Rosamma Philip

Marine yeasts — a review

Yeasts are ubiquitous in their distribution and populations mainly depend on the type and concentration of organic materials. The distribution of species, as well as their numbers and metabolic characteristics were found to be governed by existing environmental conditions. Marine yeasts were first discovered from the Atlantic Ocean and following this discovery, yeasts were isolated from different sources, viz. seawater, marine deposits, seaweeds, fish, marine mammals and sea birds. Near‐shore environments are usually inhabited by tens to thousands of cells per litre of water, whereas low organic surface to deep‐sea oceanic regions contain 10 or fewer cells/litre. Aerobic forms are found more in clean waters and fermentative forms in polluted waters. Yeasts are more abundant in silty muds than in sandy sediments. The isolation frequency of yeasts fell as the depth of the sampling site is increased. Major genera isolated in this study were Candida, Cryptococcus, Debaryomyces and Rhodotorula. For biomass estimation ergosterol method was used. Classification and identification of yeasts were performed using different criteria, i.e. morphology, sexual reproduction and physiological/biochemical characteristics. Fatty acid profiling or molecular sequencing of the IGS and ITS regions and 28S gene rDNA ensured accurate identification. Copyright

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.

Selection of marine yeasts for the generation of single cell protein from prawn-shell waste

Marine yeasts (33 strains) were isolated from the coastal and offshore waters off Cochin. The isolates were identified and then characterized for the utilization of starch, gelatin, lipid, cellulose, urea, pectin, lignin, chitin and prawn-shell waste. Most of the isolates were Candida species. Based on the biochemical characterization, four potential strains were selected and their optimum pH and NaCl concentration for growth were determined. These strains were then inoculated into prawn-shell waste and SCP (single cell protein) generation was noted in terms of the increase in protein content of the final product.

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).

Marine yeast Candida aquaetextoris S527 as a potential immunostimulant in black tiger shrimp Penaeus monodon.

A marine yeast Candida aquaetextoris S527 as a source of immunostimulant in Penaeus monodon was studied. Yeast diet was prepared by incorporating 10% C. aquaetextoris S527 biomass into a standard shrimp diet and administered in P. monodon at different frequencies (daily, once in three days, once in seven days and once in ten days) followed by challenge with white spot syndrome virus (WSSV). Immune parameters such as total protein, total hemocyte count, pro-phenoloxidase, nitroblue tetrazolium reduction, alkaline phosphatase activity and acid phosphatase activity were tested. Expression profile of antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5; immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, peroxinectin, prophenol oxidase (proPO) and transglutaminase, and WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were analyzed. The study demonstrated that marine yeast diet administered once every seven days conferred better protection to P. monodon against WSSV infection, supported by the hematological and immune gene expression profiles analyzed.

Identification and Molecular Characterization of Molluskin, a Histone-H2A-Derived Antimicrobial Peptide from Molluscs

Antimicrobial peptides are humoral innate immune components of molluscs that provide protection against pathogenic microorganisms. Among these, histone-H2A-derived antimicrobial peptides are known to actively participate in host defense responses of molluscs. Present study deals with identification of putative antimicrobial sequences from the histone-H2A of back-water oyster Crassostrea madrasensis, rock oyster Saccostrea cucullata, grey clam Meretrix casta, fig shell Ficus gracilis, and ribbon bullia Bullia vittata. A 75 bp fragment encoding 25 amino acid residues was amplified from cDNA of these five bivalves and was named “Molluskin.” The 25 amino acid peptide exhibited high similarity to previously reported histone-H2A-derived AMPs from invertebrates indicating the presence of an antimicrobial sequence motif. Physicochemical properties of the peptides are in agreement with the characteristic features of antimicrobial peptides, indicating their potential role in innate immunity of molluscs.

Weissella confusa: a rare cause of vancomycin-resistant Gram-positive bacteraemia.

We describe a case of bacteraemia caused by Weissella confusa in a 48-year-old male who was operated on for adenocarcinoma of the gastro-oesophageal junction and maintained on total parenteral nutrition. Blood cultures were positive for a vancomycin-resistant streptococcus-like organism which was identified as W. confusa by 16S rRNA gene sequencing.

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).