An inhibitory compound produced by Pseudomonas with effectiveness on Vibrio harveyi

Persistence of the antivibrio property of the potential antagonistic probiotics, Pseudomonas MCCB 102 and 103, at di¡erent temperatures, pH and in organic solvents was studied. The antivibrio compound was extracted, puri¢ed and characterized using thin-layer chromatography, high-pressure liquid chromatography, liquid chromatography-mass spectroscopy, UV^ Vis and nuclear magnetic resonance spectroscopy and identi¢ed as N-methyl-1-hydroxyphenazine, a phenazine antibiotic. The toxicity of the compound was tested in Penaeus monodon haemocyte culture and the IC50 valuewas found to be1.4 0.31mg L 1. The compound was found to be bacteriostatic at 0.5mg L 1. Its stability to varying temperature, pH, organic solvents, prolonged shelf-life and vibriostatic nature point to its suitability for prophylatic aquaculture application.

Alkali insoluble glucan extracted from Acremonium diospyri is a more potent immunostimulant in the Indian White Shrimp, Fenneropenaeus indicus than alkali soluble glucan

E¡ect of an extraction method on the structure of glucan and its immunostimulatory response in Fenneropenaeus indicus was investigated. Here we extracted alkali insoluble glucan (AIG) and alkali soluble glucan (ASG) from a ¢lamentous fungi Acremonium diospyri following alkali^acid hydrolysis and the sodium hypochlorite oxidation and dimethyl sulphoxide extraction method respectively. Structural analysis showed that 85% of glucan in AIG was a (1 !3)-b-D-glucan and it increased the prophenoloxidase and reactive oxygen intermediate activity when administered to F. indicus. On the other hand, ASG, which contained 93% (1 !3)-a-glucan, did not induce signi¢cant immune response in shrimp. Here we report that the di¡erence in immunostimulatory potential between AIG and ASG is due to the di¡erence in the percentage of (1 !3)-b-D-glucans present in each preparation, which varies with the method of extraction employed. Also our observations suggest that glucan can be used as a potential immunostimulant to shrimp, provided it contains (1 !3)-b-D-glucan as the major fraction.

Anti-lipopolysaccharide factor and crustin-III, the anti-white spot virus peptides in Penaeus monodon: Control of viral infection by up-regulation

White Spot Syndrome Virus (WSSV) is the most devastating disease affecting shrimp culture around the world. Though, considerable progress has been made in the detection and molecular characterization of WSSV in recent years, information pertaining to immune gene expression in shrimps with respect to WSSV infection remains limited. In this context, the present study was undertaken to understand the differential expression of antimicrobial peptide (AMP) genes in the haemocytes of Penaeus monodon in response to WSSV infection on a time-course basis employing semi-quantitative RT-PCR. The present work analyzes the expression profile of six AMP genes (ALF, crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5), eight WSSV genes (DNA polymerase, endonuclease, immediate early gene, latency related gene, protein kinase, ribonucleotide reductase, thymidine kinase and VP28) and three control genes (18S rRNA, β-actin and ELF) in P. monodon in response to WSSV challenge. Penaeidins were found to be up-regulated during early hours of infection and crustin-3 during late period of infection. However, ALF was found to be up-regulated early to late period of WSSV infection. The present study suggests that AMPs viz. ALF and crustin-3 play an important role in antiviral defense in shrimps. WSSV gene transcripts were detected post-challenge day 1 itself and increased considerably day 5 onwards. Evaluation of the control genes confirmed ELF as the most reliable control gene followed by 18S rRNA and β-actin for gene expression studies in shrimps. This study indicated the role of AMPs in the protection of shrimps against viral infection and their possible control through the up-regulation of AMPs

Nitrification in a packed bed bioreactor integrated into amarine recirculating maturation system under different substrate concentrations and flow rates

BACKGROUND: A packed bed bioreactor (PBBR) activated with an indigenous nitrifying bacterial consortia was developed and commercialized for rapid establishment of nitrification in brackish water and marine hatchery systems in the tropics. The present study evaluated nitrification in PBBR integrated into a Penaeus monodon recirculating maturation system under different substrate concentrations and flow rates. RESULTS:Instantnitrificationwasobservedafter integration ofPBBRinto thematuration system.TANandNO2-Nconcentrations were always maintained below0.5 mg L−1 during operation. The TANandNO2-N removalwas significant (P < 0.001) in all the six reactor compartments of the PBBR having the substrates at initial concentrations of 2, 5 and 10 mg L−1. The average volumetric TAN removal rates increased with flow rates from 43.51 (250 L h−1) to 130.44 (2500 L h−1) gTAN m−3 day−1 (P < 0.05). FISH analysis of the biofilms after 70 days of operation gave positive results with probes NSO 190 ((β ammonia oxidizers), NsV 443 (Nitrosospira spp.) NEU (halophilic Nitrosomonas), Ntspa 712 (Phylum Nitrospira) indicating stability of the consortia. CONCLUSION: The PBBR integrated into the P. monodon maturation system exhibited significant nitrification upon operation for 70 days as well as at different substrate concentrations and flow rates. This system can easily be integrated into marine and brackish water aquaculture systems, to establish instantaneous nitrification

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 immunerelated 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

Development of nitrifying bacterial consortia for immobilizing in nitrifying bioreactors designed for penaeid and non-penaeid larval rearing systems in the tropics

Two ammonia oxidizing (AMOPCU-1 and AMONPCU-1) and two nitrite oxidizing (NIOPCU-1 and NIONPCU-1) consortia for activating nitrifying bioreactors and thereby establishing nitrification in penaeid and non-penaeid hatchery systems were developed by enrichment. For further amplification of the consortia a simple medium having seawater (either salinity 30 ‰ or 15 ‰) as base, supplemented with NH4+-N/NO2--N and PO4- and pH adjusted to 8 was identified. During the amplification in a fermentor the consortia exhibited excessive wall growth and diminished their yield coefficient posing difficulty in harvesting the cells completely. The consortia consisted of both Gram negative and Gram-positive bacterial cells embedded in a mucilaginous matrix of glycocalyx - like material presumably composed of polysaccharides. The consortia besides being useful in activating nitrifying bioreactors developed for shrimp/prawn hatchery systems can also be used as bioaugmentors in the bioremediation of ammonia and nitrite toxicity in aquaculture systems.

Antimicrobial activity of chitosan against vibrios from freshwater prawn Macrobrachium rosenbergii larval rearing systems

Chitosan is a biocompatible and biodegradable natural polymer with established antimicrobial properties against specific microorganisms. The present study demonstrates its antibacterial activity against 48 isolates of Vibrio species from prawn larval rearing systems. The antibacterial activity had a positive correlation with the concentration of chitosan. This work opens up avenues for using chitosan as a prophylactic biopolymer for protecting prawn larvae from vibriosis.

A simple device for the separation of weak larvae of Macrobrachium rosenbergii (De Man)

Larvae of Macrobrachium rosenbergii (De Man) are photopositive (Ling 1969a.b) and negatively rheotactic. While investigating larval diseases of M, rosenbergii it was observed that weak larvae failed to show both these responses. It was felt that this lack of response could be used to develop a device for separating the weak larvae from the apparently healthy ones. Such a device would be a valuable tool for assessing the health of a batch in terms of the percentage of 'healthy' and 'weak' larvae. What follows is a description and mode of operation of the 'photo-flow' device developed by the authors

Anti–white spot syndrome virus activity of Ceriops tagal aqueous extract in giant tiger shrimp Penaeus monodon

White spot syndrome virus (WSSV), the most contagious pathogen of cultured shrimp, causes mass mortality, leading to huge economic loss to the shrimp industry. The lack of effective therapeutic or prophylactic measures has aggravated the situation, necessitating the development of antiviral agents. With this objective, the antiviral activity in the aqueous extract of a mangrove plant Ceriops tagal in Penaeus monodon was evaluated. The Ceriops tagal aqueous extract (CTAE) was non-toxic to shrimps at 50 mg/ml when injected intramuscularly at a dosage of 10 lL/animal (0.5 mg/animal) and showed a protective effect against WSSV at 30 mg/ml when mixed with WSSV suspension at a 1:1 ratio. When the extract was administered along with the diet and the animals were challenged orally, there was a dose-dependent increase in survival, culminating in 100 % survival at a concentration of 500 mg/kg body weight/day. Neither hypertrophied nuclei nor the viral envelope protein VP28 could be demonstrated in surviving shrimps using histology and indirect immunofluorescence histochemistry (IIFH), respectively. To elucidate the mode of action, the temporal expression of WSSV genes and shrimp immune genes, including antimicrobial peptides, was attempted. None of the viral genes were found to be expressed in shrimps that were fed with the extract and challenged or in those that were administered CTAE-exposed WSSV. The overall results suggest that the aqueous extract from C. tagal can protect P. monodon from white spot syndrome virus infection.

A new isoform of anti-lipopolysaccharide factor identified from the blue swimmer crab, Portunus pelagicus: Molecular characteristics and phylogeny

Anti-lipopolysaccharide factors are small proteins that bind and neutralize lipopolysaccharide and exhibit potent antimicrobial activities. This study presents the molecular characterization and phylogenetic analysis of the first ALF isoform (Pp-ALF1; JQ745295) identified from the hemocytes of Portunus pelagicus. The full length cDNA of Pp-ALF1 consisted of 880 base pairs encoding 293 amino acids with an ORF of 123 amino acids and contains a putative signal peptide of 24 amino acids. Pp-ALF1 possessed a predicted molecular weight (MW) of 13.86 kDa and theoretical isoelectric point (pI) of 8.49. Two highly conserved cysteine residues and putative LPS binding domain were observed in Pp-ALF1. Peptide model of Pp-ALF1 consisted of two α-helices crowded against a four-strand β-sheet. Comparison of amino acid sequences and neighbor joining tree showed that Pp-ALF1 has a maximum similarity (46%) to ALF present in Portunus trituberculatus followed by 39% similarity to ALF of Eriocheir sinensis and 38% similarity to ALFs of Scylla paramamosain and Scylla serrata. Pp-ALF1 is found to be a new isoform of ALF family and its characteristic similarity with other known ALFs signifies its role in protection against invading pathogens.

Virulence Potential And Antibiotic Susceptibility Pattern Of Motile Aeromonads Associated With Freshwater Ornamental Fish Culture Systems: A Possible Threat To public Health

Aeromonas spp. are ubiquitous aquatic organisms, associated with multitude of diseases in several species of animals, including fishes and humans. In the present study, water samples from two ornamental fish culture systems were analyzed for the presence of Aeromonas. Nutrient agar was used for Aeromonas isolation, and colonies (60 No) were identified through biochemical characterization. Seven clusters could be generated based on phenotypic characters, analyzed by the programme NTSYSpc, Version 2.02i, and identified as: Aeromonas caviae (33.3%), A. jandaei (38.3%) and A. veronii biovar sobria (28.3%). The strains isolated produced highly active hydrolytic enzymes, haemolytic activity and slime formation in varying proportions. The isolates were also tested for the enterotoxin genes (act, alt and ast), haemolytic toxins (hlyA and aerA), involved in type 3 secretion system (TTSS: ascV, aexT, aopP, aopO, ascF–ascG, and aopH), and glycerophospholipid-cholesterol acyltransferase (gcat). All isolates were found to be associated with at least one virulent gene. Moreover, they were resistant to frequently used antibiotics for human infections. The study demonstrates the pathogenic potential of Aeromonas, associated with ornamental fish culture systems suggesting the emerging threat to public health

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

Chitosan as a wall material for a microencapsulated delivery system for Macrobrachium rosenbergii(de Man) larvae

Chitosan has beenwidely accepted as awall material for preparing microcapsules of various purposes in human medicine. The possibility of using chitosan as a wall material for microencapsulating nutrients and drugs for aquaculture purposes, speci¢cally to Macrobrachium rosenbergii larvae was evaluated in this study. Two types of chitosan-coated microcapsules were prepared using either acetone (MEC-A) or NaOH (MEC-N) as the cross-linking agents. They were compared with a microbound diet relative to total leaching of nutrients and free amino acids (FAA). Among the microcapsules, MEC-N showed the lowest level of total leaching of nutrients (23.3%) during 5 h of immersion in seawater and released 65% FAA after 60min. During laboratory trials,75% larvae had accepted the MEC-N capsule. The results of the study suggest that chitosan can be used as a wall material for preparing microcapsules to deliver drugs and nutrients to M. rosenbergii larvae.

Efficacy of fermented prawn shell waste as a feed ingredient for Indian white prawn, Fenneropenaeus indicus

Prawn shell waste collected from shrimp-processing plants in Cochin, India, was subjected to fermentation using 20 chitinoclastic and proteolytic/non-proteolytic bacterial strains. The products generated were analysed for protein, lipid, total sugars, N-acetyl glucosamine, free amino acids and ash. Shrimp diets were prepared using these 20 fermented products and a control diet using raw prawn shell waste. Feeding experiment was conducted with postlarvae (PL21) of Indian white prawn, Fenneropenaeus indicus for a period of 21 days. Biogrowth parameters such as mean weight gain, feed conversion ratio, specific growth rate and protein efficiency ratio were estimated and the animals were challenged with white spot virus orally via diet. Enhanced growth could be observed in prawns fed F134 and F124, incorporated with the fermentation products generated using Bacillus spp., C134 and C124 respectively. The percentage survival of prawns after 7 days of challenge was found to be highest for groups fed diet F111 incorporated with fermentation product generated using Bacillus sp. These products of bacterial fermentation hold promise as growth enhancers and immunostimulants in aquaculture. KEY WORDS: biogrowth parameters, feed

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

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 8C. 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 (29), 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’’.