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Marine organisms represent a huge source of novel compounds for the development of effective antimicrobial drugs. The present study focus on the purification of the antimicrobial peptide crustin from the haemolymph of the blue swimmer crab, Portunus pelagicus, by blue Sepharose CL-6B matrix assisted affinity column chromatography. Crustin showed a single band with a molecular mass of 17 kDa in SDS-PAGE analysis. The XRD analysis exhibited peaks at 32° and 45° while a distinct peak with a retention time of 1.8 min resulted in high performance liquid chromatography (HPLC) pointing out the crystalline nature and purity of crustin, respectively. Crustin purified from P. pelagicus (Pp-Cru) showed immunological activities, triggering encapsulation, phagocytosis on Sepharose beads and yeast (Saccharomyces cerevisiae) respectively. Furthermore, encapsulation of GFP tagged V. parahaemolyticus in Artemia salina and challenging study were assessed under CLSM and the potential of Pp-Cru was examined in vivo. In addition, the growth reduction and biofilm inhibition potential of Pp-Cru on Staphylococcus aureus, Enterococcus faecalis (Gram- positive bacteria) and Pseudomonas aeruginosa, Escherichia coli (Gram-negative bacteria) was evidenced by inverted and confocal laser scanning microscopic analysis, revealing that 100 μg/ml of Pp-Cru can disrupt the biofilm matrix thereby the thickness of biofilm was significantly reduced. Overall, the present investigation might provide a sensitive platform to realize the significant function of Pp-Cru in crustacean immune mechanism as well as its potential to bacterial growth inhibitor. The functional properties of purified Pp-Cru antimicrobial peptide may lead to a superior understanding of innate immune response in P. pelagicus species, which suggest the promising application for drug development in aquaculture., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish (Danio rerio) challenged with GFP tagged Vibrio parahaemolyticus Dahv2 was studied. The cell free extract of probiotic B. licheniformis Dahb1 at 100 μg mL -1 showed growth inhibition of V. parahaemolyticus Dahv2 in vitro. B. licheniformis Dahb1 also inhibited the biofilm growth of GFP tagged V. parahaemolyticus Dahv2 at 100 μg mL -1 in vitro. The growth and survival of zebrafish was tested using probiotic B. licheniformis Dahb1. Weight (1.28 g) of zebrafish that received the cell free extract was much higher than in control (1.04 g). The mortality of zebrafish infected with GFP tagged V. parahaemolyticus Dahv2 at 10 7 Cfu mL -1 (Group IV) was 100%, whereas a complete survival of zebrafish that received the cell free extract of B. licheniformis Dahb1 at 10 7 Cfu mL -1 (Group VII) was observed after 30 days. The number of GFP tagged V. parahaemolyticus Dahv2 colonies in the intestine and gills significantly reduced after treatment with the cell free extract of B. licheniformis Dahb1. Furthermore, a significant decrease in the fluorescent colonies of GFP tagged V. parahaemolyticus Dahv2 was observed after treatment with the cell free extract of B. licheniformis Dahb1 under confocal laser scanning microscopy (CLSM). In conclusion, the cell free extract of B. licheniformis Dahb1 could prevent Vibrio infection by enhancing the growth and survival of zebrafish., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

β- 1, 3-glucan binding protein (β-GBP), a pattern recognition protein (PRP), plays a critical role in triggering the innate immune response by detecting β-glucan found on the surface of microbes. In the present study, β-GBP was purified from the haemolymph of rice field crab Paratelphusa hydrodromus by affinity column chromatography. The monomeric protein Ph-β-GBP appeared as a single band with a molecular weight of approximately 95 kDa in SDS-PAGE analysis and its purity was determined to be 89% by HPLC. MALDI-TOF/TOF analysis revealed that, the purified 95 kDa protein display 36% similarity with β-GBP of crayfish Astacus lepidodactylus. Purified Ph-β-GBP exhibited increased agglutination, phagocytic activity and encapsulation in a dose-dependent manner, indicating the involvement of Ph-β-GBP in cellular immune response against pathogens in crustaceans. Moreover, addition of Ph-β-GBP increased the prophenoloxidase (proPO) and serine protease activity, possibly contributing to the clearance of pathogens. The antioxidant activity of Ph-β-GBP was determined by DPPH radical scavenging activity demonstrates maximum scavenging activity of 78.4%. In addition, RBC membrane stabilization and inhibition of protein (albumin) denaturation proved anti-inflammatory property of Ph-β-GBP. Furthermore, light microscopic and confocal laser scanning microscopic analysis revealed that the reactive compound (laminarin and Ph-β-GBP) reduced the biofilm thickness of Gram-positive (Enterococcus faecalis) and Gram-negative (Vibrio parahaemolyticus) bacteria at the concentration of 25 μg/ml. Taken together, our results demonstrate that, the β-GBP triggers proPO activating system in rice field crab P. hydrodromus and plays a vital role in innate defense mechanism against invading pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The anti-Vibrio activity of essential oils (EOs) of nine medicinal plants was tested against 28 Vibrio spp. isolated from diseased Fenneropenaeus indicus. EO of Nigella sativa exhibited anti-Vibrio activity against all Vibrio spp. and greater inhibition was noted for the isolate V2 which was identified as Vibrio parahaemolyticus Dahv2. Further, EO of N. sativa effectively inhibited V. parahaemolyticus Dahv2 with an inhibition zone of 23.9mm at 101.2μgml(-1). Moreover, EO of N. sativa revealed anti-biofilm activity at 101.2μgml(-1) against V. parahaemolyticus Dahv2 and inhibited the growth of V. parahaemolyticus Dahv2 at 100μgml(-1).In vivo experimental infection studies showed that the survival of Artemia spp. infected with V. parahaemolyticus Dahv2 at 1×10(3)cfuml(-1) was only 40%. However, the survival of Artemia spp. was significantly increased after challenge with 100μgml(-1) of EO of N. sativa. EO of N. sativa showed higher anti-oxidant potential and total phenol content than other EOs tested. The anti-oxidant activity of EO of N. sativa was highly correlated to their total phenolic contents (r=0.836, P<0.05). This observation suggests that EO of N. sativa protected the Artemia spp. after experimental infection of V. parahaemolyticus Dahv2., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the present study, we synthesized and characterized a probiotic Bacillus licheniformis cell free extract (BLCFE) coated silver nanoparticles (BLCFE-AgNPs). These BLCFE-AgNPs were characterized by UV-visible spectrophotometer, XRD, EDX, FTIR, TEM and AFM. A strong surface plasmon resonance centered at 422 nm in UV-visible spectrum indicates the formation of AgNPs. The XRD spectrum of silver nanoparticles exhibited 2θ values corresponding to the silver nanocrystal. TEM and AFM showed the AgNPs were spherical in shape within the range of 18.69-63.42 nm and the presence of silver was confirmed by EDX analysis. Light and Confocal Laser Scanning Microscope (CLSM) images showed a weak adherence and disintegrated biofilm formation of Vibrio parahaemolyticus Dav1 treated with BLCFE-AgNPs compared to control. This result suggests that BLCFE-AgNps may be used for the control of biofilm forming bacterial populations in the biomedical field. In addition, acute toxicity results concluded that BLCFE-AgNPs were less toxic to the fresh water crustacean Ceriodaphnia cornuta (50 μg/ml) when compared to AgNO3 (22 μg/ml). This study also reports a short term analysis (24 h) of uptake and depuration of BLCFE-AgNPs in C. cornuta., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The pathways by which pathogens invade Fenneropenaeus indicus, the potential colonization in various tissues and the disease transmission mechanisms are unclear. The aims of the present study were to visualize the colonization and pathogenesis of GFP-tagged Vibrio parahaemolyticus, in various tissues of F. indicus to evaluate the pathogen interaction. Among the three strains isolated, a virulent strain VpDAHV2 was tagged with green fluorescent protein (GFP-VpDAHV2) and validated for both its growth characteristics and its virulence as a genuine model for F. indicus infection. VpDAHV2 was positive for toxR and tlh genes and negative for tdh genes. CLSM images revealed that maximum colonization was observed in the haemolymph of the F. indicus challenged with GFP-VpDAHV2. The haemolymph was the primary site for the colonization of GFP-VpDAHV2 in F. indicus. The enteric localization occurred independently of the flagellum or motility of GFP-VpDAHV2 through the intestinal route. The F. indicus infection model suggests that the haemolymph and the intestine represent the sites of infection by GFP-VpDAHV2, and hence are the active sites of pathogen interactions. GFP tagging of V. parahaemolyticus is a new and systemic approach to determine the presence of bacteria in vivo for the confirmation of host pathogen interactions in aquaculture studies. [ABSTRACT FROM AUTHOR]

Abstract: Alpha 2 macroglobulin (α2M) is a non-specific protease inhibitor involved in host defence mechanism in invertebrates. By c-DNA cloning and sequencing, α2M gene was identified from the haemocytes of Indian white shrimp Fenneropenaeus indicus with an open reading frame of 4599bp encoding 1532 amino acids with a signal sequence of 26 amino acids. The calculated molecular mass of the mature protein is 170.6kDa with an estimated pI of 6.13. The F. indicus α2M (Fi-α2M) sequence contains putative conserved functional domains including a GCGEQNM thioester region and a receptor-binding domain which are present in other invertebrate and vertebrate α2Ms. Sequence comparison and phylogenetic analysis revealed that Fi-α2M displays the highest similarity with Litopenaeus vannamei (96%). QRT-PCR analysis of challenge experiments by Vibrio harveyi, GFP tagged Vibrio parahaemolyticus (GFP-Vp) and WSSV infection of F. indicus significantly increased the α2M mRNA transcript up to 24h in the haemocytes. In addition, confocal laser scanning microscopy (CLSM) confirms that the GFP-Vp treated F. indicus have high proliferation of GFP-Vp in haemolymph and intestine at 24h. These results suggest that α2M is an important molecule in shrimp immune system. [Copyright &y& Elsevier]

Abstract: Penaeidins are members of a special family of antimicrobial peptide existing in penaeid shrimp and play an important role in the immunological defense of shrimp. Here, we report a penaeidin sequence cloned from the Indian white shrimp Fenneropenaus indicus (Fein-Penaeidin). The Fein-Penaeidin open reading frame encodes a 77 amino acid peptide including a 19 amino acid signal peptide. The deduced amino acid sequences of Fein-Penaeidin include a proline rich N-terminal domain and a carboxyl-domain that contains six cysteine residues. Structural analysis revealed an alpha-helix in its secondary structure and the predicted 3D structure indicated two-disulphide bridges in the alpha-helix. Phylogenetic analysis and sequence comparison with other known peaneidin suggest the gene shows high similarity to that of penaeidin from Peneaus monodon (95%), F. indicus (80%) and Fenneropenaeus chinensis (74%). Fein-Penaeidin was examined in normal and microbial challenged shrimp and was found to be constitutively expressed in haemocytes, Heart, gills, muscles, intestine, hepatopancreas and eyestalk. Bacterial challenge resulted in mRNA up-regulation, inducing expression at 6h post injection indicating the penaeidin involved in the innate immunity. [Copyright &y& Elsevier]

The study is carried out to understand the antimicrobial and immunological response of a potential immune molecule lectin, Mm Lec isolated from haemolymph of Speckled shrimp, Metapenaeus monoceros. Mm Lec was purified using mannose coupled Sepharose CL-4B affinity chromatography, which was further subjected on SDS-PAGE to ascertain the distribution of their molecular weight. Sugar binding specificity assay was conducted at various pH and temperatures to investigate the binding affinity of Mm Lec towards the specific carbohydrate molecule. Functional analysis of immune molecule Mm Lec included haemagglutination assays performed using human erythrocytes and yeast agglutination activity against Saccharomyces cerevisiae which, were analyzed using light microscopy. In order to study the antimicrobial activity, two Gram-negative (Vibrio parahaemolyticus and Aeromonas hydrophila) and two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria were treated with purified Mm Lec. Moreover, these bacterial species were also treated at different concentration of the Mm Lec to speculate the antibiofilm properties of Mm Lec which was analyzed under Light Microscopy and Confocal Laser Scanning Microscopy. In addition, other functional characterization of Mm Lec showed the uniqueness of Mm Lec in agglutination of human erythrocyte as well as the cells of yeast Saccharomyces cerevisiae. Also, the phenoloxidase activity and encapsulation assay was evaluated. MTT assay displayed that Mm Lec are potent in anticancer activity. The study will help to understand the immunological interference and antimicrobial nature of Mm Lec which would be supportive in establishing a potential therapeutic tool and to develop better and novel disease control strategies in shrimp and farmed aquaculture industries as well as in health management. • An immune molecule Mm Lec (80 kDa) was purified from haemolymph of Speckled shrimp, Metapenaeus monoceros. • The Mm Lec exhibit, haemagglutination and yeast agglutination, the phenoloxidase activity and encapsulation activity. • The antimicrobial and antibiofilm properties of Mm Lec against certain aquatic pathogens were evaluated. [ABSTRACT FROM AUTHOR]

Abstract The present study reveals purification and characterization of a C-type lectin from the serum of pearl spot, Etroplus suratensis (Es-Lec). The Es-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 75 kDa in SDS-PAGE. The surface morphology of purified Es-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 2.958 min was appeared in high performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis expresses a single peak at 31.8372̊ and MALDI-TOF peaks which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Es-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy and haemagglutination inhibition was also done. In addition, purified Es-Lec showed the broad spectrum of antibacterial activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila. Antibiofilm potential of purified Es-Lec against selected Gram-negative bacteria exhibited the disruption of biofilm architecture at the concentration of 50 μg ml−1 and also it exhibited antiviral and anticancer activity. Graphical abstract Image 1 Highlights • Es-Lec of 75 kDa was purified by mannose-sepharose column chromatography. • Its crystalline nature, quality and purity were assessed by XRD, HPLC and MALDI-TOF respectively. • Es-Lec exhibited haemagglutination and yeast agglutination activity. • They possess antibacterial, antibiofilm and antiviral activity. • They exhibited an anti-cancer potential against breast cancer cell lines MDA-MB-231. [ABSTRACT FROM AUTHOR]

Penaeid prawns are considered as most demanding fishery resources. The current study aims to purify and characterize lectin from the haemolymph of Penaeus semisulcatus. The semisulcatus-lectin was purified by affinity chromatography using mannose coupled Sepharose CL-4B column and purified lectin exhibited a single band of 66 kDa in SDS-PAGE. The purity and crystalline structure of purified lectin was confirmed by HPLC and X-ray diffraction analysis. Semisulcatus-lectin exhibited yeast agglutination activity against Saccharomyces cerevisiae and agglutinated human erythrocytes. Semisulcatus-lectin was evaluated for phenol oxidase activation and phagocytic activities. It was observed that semisulcatus-lectin had antibacterial activity against Gram-negative Vibrio parahaemolyticus and Aeromonas hydrophila , suggesting a potential therapeutic strategy in aquaculture industry for disease management. • Immune molecule lectin was purified from the P. semisulcatus by affinity chromatography. • Its crystalline nature, quality and purity were assessed by XRD and HPLC respectively. • P. semisulcatus exhibited haemagglutination, yeast agglutination and antibacterial activity. • Antibiofilm activity of lectin against Gram negative bacteria was shown. [ABSTRACT FROM AUTHOR]

In this study, the pathogenicity of GFP tagged Vibrio parahaemolyticus Dahv2 and the protective effect of the probiotic strain, Bacillus licheniformis Dahb1was studied on the Asian catfish, Pangasius hypophthalmus . The experiment was carried out for 24 days with three groups and one group served as the control (without treatment). In the first group, P. hypophthalmus was orally infected with 1 mL of GFP tagged V. parahaemolyticus Dahv2 at two different doses (10 5 and 10 7 cfu mL −1 ). In the second group, P. hypophthalmus was orally administrated with 1 ml of the probiotic B. licheniformis Dahb1 at two different doses (10 5 and 10 7 cfu mL −1 ). In the third group, P. hypophthalmus was orally infected first with 1 mL of GFP tagged V. parahaemolyticus Dahv2 followed by the administration of 1 mL of B. licheniformis Dahb1 (combined treatment) at two different doses (10 5 and 10 7 cfu mL −1 ). The growth, immune (myeloperoxidase, respiratory burst, natural complement haemolytic and lysozyme activity) and antioxidant (glutathione-S-transferase, reduced glutathione and total glutathione) responses of P. hypophthalmus were reduced after post infection of GFP tagged V. parahaemolyticus Dahv2 compared to control. However, after administration with the probiotic B. licheniformis Dahb1 at 10 5 cfu mL −1 , P. hypophthalmus showed significant increase in the growth, immune and antioxidant responses compared to 10 7 cfu mL −1 . On the otherhand, the growth, immune and antioxidant responses of P. hypophthalmus infected and administrated with combined GFP tagged Vibrio + Bacillus at 10 5 cfu mL −1 were relatively higher than that of GFP tagged V. parahaemolyticus Dahv2 and control groups but lower than that of probiotic B. licheniformis Dahb1 groups. The results of the present study conclude that the probiotic B. licheniformis Dahb1 at 10 5 cfu mL −1 has the potential to protect the P. hypophthalmus against V. parahaemolyticus Dahv2 infection by enhancing the growth, immune and antioxidant responses. The probiotic B. licheniformis Dahb1 would be effectively used in the treatment of aquatic diseases for improvement of aquaculture industry. [ABSTRACT FROM AUTHOR]

Haemocyanin (Hc) is a non-specific innate immune protein present in the haemolymph of arthropods and molluscs. In the current study, we characterized the structural and immunological properties of Hc from grooved tiger shrimp, Penaeus semisulcatus. Hc was isolated from the haemolymph of P. semisulcatus by gel filtration column chromatography using Sephadex G-100. High-performance liquid chromatography of the purified Hc emerged as a single peak through a retention time of 3.3 min demonstrating the homogeneity nature of the protein. X-ray diffraction analysis revealed a distinct peak at 31.7° indicating the crystalline character of the purified Hc. Circular dichroism spectra of the purified Hc displayed negative ellipticity bands close to 225 nm and 208 nm representing β-sheet secondary structure. The purified Hc agglutinated sheep RBCs, yeast Saccharomyces cerevisiae and fungal Candida albicans. In addition, the purified Hc displayed antibacterial activity against Gram-positive bacteria (Bacillus thuringiensis and Bacillus pumilis) and Gram-negative bacteria (Vibrio parahaemolyticus and Vibrio alginolyticus) with a minimal inhibitory concentration of 50 μg/ml. Antibiofilm activity revealed the potential of purified Hc to inhibit biofilm formation of both Gram-positive and Gram-negative bacterial pathogens. Furthermore, live/dead staining of biofilms demonstrated the reduced viability of bacterial cells after exposure to the purified Hc. In silico molecular modeling was carried out using the sequence of Hc from SwissProt and molecular docking was performed with the cell surface components found in Gram-positive and Gram-negative bacteria. Overall our study demonstrates the involvement of Hc in the native immune reaction of P. semisulcatus by eliciting pathogen recognition. Thus, Hc could enhance disease resistance against pathogenic infection in shrimp aquaculture. Image 1 • Haemocyanin was purified from the haemolymph of Penaeus semisulcatus and characterized by XRD, CD and HPLC analysis. • Immune functions of purified Haemocyanin were reported through agglutination reaction against yeast, RBC and fungi. • The haemocyanin exhibited antibacterial and antibiofilm activity against both Gram-positive and Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Here, we report the novel fabrication of ZnO nanoparticles using the Costus igneus leaf extract. Gas chromatography-mass spectrometry (GC–MS) and proton nuclear magnetic resonance (1H NMR) spectroscopy to determine the bioactive components present in the plant extract. The synthesis of Ci -ZnO NPs (C. igneus - coated zinc oxide nanoparticles) was accomplished using a cost-effective and simple technique. Ci -ZnO NPs were specified using UV–visible spectroscopy, FTIR, XRD, and TEM. Ci -ZnO NPs was authenticated by UV–Vis and exhibited a peak at 365 nm. The XRD spectra proved the crystalline character of the Ci -ZnO NPs synthesized as hexagonal wurtzite. The FTIR spectrum illustrated the presence of possible functional groups present in Ci -ZnO NPs. The TEM micrograph showed evidence of the presence of a hexagonal organization with a size of 26.55 nm typical of Ci -ZnO NPs. The α-amylase and α-glucosidase inhibition assays demonstrated antidiabetic activity of Ci -ZnO NPs (74 % and 82 %, respectively), and the DPPH [2,2-diphenyl-1-picrylhydrazyl hydrate] assay demonstrated the antioxidant activity of the nanoparticles (75%) at a concentration of 100 μg/ml. The Ci -ZnO NPs exhibited promising antibacterial and biofilm inhibition activity against the pathogenic bacteria Streptococcus mutans , Lysinibacillus fusiformis , Proteus vulgaris , and Vibrio parahaemolyticus. Additionally, the Ci -ZnO NPs showed biocompatibility with mammalian RBCs with minimum hemolytic activity (0.633 % ± 0.005 %) at a concentration of 200 μg/ml. Unlabelled Image • ZnO NPs was synthesized using leaf extract of Costus igneus. • The synthesized Ci -ZnO NPs was characterized by UV, XRD, FTIR, and TEM. • Ci -ZnO NPs shows potent anti diabetic activity and antioxidant activity. • Ci -ZnO NPs shows antibacterial and antibiofilm activity. [ABSTRACT FROM AUTHOR]