)RNA expressions of haemocytes exposed to saline, NE, and NE co-treated with various AR antagonists were observed both in vivo and in vitro. No significant differences were observed for LGBP and proPO-II mRNA expressions between haemocytes treated with saline and Pra + NE, for proPO-I mRNA expression between haemocytes treated with saline and Met + NE; or for SP and PE mRNA expressions among haemocytes treated with saline, Pra + NE, and Met + NE. These results suggest that stress-induced NE may promote the migration of circulating granulocytes to the site of the injection and the existing proPO mRNA translation which had been stored in granulocytes. NE downregulated the LGBP, proPO-I, proPO-II, SP, and PE gene transcription by haemocytes via α1-, β1-, α1-, α1- and β1-, and α1- and β1-ARs, respectively, which subsequently decreased the PO activity by α1- and β1-ARs in haemocytes of L. vannamei.

Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei

We studied the effect of two probiotic Bacillus subtilis strains on the growth performance, digestive enzyme activity, immune gene expression and disease resistance of juvenile white shrimp (Litopenaeus vannamei). A mixture of two probiotic strains, L10 and G1 in equal proportions, was administered at two different doses 105 (BM5) and 108 (BM8) CFU g−1 feed to shrimp for eight weeks. In comparison to untreated control group, final weight, weight gain and digestive enzyme activity were significantly greater in shrimp fed BM5 and BM8 diets. Significant differences for specific growth rate (SGR) and survival were recorded in shrimp fed BM8 diet as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of the feeding period, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 63.3%, whereas cumulative mortality of the shrimp that had been given probiotics was 20.0% with BM8 and 33.3% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated (P < 0.05) in the shrimp fed BM5 and BM8 diets compared to the control group. These findings demonstrate that administration of B. subtilis strains, L10 and G1, can improve growth performance and disease resistance through an enhanced immune response in shrimp.

CDNA cloning, characterization and expression of lipopolysaccharide and β-1,3-glucan binding protein (LGBP) gene from the Indian white shrimp Fenneropenaeus indicus

A 1372bp full length cDNA of an LGBP gene was identified from the Indian white shrimp, Fenneropenaeus indicus. The open reading frame (ORF) of the F. indicus LGBP (Fein-LGBP) was 1164bp long, encoding a polypeptide of 388 amino acids. The Fein-LGBP deduced amino acid has conserved potential recognition motif for β-1, 3 linkages of polysaccharides and putative RGD (Arg–Gly–Asp) cell adhesion sites. Phylogenetic analysis revealed that the Fein-LGBP was grouped together with LGBPs of other crustaceans such as F. chinensis (91%, AAZ41363), Penaeus monodon (88%, AAM21213) and Litopenaeus vannamei (88%, ABU92557). Quantitative RT-PCR results showed that Fein-LGBP gene expression was up-regulated in hemocytes following a 6h challenge in response to peptidoglycan (PG) and Vibrio parahaemolyticus. Messenger RNA transcripts of Fein-LGBP were measured in hemocytes of F. indicus from different molting stages. The highest Fein-LGBP mRNA expression was observed in pre-molting stages (D0/1) when compared to post-molt stages (A and B) and inter-molt stages (C). Fein-LGBP is involved in the regulation and activation of the prophenoloxidase cascade.

Association of CfLGBP gene polymorphism with disease susceptibility/resistance of Zhikong scallop (Chlamys farreri) to Listonella anguillarum

Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) is a pattern recognition receptor (PRR) recognizing and binding both LPS and β-1, 3-glucan, playing important roles in innate immunity. In the present study, the single nucleotide polymorphisms (SNPs) were assessed in LGBP gene from scallop Chlamys farreri (designated CfLGBP), and eight SNPs were found in its potential LPS and glucanase binding motif. The locus +7679 with the transition of G-A, which produced an amino acid substitution at codon 360 from a non polar Glycine to polar Serine, was selected to inspect their association with disease resistance/susceptibility to Listonella anguillarum. Three genotypes G/G, G/A and A/A, were revealed at locus +7679, and their frequencies were 89.7%, 7.7% and 2.6% in the resistant stock, while 63.2%, 34.2% and 2.6% in the susceptible stock, respectively. The frequency of genotypes G/G and G/A were significantly different (P < 0.05) between the two stocks. The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfLGBP (G) with G variant at locus +7679 and rCfLGBP (S) with A variant at locus +7679, were elucidated by ELISA assay. The binding affinities of both LPS and β-glucan binding affinity were varied in a dose-dependent manner, where the binding affinity of rCfLGBP (G) was significantly higher than that of rCfLGBP (S) (P < 0.05). The results collectively suggested that the polymorphism of +7679 G/G in CfLGBP possibly enhances the binding activity of LPS and β-glucan, and was associated to disease resistance of scallop against L. anguillarum, which could be a potential marker applied in future selection of scallop with enhanced resistance to L. anguillarum.

Pattern Recognition Protein Binds to Lipopolysaccharide and β-1,3-Glucan and Activates Shrimp Prophenoloxidase System

The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to β-1,3-glucan and LPS with a dissociation constant of 6.86 × 10(-7) M and 3.55 × 10(-7) M, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or β-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and β-1,3-glucan in the shrimp proPO activating system.