Isolation and characterization of a lectin-like chitinase from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii.

A lectin was isolated from the hepatopancreas of freshwater prawn, Macrobrachium rosenbergii by affinity chromatography using mucin-sepharose matrix. The purity of the isolated lectin was confirmed in native gradient PAGE that showed a single protein band of ∼37.9 kDa. In SDS-PAGE also one band of ∼43.3 kDa molecular weight was observed that indicated the protein to be a monomer. The band from the SDS-PAGE gel was identified through mass spectrometry as chitinase 1. The purified chitinase (50 µg/ml) hemagglutinated rabbit RBCs and, mucin and glucose inhibited hemagglutination with minimum concentrations of 0.1 mg/ml and 100 mM, respectively. Bacterial agglutination with Gram -ve Vibrio harveyi, Aeromonas sobria and Escherichia coli was also observed by this protein. Thus, chitinase 1 showed lectin-like properties besides its chitin hydrolytic activity. In western blot with hepatopancreas sample, rabbit antiserum against chitinase 1 cross-reacted to two additional proteins namely, chitinase 1C and obstructor E (a chitin-binding protein, CBP), besides its specific reactivity. An indirect ELISA was developed with the antiserum to quantify chitinases/CBP in hepatopancreas and serum samples of M. rosenbergii. The assay was used in samples from juvenile prawns following V. harveyi challenge. At 72 h post-challenge, significantly higher levels of chitinases/CBP were quantified in the hepatopancreas of the challenged group (1.8 ± 0.2 mg/g tissue) compared to the control (1.2 ± 0.1 mg/g tissue). This study suggests that the chitinase 1 protein with lectin-like properties is possibly induced at the protein level and can be putatively involved in the innate immune response of M. rosenbergii.

Characterization of a Lipopolysaccharide- and Beta-1,3-Glucan Binding Protein (LGBP) from the Hepatopancreas of Freshwater Prawn, Macrobrachium rosenbergii, Possessing Lectin-Like Activity.

The study focuses on the isolation, characterization, and expression analysis of a lectin from the hepatopancreas of Macrobrachium rosenbergii. The protein was isolated by affinity chromatography on a melibiose-agarose column. The molecular weight of the native protein was found to be ~120 kDa which consists of a single polypeptide of ~39.5 kDa. On mass spectrometric analysis, the protein was identified as lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP). LGBP showed hemagglutination with rabbit RBC like a lectin and its carbohydrate-binding specificity was determined by the hemagglutination inhibition test. The protein also showed antibacterial activity against two Gram-negative bacteria Vibrio harveyi and Aeromonas sobria, and one Gram positive bacteria Bacillus cereus in the disc diffusion test. Rabbit antiserum was raised against the purified LGBP and used to develop a sandwich ELISA system for quantitation of the protein in hepatopancreas and serum samples of M. rosenbergii. The expression of the LGBP transcripts in muscle, hepatopancreas, and gill tissues from M. rosenbergii juveniles at 72 h post-challenge of V. harveyi was not modulated as noticed in qPCR analysis. However, significant increases in the concentrations of LGBP protein in hepatopancreas (5.23 ± 0.45 against 3.43 ± 0.43 mg/g tissue in control) and serum (1.08 ± 0.14 against 0.61 ± 0.08 µg/ml in control) were observed in the challenged group of prawns in ELISA suggesting its putative role against bacterial infections. The study for the first time characterized the native LGBP of M. rosenbergii showing a multifunctional role in immunity.

Dietary brewer's spent yeast enhances growth, hematological parameters, and innate immune responses at reducing fishmeal concentration in the diet of climbing perch, Anabas testudineus fingerlings.

A 60-day feeding trial was conducted to optimally reduce the fishmeal level in climbing perch (Anabas testudineus) fingerling diet using a dietary brewer's spent yeast biomass (BSY) based diet. In this study, five isonitrogenous (35% CP) and isocaloric (19.15 MJ/Kg) feeds were prepared by replacing 0 (BSY0), 25% (BSY25), 50% (BSY50), 75% (BSY75) and 100% (BSY100) of fishmeal protein using BSY protein. A total of 225 numbers of uniform-sized climbing perch fingerlings (3.29 ± 0.09 g) were randomly stocked in the 15 rectangular FRP (Fiber-reinforced plastic) tanks (150 L capacity). The experimental fish were fed twice daily at 4% BW for the first fortnight and later reduced to 3% BW based on satiation. At the end of the feeding trial, the weight gain (WG) of fish increased with the increasing BSY incorporation rates corresponding to fishmeal content and peaked at 77.88%, and beyond that, WG decreased. Food conversion ratios decreased as dietary BSY levels increased and peaked at 76.28%. All other growth and feed utilization parameters followed a similar trend of weight gain. Hepatosomatic index (HSI) and viscerosomatic index (VSI), A:G ratio, serum catalase activity, and monocytes were unaffected and the total serum protein, albumin, globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), respiratory burst activity, lysozyme levels, myeloperoxidase activity, hemoglobulin, red blood cells, white blood cells, neutrophils, eosinophils, lymphocytes, and gut protease activities were increased significantly (P < 0.05) with the increasing replacement levels and peaked between 25 and 75%. The serum SOD activity and total platelets were decreased, whereas the serum uric acid and gut amylase activities were increased significantly to the increasing levels of FM replacement in the diets (P < 0.05). Among treatments, the BSY100 resulted in an overall poor growth response combined with relatively reduced values in nearly all biochemical parameters. The whole-body composition was nearly unaffected. The integrated biomarker response of various biochemical indicators from the different treatments has shown that the 50% fishmeal protein can be optimally replaced by BSY, which would cause an 18% reduction in the Economic conversion ratio (ECR) and -270.28 gCO2e- reduction in carbon footprint value per kg of climbing perch fingerlings production.

Effect of hormonal stimulation on captive broodstock maturation, induced breeding and spawning performance of striped snakehead, Channa striata (Bloch, 1793).

In the present study, there was assessment of effects of gonadotropin treatments on broodstock maturation, induced breeding, and spawning outcomes of striped snakehead in captivity. The striped snakehead (n = 128) were equally distributed in four concrete tanks (15 m2) and hormone implants (500 IU human chorionic gonadotropin (hCG)/kg body weight) were inserted intramuscularly and striped snakehead broodstock administered this treatment were confined in two tanks and striped snakehead of a non-implanted group were confined in two tanks. The hormone implanted striped snakehead had a greater (P < 0.05) gonadosomatic index (GSI) and oocyte diameter in comparison to non-implanted striped snakehead. In a subsequent experiment, hCG and carp pituitary homogenate (CPH) were evaluated for inducing breeding. Dosages of hCG used were, 2,000 (TH1), 3000 (TH2), and 4000 (TH3) IU hCG/kg body weight of females. Dosages of CPH were, 20 (TP1), 30 (TP2), and 40 (TP3) mg CPH/kg body weight of females. Males were administered 0.75 of the dosage administered to females. The values for reproductive variables were estimated. Fertilization (89.0 ±â€¯3.0 %) and hatching (92.0 ±â€¯1.0 %) rates were greater (P < 0.05) in the TH1 group of implanted striped snakehead. Relative fecundity (19,023 ±â€¯2211), as well as fertilization (96.2 ±â€¯2.4 %), and hatching (96.6 ±â€¯1.7 %) rates were greater in the TP2 group of the implanted striped snakehead. The results from the present study indicate broodstock treated with gonadotropins had greater spawning outcomes which might facilitate mass scale breeding and fertilized egg as well as juvenile production of striped snakehead in captivity.

Lectin-Like Activity of Hemocyanin in Freshwater Prawn, Macrobrachium rosenbergii.

Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of Macrobrachium rosenbergii, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0-10.0 and temperature range of 10-40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to N-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in M. rosenbergii possesses lectin-like activity.

Differential affinity of natural haemagglutinin of Macrobrachium rosenbergii towards vertebrate erythrocytes: effect of sex, size and moult stage on haemagglutination titre.

Lectins play important role in innate immunity of animals. The affinity of the natural haemagglutinin of the giant freshwater prawn Macrobrachium rosenbergii towards vertebrate erythrocytes and its level with relation to sex, size and moult stages were studied. The strongest agglutinating titres in haemolymph of prawns were marked against guinea pig, chicken, Clarias batrachus, and rabbit erythrocytes, and the weakest towards cattle, dog, horse and goat erythrocytes. A moderately agglutinating titre was evident in duck and human erythrocytes. The haemolymph of adult, male or intermoult stage prawns weighing more than 100 g had the highest haemagglutinating activity as compared to their respective counterparts with varied responses observed towards various erythrocytes.

In vivo humoral and cellular reactions, and fate of injected bacteria Aeromonas hydrophila in freshwater prawn Macrobrachium rosenbergii.

Live non-opsonized and opsonized Aeromonas hydrophila were injected into juveniles of freshwater prawn Macrobrachium rosenbergii to study the cells involved in phagocytosis, distribution of bacteria, cellular reactions and clearance of both forms of bacteria from the system. The bacteria were rapidly distributed to various tissues viz., gills, heart, hepatopancreas within 1h, and the tissues revealed haemocytic nodule formation after 3 h of injection. There was rapid clearance of both the forms of bacteria from the circulation. However, clearance efficiency was significantly (P < 0.05) faster in the case of opsonized bacteria at 12 h after injection. Similarly, the nodule formation, that was prominent in cardiac musculature, was rapidly eliminated from the tissues of the group injected with opsonized bacteria as compared to non-opsonized bacteria injected group, thus confirming the existence of opsonic factors in haemolymph of this prawn. In another experiment, various dose levels of bacteria were injected intramuscularly into prawns and haemolymph was collected after 1, 6, 24, 72 h and 7 days of injection to study various immune parameters. Although, no major alterations in the total and differential haemocyte counts were observed in bacteria injected prawns compared to control, there was a significant decline in phenoloxidase activity in the highest dose bacteria injected group at the earlier phase and a rise in agglutinin levels at the later phase of the experimental period in the higher dose groups.

Dietary administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress.

Haemocyte count, phenoloxidase activity, agglutinin levels, total protein content, bacterial clearance efficiency, resistance to the pathogen Aeromonas hydrophila and nitrite stress were measured in the giant freshwater inter-moult sub-adult prawn Macrobrachium rosenbergii (15-20 g) which had been fed diets containing bovine lactoferrin (Lf) at 50, 100, 200mg kg(-1) feed for 7 or 14 days. M. rosenbergii fed a diet containing 100mg Lf kg(-1) diet for 7 days showed significant (P<0.05) increase in total protein levels, agglutination titres against bacteria A. hydrophila and rabbit RBC, phenoloxidase activity, bacterial clearance (as observed through reduced number of circulating bacteria) as well as survival against A. hydrophila challenge. Increased bacterial clearance was also noticed in prawns fed Lf at 50 or 200mg kg(-1) for 14 days compared to control. Feeding of Lf at 50mg kg(-1) diet for 7 or 14 days was able to enhance only PO activity and reduce percent mortality against A. hydrophila challenge compared to its control. Total haemocyte count was higher in the lowest dose of Lf feeding, i.e. 50mg kg(-1) for 7 days. However, there was no significant alteration in the differential haemocyte population with respect to graded levels of Lf feeding for 7 or 14 days. A notable reduction in mortality percent after 120 h of nitrite stress was observed in prawn fed Lf at 100mg kg(-1) diet for 14 days. On the contrary, feeding of the highest dose of Lf, i.e. 200mg kg(-1) diet for 14 days failed to stimulate most of the innate immune parameters or reduce the percent mortality against A. hydrophila challenge or nitrite stress. It is therefore concluded that administration of Lf in the diet at 100mg kg(-1) for 7 days could enhance the immune ability of M. rosenbergii and increase its resistance to A. hydrophila infection or nitrite stress.

Immunomodulation by 'Immuplus (AquaImmu)' in giant freshwater prawn, Macrobrachium rosenbergii (De Man).

ImmuPlus, a polyherbal commercial formulation was used to modulate the immune system of commercially important giant freshwater prawn M. rosenbergii. The prawns were fed with basal diet supplemented with ImmuPlus at 1g/kg feed for 4 weeks. Results showed that the phenoloxidase activity (PO), haemagglutination and lysozyme activities were significantly elevated in ImmuPlus-fed prawn up to 3 weeks of feeding and declined after 4 weeks of feeding. The total protein level in ImmuPlus-fed prawn raised up to 2nd week of feeding. Incorporation of ImmuPlus at the rate of 1g/kg feed in the diet of prawn for 3 weeks may be beneficial in raising the immune status of prawn.