I.S. Bright Singh

Molecular characterization of a crustin­like antimicrobial peptide in the giant tiger shrimp, Penaeus monodon, and its expression profile in response to various immunostimulants and challenge with WSSV

A crustin-like antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustin-like AMP from other shrimp crustins. The differential expression of the crustin-like AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two β-glucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two gram-positive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded pre- and post-challenge white spot syndrome virus (WSSV) by semi-quantitative RT-PCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustin-like AMP was found to be constitutively expressed in the animal and a significant down-regulation could be noted post-challenge WSSV. Remarkable down-regulation of the gene was observed in the immunostimulant fed animals pre-challenge followed by a significant up-regulation post-challenge WSSV. Tissue-wise expression of crustin-like AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustin-like AMP and confer significant protection to P. monodon against WSSV infection.

Optimization of carbon and nitrogen sources and growth factors for the production of an aquaculture probiotic (Pseudomonas MCCB 103) using response surface methodology

Aim:  To develop a new medium for enhanced production of biomass of an aquaculture probiotic Pseudomonas MCCB 103 and its antagonistic phenazine compound, pyocyanin.

Development of a cell culture system from the ovarian tissue of African catfish Clarias gariepinus

Abstract A growth medium with Leibovitz-15 (L-15) as the base, supplemented with foetal bovine serum (10% v/v), fish muscle extract (10% v/v), prawn muscle extract (10% v/v), lectin (concanavalin A) (0.02 μg ml −1 ), lipopolysaccharide (0.02 μg ml −1 ), glucose D (0.2 mg ml −1 ), ovary extract (0.5% v/v) and prawn haemolymph (0.5%) has been formulated with 354±10 mOsm for the development and maintenance of a cell culture system from the ovarian tissue of African catfish, Clarias gariepinus . For its subculturing, a cell dissociation/extracting solution, composed of equal portions of trypsin phosphate versene glucose (TPVG) (containing 0.0125% (w/v) trypsin and 25% (v/v) non-enzymatic cell dissociation solution 1 and 2, has also been developed with which the cell culture can be passaged 15 times after which they cease to multiply and consequently perish. The cell cultures can be maintained for 12–15 days without fluid change between the passages. This is the first report of a cell culture system from the ovarian tissues of African catfish.

Molecular characterization of a crustin-like, putative antimicrobial peptide, Fi-crustin, from the Indian white shrimp, Fenneropenaeus indicus

Antimicrobial peptides are important innate immune defense, especially in those animals which lack adaptive immunity [1e8]. Due to their small size, amphipathic structure and cationic character they can rapidly diffuse to the point of infection [9], a mechanism that presumably makes it easier to circumvent microbial resistance against the peptides [10]. Besides providing an immediate and broad-spectrum microbicidal activity, AMPs can kill bacteria in micromolar range, are promptly synthesized at low metabolic cost, and are easily stored in large amounts and readily available shortly after an infection [11e13]. Many AMPs show a remarkable specificity for prokaryotes with low toxicity for eukaryotic cells; a phenomenon which has favored their investigation and exploitation as potential new antibiotics [14]. AMP gene expression and distribution are regulated through haemocyte reactions [15]. Transcripts of crustin-encoding genes have also been observed in gills, heart and intestine [16e18] but as these tissues are highly vascularised, it is assumed the transcripts from these organs are due primarily to the haemocytes. In penaeid shrimps, four main families of AMPs have been currently described and characterized from the haemocytes: penaeidins, crustins, anti-lipopolysaccharide factors (ALFs) and lysozymes. Penaeidins are mainly active against Gram-positive bacteria, filamentous fungi [19], viruses and protozoans [20] whereas ALFs have a broader antimicrobial spectrum including Gram-negative bacteria [21,22]. Conversely, crustins are reported to have a more-restricted activity spectrum, affecting mainly marine Gram-positive bacteria [17,23,24] Crustins, a widely distributed family of AMPs was first isolated from the shore crab, Carcinus maenas as an 11.5 kDa peptide [23]. Crustins are cationic, cysteinerich antimicrobial AMPs having molecular weight of 7e14 kDa, with an isoelectric point in the range of 7.0e8.7, and contain one whey-acidic protein (WAP) domain at the carboxy terminus [25]. Crustins have been proved to be an important antimicrobial protein in the plasma and haemocyte granules of crustaceans and described as a component of the innate immune system [8]. These AMPs are dominantly synthesized and stored in haemocytes [4,8,16,18,23,24,26e30] and their release from haemocytes is induced by bacterial infection [15,27,31]. Crustin mechanisms of action and function are still largely unknown, although they contain a whey-acidic protein (WAP) domain common to proteinase inhibitory activities as well as antimicrobial activities [8]. Many full-length cDNA and several ESTs of crustins have been described in a wide range of penaeid prawns including Litopenaeus vannamei [8,24,30,32], Litopenaeus setiferus [24,32,33], Penaeus monodon [16,17,29,30,34e37], Marsupenaeus japonicus [17,38], Litopenaeus schmitti [33], Fenneropenaeus chinensis [17,29], Farfantepenaeus brasiliensis [33], Farfantepenaeus paulensis [33] and Farfantepenaeus subtilis [33]. However, no antimicrobial peptide sequences have been reported from Fenneropenaeus indicus. In the current study a crustin cDNA has been characterized from the Indian White Shrimp, F. indicus. Healthy adult F. indicus (8e10 g body weight) were purchased from a local shrimp farm in Vypeen, Kochi. Theywere transferred to aquaria of 500 l capacity and acclimatized for one week under laboratory conditions. Prawns were fed with a standard feed (Higashimaru, India). Aeration was provided in all tanks during the experiment and bioreactorwas set in all the aquaria for the removal of ammonia and nitrate. Only shrimps in the intermoult stage were sampled during the study. Haemolymph was collected from the rostral sinus using specially designed capillary tubes (RNase-free) rinsed using precooled anticoagulant solution (RNase-free, 10% sodium citrate, pH 7.0). Total RNA was extracted from the haemocytes using TRI * Corresponding author. Tel.: þ91 484 2368120; fax: þ91 484 2381120. E-mail addresses: rose@cusat.ac.in, rosammap@gmail.com (R. Philip).

Optimization of medium for the production of a novel aquaculture probiotic, Micrococcus MCCB 104 using central composite design

A marine isolate ofMicrococcus MCCB 104 has been identified as an aquaculture probiotic antagonistic toVibrio. In the present study different carbon and nitrogen sources and growth factors in a mineral base medium were optimized for enhanced biomass production and antagonistic activity against the target pathogen,Vibrio harveyi, following response surface methodology (RSM). Accordingly the minimum and maximum limits of the selected variables were determined and a set of fifty experiments programmed employing central composite design (CCD) of RSM for the final optimization. The response surface plots of biomass showed similar pattern with that of antagonistic activity, which indicated a strong correlation between the biomass and antagonism. The optimum concentration of the carbon sources, nitrogen sources, and growth factors for both biomass and antagonistic activity were glucose (17.4 g/L), lactose (17 g/L), sodium chloride (16.9 g/L). ammonium chloride (3.3 g/L), and mineral salts solution (18.3 mL/L).

Immune gene expression profile of Penaeus monodon in response to marine yeast glucan application and white spot syndrome virus challenge.

Immunostimulant potential of eight marine yeast glucans (YG) from Candida parapsilosis R20, Hortaea werneckii R23, Candida spencermartinsiae R28, Candida haemulonii R63, Candida oceani R89, Debaryomyces fabryi R100, Debaryomyces nepalensis R305 and Meyerozyma guilliermondii R340 were tested against WSSV challenge in Penaeus monodon post larvae (PL). Structural characterization of these marine yeast glucans by proton nuclear magnetic resonance (NMR) indicated structures containing (1-6)-branched (1-3)-β-D-glucan. PL were fed 0.2% glucan incorporated diet once in seven days for a period of 45 days and the animals were challenged with white spot syndrome virus (WSSV). The immunostimulatory activity of yeast glucans were assessed pre- and post-challenge WSSV by analysing the expression profile of six antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5 and 13 immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, caspase, catalase, glutathione peroxidase, glutathione-s-transferase, haemocyanin, peroxinectin, pmCathepsinC, prophenol oxidase (proPO), Rab-7, superoxide dismutase and transglutaminase. Expression of seven WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, thymidine kinase and VP28 were also analysed to detect the presence and intensity of viral infection in the experimental animals post-challenge. The study revealed that yeast glucans (YG) do possess immunostimulatory activity against WSSV and also supported higher survival (40-70 %) post-challenge WSSV. Among the various glucans tested, YG23 showed maximum survival (70.27%), followed by YG20 (66.66%), YG28 (60.97%), YG89 (58.53%), YG100 (54.05%), YG63 (48.64%), YG305 (45.7%) and YG340 (43.24%).

Isolation and characterization of virulent Aeromonas veronii from ascitic fluid of oscar Astronotus ocellatus showing signs of infectious dropsy.

The cichlid oscar Astronotus ocellatus has worldwide commercial value in the pet fish industry because of its early maturation, relatively high fecundity, ability to identify its caretaker and also to alter colouration amongst conspecifics. Pathogenic strains of Aeromonas veronii resistant to multiple antibiotics were isolated from A. ocellatus individuals showing signs of infectious abdominal dropsy. The moribund fish showed haemorrhage in all internal organs, and pure cultures could be obtained from the abdominal fluid. The isolates recovered were biochemically identified as A. veronii biovar sobria and genetically confirmed as A. veronii based on 16S rRNA gene sequence analysis (GenBank accession no. FJ573179). The RAPD profile using 3 primers (OPA-3, OPA-4 and OPD-20) generated similar banding patterns for all isolates. They displayed cytotoxic and haemolytic activity and produced several exoenzymes which were responsible for the pathogenic potential of the isolates. In the representative isolate MCCB 137, virulence genes such as enterotoxin act, haemolytic toxin aerA, type 3 secretion genes such as aexT, ascVand ascF-ascG, and gcat (glycerophospholipid-cholesterol acyltransferase) could be amplified. MCCB 137 exhibited a 50% lethal dose (LD50) of 10(5.071) colony-forming units ml(-1) in goldfish and could be subsequently recovered from lesions as well as from the internal organs. This is the first description of a virulent A. veronii from oscar.

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.

Nitrification in brackish water recirculating aquaculture system integrated with activated packed bed bioreactor

Recirculation aquaculture systems (RAS) depend on nitrifying biofilters for the maintenance of water quality, increased biosecurity and environmental sustainability. To satisfy these requirements a packed bed bioreactor (PBBR) activated with indigenous nitrifying bacterial consortia has been developed and commercialized for operation under different salinities for instant nitrification in shrimp and prawn hatchery systems. In the present study the nitrification efficiency of the bioreactor was tested in a laboratory level recirculating aquaculture system for the rearing of Penaeus monodon for a period of two months under higher feeding rates and no water exchange. Rapid setting up of nitrification was observed during the operation, as the volumetric total ammonia nitrogen removal rates (VTR) increased with total ammonia nitrogen (TAN) production in the system. The average Volumetric TAN Removal Rates (VTR) at the feeding rate of 160 g/day from 54-60th days of culture was 0.1533+/-0.0045 kg TAN/m(3)/day. The regression between VTR and TAN explained 86% variability in VTR (P<0.001). The laboratory level RAS demonstrated here showed high performance both in terms of shrimp biomass yield and nitrification and environmental quality maintenance. Fluorescent in-situ Hybridization analysis of the reactor biofilm ensured the presence of autotrophic nitrifier groups such as Nitrosococcus mobilis lineage, Nitrobacter spp and phylum Nitrospira, the constituent members present in the original consortia used for activating the reactors. This showed the stability of the consortia on long term operation.

Production and characterization of polyhydroxybutyrate from Vibrio harveyi MCCB 284 utilizing glycerol as carbon source

Production and characterization of polyhydroxybutyrate (PHB) from moderately halophilic bacterium Vibrio harveyi MCCB 284 isolated from tunicate Phallusia nigra.