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

Immune responses and histopathological analyses of giant river prawn (Macrobrachium rosenbergii, De Man 1879) challenged with a sub-lethal dose of decapod iridescent virus 1 (DIV1) and chemical control investigation.

Decapod iridescent virus 1 (DIV1) is a lethal virus that has a significant influence on the shrimp and prawn culture industries. The mechanism through which infected prawns respond to the DIV1 virus is currently unknown. Here, we examined in detail the clinical signs, histopathology, and humoral, cellular, and immune-related gene responses after a sub-lethal dose of DIV1 during the acute infection period of 0-120h post infection (hpi). Interestingly, at the end of the experiment, DIV1-infected prawns had black lesions on several external regions. The DIV1-infected prawns also exhibited few karyopyknotic nuclei in the gills and intestine tissues and exhibited increasing immunological responses, as revealed by significant increases in all examined parameters, including total hemocytes, phagocytosis, lysozyme, and overall bactericidal activity, from 6 to 48 hpi. In addition, between 72 and 120 hpi, all immune response activities of DIV1-infected prawn were impaired compared with those of normal prawns, indicating negative impacts on immunological parameters. A viral load analysis of various tissues by qPCR indicated that hemocytes were the dominant initial viral target tissues, followed by the gills and hepatopancreas. An expression analysis of crucial immune-related genes by qRT‒PCR revealed various expression patterns in response to DIV1 infection; in particular, fold changes in the relative expression of anti-lipopolysaccharide factors (ALFs), prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan binding protein (LGBP) were observed. Additionally, five common chemicals, calcium hypochlorite [Ca(OCl)2] at 16.25-130ppm, hydrogen peroxide (H2O2) at 8.75-70ppm, povidone iodine (PVP-I) at 3-24ppm, benzalkonium chloride (BKC) at 20-160ppm, and formalin at 25-200ppm, had a significant effect on the killing of DIV1 particles in vitro within 24h after exposure. These data will be helpful for determining the health status and immune defense mechanisms of giant river prawns during DIV1 infection periods. The study performed the first application of very common disinfectants, and the obtained information will be useful for implementing effective strategies to prevent and control DIV1 infection in both hatchery and grow-out ponds.

Immune response, gene expression, and intestinal microbial composition of Pacific white shrimp fed with multispecies synbiotic for the prevention of coinfection disease.

This study aimed to evaluate the application of synbiotic containing multispecies of probiotics with different cell densities in white shrimp rearing against infectious myonecrosis virus (IMNV) and Vibrio parahaemolyticus coinfection. This study used a completely randomized design with five treatments and three replications. One additional replication of each treatment was provided for the lethal sampling. Pacific white shrimp were fed with three dosages of synbiotic multispecies for 30 days, namely 103 CFU mL−1 (Sin 3), 106 CFU mL−1 (Sin 6), and 109 CFU mL−1 (Sin 9), and the controls without synbiotic administration consisted of the positive control (K +) and the negative control (K −). Pacific white shrimp from all treatments, except for the K − , were challenged with IMNV a dose of 100 µL and 106 CFU mL−1 V. parahaemolyticus, injected intramuscularly. Infected Pacific white shrimp showed clinical signs like anorexia, melanosis, empty gut, colorless hepatopancreas, and white necrotic areas in striated skeletal muscles, especially of the distal abdominal segments and uropod. The results showed that administration of synbiotic for 30 days resulted in higher immune parameters, such as total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RB), and total viable bacterial count (TBC) compared to K + /K − . After coinfection, they showed significantly higher levels for THC, PO, RB, gene expression prophenoloxidase (ProPO), and lipopolysaccharide and β-1.3-glucan-binding protein (LGBP), better clinical signs, and lower mortalities. Sin 9 treatment significantly showed the highest survival rate (SR) compared to the other treatments.

Induction of transgenerational innate immune memory against Vibrio infections in a brine shrimp (Artemia franciscana) model

The occurrence of transgenerational innate immune memory in invertebrates possibly offers an opportunity for the creation of offspring with increased resistance against infectious diseases. Shrimps, being invertebrates rely on their innate immune system as the main defence mechanism to combat invading pathogens. Thus, creating transgenerational innate immune memory could be used for disease prevention in shrimp farming. However, the mechanisms behind the induction of transgenerational innate immune memory and the creation of a broad spectrum protective immune memory are still not fully understood. Here, the creation of transgenerational innate immune memory and the mechanisms involved were studied using the brine shrimp (Artemia franciscana) as a model for shrimps. Hereto, a parental generation was primed by exposure to a live or dead Vibrio parahaemolyticus PV1 while another parental generation was primed with live or dead Vibrio campbellii LMG21363. Both Vibrio strains are known to cause diseases in farmed shrimps, resulting in significant economic losses. Axenic larvae of three subsequent generations were exposed either to the homologous or heterologous (viable) Vibrio species and their survival was recorded. In general, progenies from primed F0 parents developed innate immune memory as compared to offspring of non-primed control F0 parents as they were significantly protected against a subsequent Vibrio infection. Protection in the infected offspring was more pronounced when they were infected with the homologous Vibrio strain. Disease resistance was associated with significantly increased transcription of especially peroxinectin (pxn), prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), high mobility group box 1 (HMGB1) and heat shock protein 90 (hsp90) genes and significant changes in histone H3 and H4 acetylation and m6A RNA methylation. Overall, results might lead to procedures at shrimp brood stock level for parental conditioning and creation of transgenerational innate immune memory in offspring.

Synergic degradation of yeast β-glucan with a potential of immunostimulant and growth promotor for tiger shrimp

The synergic effect using γ-irradiation in combination with hydrogen peroxide was applied for preparation of water-soluble and low molecular weight (Mw) β-glucan from yeast water-insoluble β-glucans. The structural characterization by gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) suggested that the γ-irradiation did not cause any change in basic structure of β-glucans, except for the reduction in degree of polymerization. The suitable degradation conditions were determined at 10% β-glucans in pH~9% and 1% hydrogen peroxide. The water-soluble oligoβ-glucan with Mw~15 kDa showed a significantly effect on the increase of body weight and survival rate of Penaeus monodon shrimps challenged with Vibrio parahaemolyticus and White spot syndrome virus (WSSV). The oral administration of 15 kDa-oligoβ-glucan with a daily dose of 1000 ppm strongly stimulated immune factors in tested shrimps such as phagocytosis activity (PA), phenoloxidase (PO) and superoxide dismutase (SOD). Furthermore, dietary of 1000 ppm 15 kDa-oligoβ-glucan also significantly up-regulated the gene expressions of lipopolysaccharide and β-1,3-glucan-binding protein (LGLP) and prophenoloxidase (proPO). The relative percent of survivals (RPS) of shrimps fed with 1000 ppm 15 kDa-oligoβ-glucan were 56.2% and 38.1% post the challenge with V. parahaemolyticus and WSSV, respectively. These results demonstrated that the water-soluble oligoβ-glucan with Mw~15 kDa prepared by synergic degradation method showed a very promising potential for application as a natural growth promotor and immunostimulant in shrimp culture.

Effect in white shrimp Litopenaeus vannamei of Eleutherine bulbosa (Mill.) Urb. Powder on immune genes expression and resistance against Vibrio parahaemolyticus infection

This study investigated the influence of Eleutherine bulbosa (Mill.) Urb. on the immune responses, bacterial population in the intestines, and resistance of white shrimp, Litopenaeus vannamei, against infection with Vibrio parahaemolyticus. Shrimp were fed with three dosages of powder, at 6.25 g kg–1 (P6.25), 12.5 g kg–1 (P12.5), and 25 g kg–1 (P25). One dosage of the crude extract was provided, 1.25 g kg–1 (E1.25), and the controls without administration of E. bulbosa consisted of a positive control (PC) and a negative control (NC). Feed supplementation was carried out for 30 days; then shrimp from all treatments were challenged by intramuscular injection with V. parahaemolyticus (106 cfu/mL), except for the NC. The results showed that supplementation with the powder and extract of E. bulbosa for 30 days resulted in significantly higher (P < 0.05) immune responses (total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RBs)), gene expression (prophenoloxidase (proPO), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP)), and total bacterial count (TBC) compared to PC/NC. In post challenge testing, there were significantly higher levels for THC, PO, RBs, proPO, LGBP, and PE (peroxinetin), and the treatments were able to suppress V. parahaemolyticus in the intestines, hepatopancreas, and muscles and to reduce damage to the muscles and hepatopancreas. The survival rate with P12.5 was significantly higher compared to the other treatments. It was concluded that the shrimp receiving supplementation with the powder and extract of E. bulbosa had increased immunity and resistance against V. parahaemolyticus infection, with the best dosage being the P12.5 treatment.

Effect of Amino Acid Substitution in the Penaeus monodon LGBP and Specificity Through Mutational Analysis

Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) is a pattern recognition protein (PRP) purified from the Penaeus monodon by Blue-Sepharose, Phenyl-Sepharose followed by Sephadex G-100 chromatography. P. monodon LGBP consist of 36 and 48 kDa subunits on 10% SDS-PAGE under reducing and non-reducing conditions respectively. Purified P. monodon LGBP agglutinates the fungal pathogen Candida glabrata, due to the presence of β-glucan (βG) on its surface. This agglutination was cross checked with the in silico docking analysis of LGBP-βG and LGBP-laminarin (isomeric form βG) interaction. As part of a strategy, to determine the precise role of P. monodon LGBP (Pm-LGBP) in pattern recognition mechanism mutations were introduced by in silico approach. In crustacean LGBP, RGD motif (Arg, Gly, Asp) plays the vital role in the cell adhesion and pattern recognition mechanism. Role of Asp in RGD motif was determined through amino acid substitution, introduction of a specific mutation D134K into a central area of the sugar-binding (βG) site resulted in complete loss of pathogen recognition and binding of Pm-LGBP to βG. These results demonstrate that, the RGD motif of Asp134 is essential for sugar binding in P. monodon. To our knowledge, P. monodon D134K is the first mutant shrimp LGBP which is unable to bind with the sugar residues. This mutant could be useful in the discovery of actual function of Pm-LGBP in the recognition of homologous symbionts. Docking analysis of LGBP binding towards β-glucan (i) LGBP without mutationv (ii) LGBP with mutation.

Effect of dietary Tenebrio molitor protein on growth performance and immunological parameters in Macrobrachium rosenbergii

Five diets were formulated with 0% (control), 4% (D4), 8% (D8), 12% (D12), and 16% (D16) Tenebrio molitor protein in prepared aquafeed to investigate the effects of T. molitor protein on growth performance, immunological parameters, and resistance against Lactococcus garvieae and Aeromonas hydrophila in Macrobrachium rosenbergii for 10 weeks. The weight gain (WG), percentage of weight gain (PWG), daily growth rate (DGR), specific growth rate (SGR), and protein efficiency ratio (PER) of D12 were significantly higher than those of the control, D4, and D8, but not significantly different from those of D16. The condition factor (K), feed conversion ratio (FCR), and survival rate (SR) did not differ significantly among groups. Lipid content decreased and protein content increased in the carcass and muscle of prawns as T. molitor protein contents increased. The total haemocyte count (THC) and numbers of semigranular cells (SCs), granular cells (GCs), and hyaline cells (HCs) significantly increased from the control to D16. Increased activities of superoxide dismutase (SOD), phenoloxidase (PO), lysozyme (LZ), and alkaline phosphatase (ALP) were detected as T. molitor protein contents increased. mRNA expression of prophenoloxidase (proPO), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), and α2-macroglobulin (α2-M) increased in haemocytes of M. rosenbergii from the control to D16. Phagocytosis of L. garvieae and the relative survival rate (RPS) against A. hydrophila were positively correlated with activities of protective enzymes and expression of proPO system immune-related genes in prawns fed diets with T. molitor protein. These results demonstrated that dietary inclusion of 12% T. molitor protein had the best effects on growth performance, immunological parameters, and resistance against L. garvieae and A. hydrophila, when balanced against the additional feed cost. This study supports the inclusion of T. molitor protein in functional aquafeed.

Long-term administration of diets containing Gracilaria tenuistipitata extract induce the expression of immune-related genes and increase the immune response and resistance against Vibrio harveyi in white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei juveniles (2.15 ± 0.16 g) fed diets containing Gracilaria tenuistipitata extract (GTE) at 0, 0.5, 1.0, and 2.0 g kg−1 for 16 weeks were examined for transcripts of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), integrin β (IB), prophenoloxidase (proPO) I, proPO II, α-2 macroglobulin (α-2 M), cytMnSOD, mtMnSOD, glutathione peroxidase (GPx), transglutaminase (TG) I, TG II, lysozyme, and heat shock protein 70 (HSP70) as well as immune parameters. Shrimp fed the 2.0 g kg−1 GTE diet significantly exhibited up-regulated expressions of these genes, increased hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, and earlier hemolymph clotting time. In a separate experiment, shrimp fed different diets for 16 weeks were challenged with Vibrio harveyi at 2 × 104 cfu shrimp−1 and then placed in seawater. The survival rate of shrimp fed the 2.0 g kg−1 GTE diet was significantly higher over 24–168 h post challenge. In conclusion, shrimp following long-term oral administration of a GTE-containing diet at 2.0 g kg−1 induced the expression of immune-related genes and exhibited increased their immune response and resistance against V. harveyi.

Endocrine-disrupting chemicals impair the innate immune prophenoloxidase system in the intertidal mud crab, Macrophthalmus japonicus

Endocrine-disrupting chemicals (EDCs), xenobiotics that interfere with endogenous hormone function, have been studied for their impacts in aquatic environments. However, there is limited information about the potentially hazardous impact of bisphenol A (BPA) and di-(2-ethylhexyl) phthalate (DEHP) on the marine environment. The aim of this study was to investigate the effects of BPA and DEHP on the immune response of the intertidal mud crab, Macrophthalmus japonicus. In order to examine immunological responses involving the prophenoloxidase (proPO) system, mRNA transcript and activity levels of six immune-related genes, including lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), proPO, phenoloxidase (PO), peroxinectin (PE), serine protease inhibitor (Serpin), and trypsin (Tryp), were assessed in M. japonicus hepatopancreas and gills exposed to BPA or DEHP. Expression of immune genes generally decreased in M. japonicus hepatopancreas and gills exposed to all concentrations of BPA by days 4 and 7. However, at day 1, expression of Serpin and Tryp genes was significantly increased in M. japonicus hepatopancreas and gills exposed to BPA. For DEHP exposure, all genes, with the exception of Serpin, were significantly downregulated in M. japonicus gills. In the hepatopancreas, gene expression of PO, proPO, and LGBP increased at day 1, and then decreased by day 7, while mRNA expression of Serpin and Tryp exhibited up-regulation over all exposure periods. In addition, PE gene expression was upregulated in hepatopancreas at day 7 in a dose-dependent manner. Taken together, these results indicated that the crab immune responses were perturbed by exposure to BPA, and, in particular, DEHP.

Effects of dietary Bacillus subtilis and Shewanella algae in expression profile of immune-related genes from hemolymph of Litopenaeus vannamei challenged with Vibrio parahaemolyticus

B. subtilis and S. algae effects in growth, survival and innate immunity were assessed on L. vannamei juveniles. During 60 days, shrimp were reared in three treatments: Bs, fed with 106 CFU of B. subtilis per gram of commercial feed, Sa, fed with 106 CFU of S. algae per gram of commercial feed and Control (without bacterial addition). Then, the animals were subjected to a V. parahaemolyticus challenge. For this purpose, four treatments were established: Control (shrimp not submitted to probiotic treatments), Vibrio (Vibrio challenged shrimp), Vibrio + Bs (Bs challenged shrimp) and Vibrio + Sa (Sa challenged shrimp). Shrimp hemolymph was sampled 45-days after rearing and 24 h post-challenge for quantification of prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) and hemocyanin (HEM) transcripts by qPCR. Moreover, shrimp final weight and survival were also verified. B. subtilis administration enhanced shrimp growth and improved proPO, LGBP and HEM expression levels before and after challenge. After 60-days of feeding, Sa final weight was higher than the Control, whereas Vibrio + Sa cumulative mortality after 48 h of Vibrio challenge was lower than Vibrio group. These results could be correlated with the proPO and LGBP up regulation in Vibrio + Sa compared to Vibrio group, protecting L. vannamei from the bacterial infection. Together, these results suggest the probiotic potential of B. subtilis e S. algae in the modulation of immune-related genes as a tool to control V. parahaemolyticus infection inside shrimp.

Molecular characterization of a pattern recognition protein LGBP highly expressed in the early stages of mud crab Scylla paramamosain

The early developmental stages of the mud crab Scylla paramamosain suffer from high mortality caused by pathogen infections; however, few immune associated factors are known. Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) functions as a typical pathogen recognition receptor and plays an important role in the innate immune system of invertebrates. In this study we characterized a LGBP gene (SpLGBP) which was highly expressed in the late embryonic, zoea I larval stage and hepatopancreas of S. paramamosain.. It encodes 364 amino acids, composed of several conserved domains like the bacterial glucanase motif. The recombinant SpLGBP protein (rSpLGBP) was obtained through the E.coli expression system, in which two 6◊His-tags were added to both C and N terminals during vector construction for the improvement of purification efficiency. In vivo the study showed that the SpLGBP mRNA was significantly up-regulated under Vibrio parahaemolyticus and a lipopolysaccharide (LPS) challenge in the hemocytes and hepatopancreas. The ELISA binding assay in vitro indicated that the rSpLGBP was capable of binding to LPSs and peptidoglycan (PGN). The rSpLGBP could agglutinate both G+ and G- bacteria in the presence of Ca2+. Our results suggest that SpLGBP may play an immunological role against pathogenic infection in the early developmental stages of S. paramamosain.

Expression of Macrobrachium rosenbergii lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) in Saccharomyces cerevisiae and evaluation of its immune function

Pattern recognition proteins (PRPs) activate the innate immune system in invertebrates, and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) is an important PRP with various biological functions. Here, the open reading frame (ORF) of Macrobrachium rosenbergii LGBP (MrLGBP) was cloned into plasmid vector pHAC181, then integrated into downstream of the GAL1 promoter of Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination, followed by its expression in the yeast eukaryotic system. The resulting recombinant LGBP contained a 3 × HA-tag at its C terminus and had a molecular weight of about 45 kDa, as evaluated by western blot analysis. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were ranged from 0.340 to 0.802 and 1.189–1.810 μM, respectively. The recombinant MrLGBP protein agglutinated almost all tested bacteria except Bacillus thuringiensis and Staphylococcus aureus. These results revealed that this recombinant protein exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. M. rosenbergii prawns were fed with the recombinant yeast strain MrLGBP for 1 month and challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. These prawns showed lower mortality and higher enzymatic activity and expression levels of immunity genes than did the control groups. All these results suggest that MrLGBP may play important roles in the innate immunity of crustaceans, and recombinant strain S. cerevisiae MrLGBP may be useful for the development of an effective immune feed additive in the future.

Identification of a crustacean β-1,3-glucanase related protein as a pattern recognition protein in antibacterial response

Prophenoloxidase (proPO) activating system is an important immune response for arthropods. β-1, 3-glucanase related protein (previously named as lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) in crustaceans) is a typical pattern recognition receptor family involved in the proPO activation by recognizing the invading microbes. In this study, we pay special attention to a bacteria-induced β-1,3-glucanase related protein from red swamp crayfish Procambarus clarkii, an important aquaculture specie in China. This protein, designated PcBGRP, was found a typical member of crustacean BGRP family with the glucanase-related domain and the characteristic motifs. PcBGRP was expressed in hemcoyes and hepatopancreas, and its expression could be induced by the carbohydrate and bacteria stimulants. The induction by lipopolysaccharide (LPS) and β-1,3-glucan (βG) was more significant than by peptidoglycan (PG). The response of PcBGRP to the native Gram-negative bacterial pathogen Aeromonas hydrophila was more obvious than to Gram-positive bacteria. Using RNA interference and recombinant protein, PcBGRP was found to protect crayfish from A. hydrophila infection revealed by the survival test and morphological analysis. A mechanism study found PcBGRP could bind LPS and βG in a dose-dependent manner, and the LPS recognizing ability determined the Gram-negative bacterium binding activity of PcBGRP. PcBGRP was found to enhance the PO activation both in vitro and in vivo, and the protective role was related to the PO activating ability of PcBGRP. This study emphasized the role of BGRP family in crustacean immune response, and provided new insight to the immunity of red swamp crayfish which suffered serious disease during the aquaculture in China.

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

Lipopolysaccharide- and β-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 β-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 β-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 β-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 β-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 sulfated galactans supplemented diet enhances the expression of immune genes and protects against Vibrio parahaemolyticus infection in shrimp

A sulfated galactans (SG) supplemented diet was evaluated for the potential to stimulate immune activity in shrimp Penaeus vannamei (P. vannamei). Shrimp given the SG supplemented diet (0.5, 1 and 2% w/w) for 7 days showed enhanced expression of the downstream signaling mediator of lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and immune related genes including p-NF-κB, IMD, IKKβ and IKKε, antimicrobial peptide PEN-4, proPO-I and II. Following immersion with Vibrio parahaemolyticus (V. parahaemolyticus) for 14 days, the shrimp given the SG supplemented diet (1 and 2% w/w) showed a decrease in bacterial colonies and bacterial toxin gene expression, compared to shrimp given a normal diet, and they reached 50% mortality at day 14. However, shrimp given the normal diet and challenged with the bacteria reached 100% mortality at day 6. SG-fed shrimp increased expression of immune genes related to LGBP signaling at day 1 after the bacterial immersion compared to control (no immersion), which later decreased to control levels. Shrimp on the normal diet also increased expression of immune related genes at day 1 after immersion which however decreased below control levels by day 3. Taken together, the results indicate the efficacy of the SG supplemented diet to enhance the immune activity in shrimp which could offer protection from V. parahaemolyticus infection.

Physiological and immune response of Litopenaeus vannamei undergoing the acute phase of the necrotizing hepatopancreatitis disease and after being treated with oxytetracycline and FF

The physiological and immune responses of adult shrimp (Litopenaeus vannamei) undergoing the acute phase of the necrotizing hepatopancreatitis (NHP) disease and the efficiency of oxytetracycline (OXI) and florfenicol (FF) to eliminate the pathogen were evaluated. Four shrimp groups were considered: three groups infected with necrotizing hepatopancreatitis bacteria (NHP-B) (two treated with antibiotics and a positive control) and one group non-infected (negative control). Hemolymph concentration of glucose, lactate, acylglycerides, cholesterol, total protein, aminotransferases, superoxide dismutase, and the transcriptional expression of several immune related genes were monitored at the acute phase of the disease, and at 15 and 20 days after administration of antibiotics (daa). Shrimp from the positive control registered a mortality of 100%. NHP-B infection affected the immu-nophysiological response of shrimp; herein, most of the parameters were significantly up regulated in infected shrimp before the use of antibiotics, compared to the negative control. Increased transcriptional levels of clotting protein, lipopolysaccharide and β-1-3-glucan binding protein (LGBP), serine protease, peroxinectin, lysozyme, heat shock proteins (HSP) 60 and 70 were detected in shrimp treated with OXI. At 20 daa NHP-B was still detected in FF-treated shrimp, but not in OXI-treated shrimp. It is hypothesized that despite none of the antibiotics per se eliminated the bacterium, both had a negative effect on its virulence. OXI seems to have a greater effect, allowing shrimp to integrate a better immune response at 15 daa.

Antimicrobial and Antitumor Activities of Novel Peptides Derived from the Lipopolysaccharide- and β-1,3-Glucan Binding Protein of the Pacific Abalone Haliotis discus hannai.

Antimicrobial peptides are a pivotal component of the invertebrate innate immune system. In this study, we identified a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) gene from the pacific abalone Haliotis discus hannai (HDH), which is involved in the pattern recognition mechanism and plays avital role in the defense mechanism of invertebrates immune system. The HDH-LGBP cDNA consisted of a 1263-bp open reading frame (ORF) encoding a polypeptide of 420 amino acids, with a 20-amino-acid signal sequence. The molecular mass of the protein portion was 45.5 kDa, and the predicted isoelectric point of the mature protein was 4.93. Characteristic potential polysaccharide binding motif, glucanase motif, and β-glucan recognition motif were identified in the LGBP of HDH. We used its polysaccharide-binding motif sequence to design two novel antimicrobial peptide analogs (HDH-LGBP-A1 and HDH-LGBP-A2). By substituting a positively charged amino acid and amidation at the C-terminus, the pI and net charge of the HDH-LGBP increased, and the proteins formed an α-helical structure. The HDH-LGBP analogs exhibited antibacterial and antifungal activity, with minimal effective concentrations ranging from 0.008 to 2.2 μg/mL. Additionally, both were toxic against human cervix (HeLa), lung (A549), and colon (HCT 116) carcinoma cell lines but not much on human umbilical vein cell (HUVEC). Fluorescence-activated cell sorter (FACS) analysis showed that HDH-LGBP analogs disturb the cancer cell membrane and cause apoptotic cell death. These results suggest the use of HDH-LGBP analogs as multifunctional drugs.

White shrimp Litopenaeus vannamei that have received fucoidan exhibit a defense against Vibrio alginolyticus and WSSV despite their recovery of immune parameters to background levels

White shrimp Litopenaeus vannamei receiving fucoidan at 2, 6, and 10 μg g−1 after 0–144 h or 0–120 h were examined for immune parameters (haemograms, phenoloxidase activity, respiratory burst, and superoxide dismutase activity), proliferation of haemocyte in the haematopoietic tissue (HPT), gene expression, and phagocytic activity and clearance efficiency to Vibrio alginolyticus. Immune parameters and mitotic index of HPT increased after 3–24 h, reached their maxima after 48–72 h, and returned to background values after 144 h. Transcripts of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), prophenoloxidase (proPO) I, proPO II, astakine, and haemocyte homeostasis-associated protein (HHAP) were up-regulated to a maximum after 48–72 h and returned to background values after 144 h. Phagocytic activity and clearance efficiency to V. alginolyticus increased after 12 h, reached its maximum after 48 h, and continued to remain higher after 120 h. In another experiment, shrimp receiving fucoidan after 48 h and 144 h were respectively challenged with V. alinolyticus at 6 × 106 colony-forming units (cfu) shrimp−1 or challenged with WSSV at 1.2 × 105 copies shrimp−1 and then placed in seawater. The survival rate of shrimp receiving fucoidan was significantly higher than in controls. In conclusion, shrimp receiving fucoidan showed a proliferation of HPT, increased immune parameters, and up-regulated transcripts of LGBP, PX, proPO I, proPO II, astakine, and HHAP after 48 h. Shrimp receiving fucoidan exhibited a defense against V. alginolyticus and WSSV, even after immune parameters recovered to background levels.

Lipopolysaccharide- and β-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 β-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 β-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, β-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 β-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.

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.