Showing papers by "Rosamma Philip published in 2013"

Characterization and virulence potential of phenotypically diverse Aeromonas veronii isolates recovered from moribund freshwater ornamental fishes of Kerala, India

Abstract: In the present study, we investigated the involvement of Aeromonas spp. in eliciting disease outbreaks in freshwater ornamental fishes across the state of Kerala, India. We investigated three incidences of disease, in which the moribund fishes exhibited clinical signs such as haemorrhagic septicemia (in gouramy, Trichogaster sp.), dropsy (in Oscar, Astronotus ocellatus) and tail rot/fin rot (in gold fish, Carassius carassius). Pure cultures (n = 20 from each fish; 60 in total) of Aeromonas spp. were recovered from the abdominal fluid as well as from internal organs of affected fishes, although they could not be identified to species level because of the variations in their phenotypic characters. The molecular fingerprinting of the isolates using Enterobacterial Repetitive Intergenic Consensus PCR proved the genetic diversity of the isolates from the three sites. The phylogenetic trees constructed using concatenated sequences (using 16S rRNA, gyrA, gyrB and rpoD genes) indicated that they were related to Aeromonas veronii. They exhibited marked cytotoxic and haemolytic activity, which were responsible for the pathogenic potential of the isolates. The isolates possessed multiple virulence genes such as enterotoxins (act and alt), haemolytic toxins (aerA and hlyA), genes involved in type III secretion system (ascV, aexT and ascF–ascG), glycerophospholipid-cholesterol acyltransferase (gcat) and a type IV pilus (tapA) gene, as determined by PCR. Virulence of representative isolates to goldfish was also tested, and we found LD50 values of 104.07–105.35 cfu/fish. Furthermore, the organisms could be recovered as pure cultures from the lesions as well as from the internal organs.

A novel medium for the development of in vitro cell culture system from Penaeus monodon.

Abstract: Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg−1 and temperature of incubation was 25 oC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”.

Characterization of Histone H2A Derived Antimicrobial Peptides, Harriottins, from Sicklefin Chimaera Neoharriotta pinnata (Schnakenbeck, 1931) and Its Evolutionary Divergence with respect to CO1 and Histone H2A.

Abstract: Antimicrobial peptides (AMPs) are humoral innate immune components of fishes that provide protection against pathogenic infections. Histone derived antimicrobial peptides are reported to actively participate in the immune defenses of fishes. Present study deals with identification of putative antimicrobial sequences from the histone H2A of sicklefin chimaera, Neoharriotta pinnata. A 52 amino acid residue termed Harriottin-1, a 40 amino acid Harriottin-2, and a 21 mer Harriottin-3 were identified to possess antimicrobial sequence motif. Physicochemical properties and molecular structure of Harriottins are in agreement with the characteristic features of antimicrobial peptides, indicating its potential role in innate immunity of sicklefin chimaera. The histone H2A sequence of sicklefin chimera was found to differ from previously reported histone H2A sequences. Phylogenetic analysis based on histone H2A and cytochrome oxidase subunit-1 (CO1) gene revealed N. pinnata to occupy an intermediate position with respect to invertebrates and vertebrates.

Marine yeasts in the slope sediments of Arabian sea and bay of Bengal

Abstract: Yeasts are ubiquitous in distribution and are found throughout the aquatic environment. Their distribution and population depends mainly on the surrounding environment. This study is about the occurrence and distribution of yeasts from the slope sediments of Arabian Sea and Bay of Bengal. Sediment samples were collected from 200, 500 and 1000 m depths along the west and east coast of Indian peninsula. Majority of sampling stations showed a clayey silt sediment texture. Organic matter was found to be comparatively higher in the Arabian Sea. Yeast population varied between stations and found to be more in Arabian Sea. Candida was the predominant genera identified from both Arabian Sea and Bay of Bengal. Diversity was maximum at 500 m in Arabian Sea and at 1000 m in Bay of Bengal. Data analysis was carried out using PRIMER 5.1 and SPSS 10.0. Correlation studies using PRIMER software showed a positive correlation between organic matter and yeast population.

Molecular Characterization and Phylogenetic Analysis of a Histone-Derived Antimicrobial Peptide Teleostin from the Marine Teleost Fishes, Tachysurus jella and Cynoglossus semifasciatus.

Abstract: Antimicrobial peptides (AMPs) are host defense peptides that are well conserved throughout the course of evolution. Histones are classical DNA-binding proteins, rich in cationic amino acids, and recently appreciated as precursors for various histone-derived AMPs. The present study deals with identification of the potential antimicrobial peptide sequence of teleostin from the histone H2A of marine teleost fishes, Cynoglossus semifasciatus and Tachysurus jella. A 245 bp amplicon coding for 81 amino acids was obtained from the cDNA transcripts of these fishes. The first 52 amino acids from the N terminal of the peptide were identical to previously characterized histone-derived antimicrobial peptides. Molecular and physicochemical characterizations of the sequence were found to be in agreement with previously reported histone H2A-derived AMPs, suggesting the possible role of histone H2A in innate defense mechanism in fishes.

Salinity a significant environmental factor for Vibrio harveyi virulence in Fenneropenaeus indicus

Abstract: The effect of ambient salinity on the haemolymph variables of Fenneropenaeus indicus and its susceptibility to Vibrio harveyi infection under salinity stress has been studied. Adult shrimps were acclimated to 5‰ (hypo osmotic), 25‰ (iso osmotic) and 35‰ (hyper osmotic) salinity levels and the animals were injected with a mid logarithmic culture of V. harveyi at sub lethal level and haemolymph parameters were analysed. Haemolymph proteins, intracellular superoxide anion production, phenoloxidase (PO), alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were found to be at elevated level both at 5‰ and 35‰ post challenge. The haematological responses showed a progressive increase (P < 0.05) up to post challenge day 5 (PCD 5) followed by a considerable decline at all salinities with the lowest being at 35‰. The alterations in the variables were higher in shrimps held at 5‰. However, the V. harveyi infection was severe in animals held at 35‰. The reduction in the parameters could be correlated with the decrease in survival rate of shrimps at 35‰ with a concurrent increase in V. harveyi at this salinity. Multiple regression analysis revealed that ACP (P < 0.001), haemocyte protein HCP (P < 0.001) and PO (P < 0.05) could explain 91% variability in the shrimp survival. These parameters may be used as effective shrimp health indicators. It is evident from the study that ambient salinity alters the haemolymph variables, modulates the virulence in V. harveyi and makes the shrimps more vulnerable to infection at higher salinity. The virulence of V. harveyi is increased at 35‰ salinity as being evidenced from the high mortality at this salinity. The study emphasizes the importance of salinity as an important environmental factor both in terms of host susceptibility and virulence of the pathogen.

Actinobacteria from sediment samples of Arabian Sea and Bay of Bengal: Biochemical and physiological characterization

Abstract: Actinobacteria isolated from the sediment samples of Arabian Sea and Bay of Bengal were used for the study. Morphological and biochemical characterization of the isolates revealed Streptomyces (76%) as the dominant genus followed by Nocardiopsis (24%). Among the carbon sources tested, the hexose sugar glucose and pentose sugar rhamnose were utilized by all the isolates and the least preferred carbon sources were sorbitol (85.22%), lactose (81.3%) and xylose (76.96%). Acid production from carbohydrates varied significantly among the isolates. Hydrolytic enzyme profile revealed that most of the marine actinomycete isolates were capable of gelatinase production (99.13%) followed by DNase (96.09%), lipase (86.96%), phosphatase (84.78%), chitinase (63.48%), pectinase (22.17%) and ligninase (15.22%) producing forms. Melanin production was exhibited by 4% of the Streptomyces isolates. The present study has unraveled the metabolic potential of marine Streptomyces and Nocardiopsis, expanding the scope for the discovery of novel metabolites of marine origin.

Original Article A MOLASSES BASED FERMENTATION MEDIUM FOR MARINE YEAST BIOMASS PRODUCTION

Abstract: Four marine yeasts were used for the study based on their performance in a feeding experiment in Fenneropenaeus indicus viz.,1. Debaryomyces hansenii (S8) 2. Debaryomyces hansenii (S100), 3.Candida sake (S165) and 4.Candida tropicalis (S186). Molasses was the most preferred carbon source by the marine yeasts compared to glucose, sucrose and rice water. Molasses (amount of total sugars 9mg/ml) supplemented with peptone (0.75%), yeast extract (0.5%) and MgSO4 (0.25%) was found to be favouring maximum growth of the four yeast strains tested. Two yeast strains (S8 & S186) showed their maximum growth at 30ppt salinity and the other two (S100 & S165) at 25ppt and 20ppt respectively. pH6 was found to be most favourable for growth. This study shows that molasses supplemented with peptone and yeast extract could be used as a good production medium for large scale production of yeast biomass.

A Novel Isoform of the Hepatic Antimicrobial Peptide, Hepcidin (Hepc-CB1), from a Deep-Sea Fish, the Spinyjaw Greeneye Chlorophthalmus bicornis (Norman, 1939): Molecular Characterisation and Phylogeny

Abstract: Hepcidin is cysteine-rich short peptide of innate immune system of fishes, equipped to perform prevention and proliferation of invading pathogens like bacteria and viruses by limiting iron availability and activating intracellular cascades. Hepcidins are diverse in teleost fishes, due to the varied aquatic environments including exposure to pathogens, oxygenation and iron concentration. In the present study, we report a 87-amino acid (aa) preprohepcidin (Hepc-CB1) with a signal peptide of 24 aa, a prodomain of 39 aa and a bioactive mature peptide of 24 aa from the gill mRNA transcripts of the deep-sea fish spinyjaw greeneye, Chlorophthalmus bicornis. Molecular characterisation and phylogenetic analysis categorised the peptide to HAMP2-like group with a mature peptide of 2.53 kDa; a net positive charge (+3) and capacity to form β-hairpin-like structure configured by 8 conserved cysteines. The present work provides new insight into the mass gene duplication events and adaptive evolution of hepcidin isoforms with respect to environmental influences and positive Darwinian selection. This work reports a novel hepcidin isoform under the group HAMP2 from a non-acanthopterygian deep-sea fish, C. bicornis.

Identification of haematological markers in shrimp health assessment from the immune profile of Fenneropenaeus indicus on β-1,3-glucan administration and white spot syndrome virus challenge

Abstract: Haematological alterations in Fenneropenaeus indicus fed marine yeast glucan-incorporated diet and challenged with white spot syndrome virus were analysed. Adult F. indicus 16.45 ± 2.12 g (mean ± SD) were reared in 25 ‰ sea water and allowed to acclimate for a period of 7 days. Diet was prepared incorporating glucan (0.2 %) extracted from marine yeast, Debaryomyces hansenii S169. The shrimps were reared on experimental diet for 28 days and then challenged with white spot syndrome virus through oral administration. Random sampling was done (n = 6 shrimps) on 1st, 15th and 28th day of the experiment and on post-challenge day 2 (PCD 2) and PCD 7 for haematological analysis. The immune parameters viz. plasma protein, total haemocyte count, phenoloxidase activity, superoxide anion production, alkaline phosphatase and acid phosphatase activities were estimated in the haemolymph. Superoxide dismutase, catalase, glutathione peroxidase, hydroperoxide, conjugated dienes and malondialdehyde concentrations in the haemolymph of shrimps were also analysed. Analysis of variance showed that there are significant differences (p < 0.05) in the immune and antioxidant parameters in different treatment groups of F. indicus. The shrimps fed glucan-incorporated diet showed higher survival rate with comparatively lower accumulation of lipid peroxidation products. Correlation coefficients showed that all haematological variables except hydroperoxides and conjugated dienes exhibited positive correlation with the survival rate. The immune variables and antioxidant parameters exhibited a greater degree of correlation with each other. When multiple regression of survival rate on all immune parameters was considered, the amount of variability explained was 93 % (R Square = 0.932). When significant regression coefficients among the immune parameters were taken into account, it was found that total haemocyte count (p < 0.001), phenoloxidase activity (p < 0.05), alkaline phosphatase activity (p < 0.05) and plasma protein concentration (p < 0.05) together are explaining 84 % (R 2 = 0.842) of the variability, indicating that these four parameters are mainly supporting the survival. The amount of variability explained by the antioxidant parameters was 94 % (R 2 = 0.938). When significant regression coefficients among the antioxidant parameters were taken into account, it was found that superoxide dismutase activity (p < 0.01) and catalase activity (p < 0.05) together are explaining 87.4 % (R 2 = 0.874) of the variability, indicating that these two antioxidants act as the major immune effectors supporting the survival in shrimps.

Phylogeny and genetic divergence of three phenotypic variants of the ornamental goldfish, Carassius auratus (Linnaeus, 1758) based on CO1 gene

Abstract: Carassius auratus is one of the most popular aquarium fishes having wide distribution in Eurasian continents. Artificial selection has led to the formation of a number of morphological variants of this fish. The present study is aimed at determining the genetic variation among the morphological variants of C. auratus based on cytochrome oxidase subunit-1 (CO1), mitochondrial gene. The intraspecific and interspecific genetic divergence for C. auratus based on CO1 gene was also assessed. Analysis of the CO1 nucleotide sequences obtained from three morphological variants of goldfish confirmed that the specimens belonged to species C. auratus and no genetic divergence exist among the phenotypic variants of goldfish. The mean interspecific genetic distances (Kimura 2 parameter) calculated for C. auratus was found to be 36 fold higher than the intraspecific mean distance calculated for Cyprinidae fishes.The phylogenetic tree constructed (NJ method) based on CO1 gene showed C. auratus to align with the Cyprinidae clade. The mean interspecific distance calculated for Cyprinidae fishes indicated that with regard to genetic variations, family Cyprinidae is more stable in comparison to other Cypriniformes families. High efficiency in species identification and phylogenetic analysis was exhibited by CO1 gene in the present study highlighting the proficiency of mitochondrial genome based molecular markers in taxonomic and evolutionary studies.

Antioxidant defence profile of hepatopancreas, gill and muscle tissue of Fenneropenaeus indicus subjected to acute salinity change and WSSV challenge

Abstract: The present study describes the tissue antioxidant profile of adult Fenneropenaeus indicus subjected to acute salinity alteration followed by challenge with white spot syndrome virus. Shrimps were initially reared in 25 ‰ sea water and after seven days, the hepatopancreas, gills and muscle samples were tested for antioxidant profile and oxidation stress viz., Super oxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione –S- transferase, total reduced glutathione, hydroperoxides, conjugated dienes and malondialdehyde. Then the salinity of rearing water (25‰) was adjusted to two different salinities i.e., 5‰ and 35‰ followed by WSSV challenge. The antioxidant enzyme level was significantly (p<0.05) high in the hepatopancreas of WSSV challenged F. indicus compared to gills and muscle tissues. Hydroperoxides, conjugated dienes and malondialdehyde contents increased significantly (p<0.05) after WSSV challenge. Lipid peroxidation was also found higher in the hepatopancreas of shrimps compared to gills and muscle tissues. The study revealed the antioxidant stress and the elevated antioxidant defence profile of shrimps at lower salinity (5 ‰) followed by 35 ‰ compared to the animals maintained at 25 ‰ on WSSV challenge.

Heterotrophic bacterial and fungal diversity in the inner shelf sediments ofcentral west coast of India

Abstract: Vertical and horizontal distribution of culturable heterotrophic bacteria and fungi in the inner shelf sediments of central west coast of India (Arabian Sea) were examined. Physico-chemical parameters of the sediment and its influence on microbial diversity and abundance were evaluated. Microbial abundance displayed a significant positive correlation with sediment texture and organic matter. Bacterial population in the inner shelf regions increased towards northern transects and showed a decreasing pattern as core depth increased. A total of 221 bacterial cultures were isolated, out of which 63% gram negative and 37% were gram positive. Alteromonas (29%) was found to be the dominant genus in the sediment core samples followed by Micrococcus (18%) and Bacillus (10%). Other identified groups include Psychrobacter (10%), Moraxella (6%), Acinetobacter (5%), Flexibacter (5%), Corynebacterium (5%), Pseudomonas (3%), Alcaligenes (2%), Staphylococcus (2%), Enterobacteriaceae (1%) and Vibrio (1%). Out of the 47 fungal isolates, 34% were belonging to genus Aspergillus and 25% to Scopulariopsis

Molecular Characterisation and Phylogenetic Analysis of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Zc-hepc1), from the Coral Fish Moorish idol, Zanclus cornutus (Linnaeus, 1758)

Abstract: Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of +1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a β-hairpin-like structure with two antiparallel β-sheets. This is the first report of an AMP from the coral fish Z. cornutus.

Bathymetric and latitudinal shift in sedimentary parameters along the shelfsediments of Bay of Bengal: Influence on heterotrophic bacteria

Abstract: Bathymetric and spatial alterations of sedimentary parameters and its influence on heterotrophic bacterial population in the shelf sediments of south east coast of India (Bay of Bengal) were analysed. The hydrographical parameters did not show significant (p<0.05) spatial variation though it varied with depth. Sediment was fine sand at 50 -100m depth and clayey silt at 200m depth. Organic matter in the sediment ranged from 0.95 - 3.76% showing significant depth wise variation (p<0.05). Total heterotrophic bacterial population ranged from 4.87 × 103 – 2.32 × 105 CFU g-1 dry wt. and the abundance was greater towards the northern latitudes. Bacillus, Vibrio and Alteromonas were the dominant genera in the shelf sediments followed by Alcaligenes, Enterobacteriaceae, Acinetobacter, Flexibacter and Moraxella. Significant positive correlation of bacterial abundance with sediment texture and organic matter was evident from the spearman rank correlation analysis. BIOENV identified dissolved oxygen, clay, silt and total nitrogen as a combination of environmental parameters that best explained the distribution patterns of heterotrophic bacteria (ρ = 0.644). The Principal component Analysis (PCA) displayed higher similarity of environmental characteristics within respective depth regions. First two components together explain 90% of the data variance between stations. The canonical correspondence analysis (CCA) further substantiated that major environmental parameters (such as temperature, dissolved oxygen, silt, sand and organic matter), had significant effects on the spatial distribution of culturable heterotrophic bacteria in the shelf sediments of Bay of Bengal.

Phylogenetic relationship of Sailfin (Poecilia latipinna) and Shortfin (Poecilia sphenops) mollies (Order: Cyprinodontiformes) as inferred from the mitochondrial cytochrome gene sequence

Abstract: Poeciliid fishes are used as model organisms for experimental studies on natural and sexual selection. The present study is aimed at phylogenetic analysis of Poeciliid fishes, focussing on P. latipinna (Sailfin) and P. sphenops (Shortfin). This paper reports result of phylogenetic analyses based upon nucleotide sequence data from the mitochondrial COI gene of Poeciliids. Briefly, PCR amplification of DNA was carried out using the COI gene primers and sequences were analysed using BioEdit, ExPASy, GeneDoc and Mega 5.0. Partial COI amplicon of 595 bp could be recovered from P. sphenops (JF752336) and 645 bp amplicon from P. latipinna (JF752337) respectively. Phylogenetic relationship of 13 species of Poeciliid fishes, were constructed employing both neighbor joining and maximum likelihood methods. The results of phylogenetic analysis confirmed the basal placement of mollies in the fish phylogeny. The phylogenetic relationships among the Poeciliid fishes in the ML tree were virtually identical to those in the NJ tree. The present study shows that P. sphenops and P. latipinna are more closely related to each other than to other Poeciliid fishes. Molecular phylogenetic arrangements as inferred from the present study also suggest the presence of single origin of Sailfin species from a Shortfin ancestor.

Original Article Filamentous fungal isolates from the continental shelf and slope sediments of

Abstract: The occurrence and diversity of fungal species in the Arabian Sea, one of the most dynamic and productive regions of world oceans, were investigated. Sediment samples were collected during six voyages of FORV Sagar Sampada. A total of 302 fungal isolates, comprising 27 different species belonging to Ascomycota and Zygomycota, were isolated from 95 samples. Among the isolates, Aspergillus sp. ranked first (37%), followed by Penicillium sp. and Cladosporium also showed significant rate of isolation. Other filamentous fungi isolated were Paecilomyces, Fusarium, Scopulariopsis, Curvularia, Epicoccum and Oedocephalum. Zygomycetes obtained were Mucor and Mortierella. A yeast-like fungi Geotrichum candidum was obtained from the slope sediments. Depth wise analysis of fungal isolates showed that the occurrence of fungi decreases with increase in depth. Diversity indices were calculated and species richness (d) varied from 2.0665 to 3.4151; evenness (J'), from 0.7611 to 0.8513 and Shannon-Weiner diversity index (H' log 2), from 2.4128 to 3.3160. The diversity was found to be lowest at a depth of 50m and showed more or less similar trend in other stations. 95% confidence interval and ellipse showed that the stations were lying well within the funnel. Cluster analysis and MDS grouped the continental shelf and slope regions separately. © 2011 Universal Research Publications. All rights reserved