Development of antiviral gene therapy for Monodon baculovirus using dsRNA loaded chitosan-dextran sulfate nanocapsule delivery system in Penaeus monodon post-larvae.

In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon (P. monodon) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3-25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon. Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp.

Optimization of Culture Conditions for Mass Production of the Probiotics Pseudomonas MCCB 102 and 103 Antagonistic to Pathogenic Vibrios in Aquaculture.

Rapid growth of shrimp farming industry is affected by the recurrence of diverse diseases, among which vibriosis is predominant. Eco-friendly disease management strategy by the application of antagonistic probiotics is widely accepted. In the present study, culture conditions of antagonistic probiotics, Pseudomonas MCCB 102 and 103, were optimized to enhance their biomass production and antagonistic activity against the shrimp pathogen V. harveyi MCCB 111. Primarily, one-dimensional screening was carried out to fix the optimum range of sodium chloride concentration, pH and temperature. The second step optimization was done using a full-factorial central composite design of response surface methodology. As per the model, 12.9 g/L sodium chloride and pH 6.5 for Pseudomonas MCCB 102, and 5 g/L sodium chloride and pH 7 for Pseudomonas MCCB 103 were found to be ideal to maximize antagonistic activity. However, optimum temperature was the same (25 °C) for both isolates. Finally, the models were experimentally validated for enhanced biomass production and antagonistic activity. The optima for biomass and antagonistic activity were more or less the same, suggesting the possible influence of biomass on antagonistic activity.

Development of SYBR Green based real time PCR assay for detection of monodon baculovirus in Penaeus monodon.

Shrimp farming is one of the most important aquaculture activities. Expansion and intensification of shrimp farming has been accompanied with the outbreak of diseases, which threaten the development and sustainability of the industry. Viral diseases are the major challenges faced by shrimp farming industries. The prevention/control of such diseases have become critical in determining the viability of the shrimp farming. The disease caused by monodon baculovirus (MBV) is the major limiting factor especially in shrimp hatchery. There are no therapeutic measures to control the viral diseases. So the detection of the disease is crucial in the prevention and spread of the disease. Hence, in this study, SYBR Green based real time polymerase chain reaction (PCR) assay was developed for the detection of MBV. The sensitivity of the real time PCR was determined using 10-fold dilutions of purified plasmid DNA with the concentration in the range of 10(1)-10(5) copies per reaction. The assay could detect as low as 12 copies indicating that the assay was sensitive and could be effectively used for the quantification of MBV. The real time PCR assay was found to be specific and did not show any amplification with P. monodon infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and hepatopancreatic parvo-like virus (HPV). The novelty of this assay is that it could be employed for diagnosis of low level MBV infection in broodstock using fecal matter of shrimp, a non-invasive diagnostic tool.

Comparative efficacy of double-stranded RNAs targeting WSSV structural and nonstructural genes in controlling viral multiplication in Penaeus monodon.

RNA interference (RNAi) is a potential strategy to control shrimp viral diseases, including the white spot disease caused by White Spot Syndrome Virus (WSSV). Selection of genes for targeting is an important criterion. We have compared the efficacy of dsRNAs targeting structural (vp28 and vp281) and nonstructural genes (rr1 and dnapol) of WSSV in controlling viral multiplication in Penaeus monodon. Targeting the rr1 and vp28 genes provided better protection (93.3% and 90% survival respectively) compared to vp281 and dnapol in experimentally infected shrimp. Temporal transcriptional analysis of the corresponding genes and PCR-based diagnosis of WSSV in samples collected at different time points in the experiment supported this observation, thereby indicating that targeting a combination of rr1 and vp28 would be effective in limiting WSSV multiplication.

Loose shell syndrome of farmed Penaeus monodon in India is caused by a filterable agent.

Loose shell syndrome (LSS) of farmed black tiger shrimp Penaeus monodon has been reported from Indian shrimp farms since 1998 and is recognized as a major disease problem causing significant economic loss to the shrimp aquaculture sector. Unlike the rapid mortalities associated with viral pathogens such as white spot syndrome virus and yellow head virus, progression of LSS is gradual, leading to low-level progressive mortalities. The signs of LSS include a flaccid spongy abdomen due to muscular dystrophy, space between the exoskeleton and muscle, and a shrunken hepatopancreas. The feed conversion efficiency is reduced, and shrimp have poor meat quality, caused by impairment of the hepatopancreatic functions such as digestion and absorption as evidenced by the atrophy of the hepatopancreas. Histopathological investigations on LSS-affected shrimp showed shrinkage of extensor and flexor muscles with occasional hemocytic infiltration. The hepatopancreas showed inflammation of hepatopancreatic tubules with enlargement of intertubular spaces, hemocytic infiltration, and low levels of lipid reserves in the R cells. In advanced stages of LSS, many tubules were in highly necrotic condition with a sloughed epithelium, reflecting the dysfunction of the digestive gland. LSS could be induced in healthy tiger shrimp by challenge studies using membrane-filtered LSS-affected shrimp tissues, suggesting involvement of a filterable infectious agent.

Anti-oxidant status in embryonic, post-hatch and larval stages of Asian seabass (Lates calcarifer).

The concentrations of anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and selenium-dependent glutathione peroxidase (SeGPx), and low molecular weight free-radical scavengers such as reduced glutathione (GSH) and ascorbic acid (vitamin C) were evaluated during the period from gastrulation (GS) to 25 days post-hatch (dph) in the larvae of Asian Seabass, Lates calcarifer. Oxidative damage due to lipid peroxidation (LPO) was also assessed, by evaluation of the formation of malondialdehyde (MDA). All the three anti-oxidant enzymes, SOD, CAT and GPx, showed high activities during gastrulation, suggesting an increased metabolic rate during the period of embryonic development. Though the SOD activity apparently decreased progressively during 3-20 dph of larval development, the difference was not significant. CAT showed high activity during gastrulation and remained constant up to 3 dph, suggesting an increased need to metabolise hydrogen peroxide (H2O2) and organic peroxides. In contrast, SeGPx activity increased progressively from 5 dph to 25 dph during larval development, indicating an increased need to detoxify lipid peroxides. This is evident from the observation of increased lipid peroxidation from 10 dph to 25 dph during larval development. GSH levels were low at gastrulation, indicating increased metabolic rate and formation of lipid radicals during this period, corresponding to the decrease in the level of ascorbic acid, which is consumed for regeneration of GSH.

Involvement of Enterobacter cloacae in the mortality of the fish, Mugil cephalus.

AIMS: To identify the causative agent of the mortality in the fish, Mugil cephalus, in Muttukadu lagoon. METHODS AND RESULTS: An enteric bacterium from the kidneys of moribund fish M. cephalus, was isolated and identified as Enterobacter cloacae (MK). Mugil cephalus was experimentally infected by this isolate and was re-isolated from the kidneys of the moribund fish. Enterobacter cloacae isolates from the lagoon water (MW1, MW2 and reference strain ATCC 13047) and the reference strain were not able to induce similar pathogenesis. The putative factor imparting pathogenicity to the MK isolate was identified as a cationic molecule, which migrated towards the cathode on agarose gel electrophoresis. CONCLUSIONS: The Ent. cloacae (MK) isolate harbouring a cationic factor was the causative agent for the mortality of M. cephalus, found in Muttukadu lagoon. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that human enteric bacteria MK which is considered as nonpathogenic to fish, may become pathogenic to fish when it harbours this cationic factor. This cationic factor is found to be pathogenic to the fish M. cephalus leading to mortality. It was also found to be pathogenic to mice. Therefore, the shuttling of Ent. cloacae, harbouring cationic factor, between human and fish may be of human health importance.

Phenotypic and molecular typing of Vibrio harveyi isolates and their pathogenicity to tiger shrimp larvae.

AIMS: The objective of the present study was to identify the biotype(s) and molecular type(s) of Vibrio harveyi associated with pathogenicity in tiger shrimp (Penaeus monodon) larvae. METHODS AND RESULTS: Five luminescent and four nonluminescent V. harveyi isolates were subjected to phenotyping and random amplified polymorphic DNA (RAPD) fingerprinting, and pathogenicity testing to P. monodon mysis. Four isolates induced 34-41% mortality of P. monodon mysis when challenged at the rate of 10(6) CFU ml(-1) within 60 h. Sucrose-fermenting biotypes of V. harveyi appeared to be associated with pathogenicity to larval shrimp. Higher temperature and salinity appeared to play a role on the onset of vibriosis and mortality in the challenged larval shrimp. Pathogenic isolates of V. harveyi could be demarcated as revealed by their clustering in the dendrogram constructed based on the RAPD fingerprints. CONCLUSIONS: Nonluminescent V. harveyi also appear to be important aetiological agents of vibriosis of shrimp larvae. Sucrose-fermenting biotypes are likely to be pathogenic. High temperature may trigger onset of vibriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Biotyping of V. harveyi isolates and looking for traits, such as ability to ferment sucrose may be helpful in identifying the pathogenic forms, and such approach requires to be investigated further with larger number of isolates.

Polychaete worms--a vector for white spot syndrome virus (WSSV).

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.

Evaluation of Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 on immune indices of tiger shrimp, Penaeus monodon.

Occurrence of widespread epizootics among cultured stock of shrimp has put research programmes on preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study two bacteria, Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 were selected as candidate probionts from a pool of bacteria isolated from gut of farm reared sub-adult shrimp and tested for their effect on the immunity indicators of tiger shrimp. Sub-adult shrimp, weighing 14 to 22 g were treated in separate experiments with Pseudomonas sp. PM 11 and V. fluvialis PM 17 at 10(3) bacterial cells ml(-1) in the experimental shrimp culture tanks. One set of experimental animals was treated every 3 days and another set of animals every 7 days with each of the candidate probionts. Estimation of immunological indicators such as haemocyte counts, phenol oxidase and antibacterial activity showed declining trends. The haemocyte counts dropped from 31 x 10(3) to 65 x 10(3) ml(-1) on the first day to 4-16 x 10(3) ml(-1) on the 45th day. Similarly, the phenol oxidase activity declined from 12-32 units on the first day to 11-14 units on 45th day of the experiment. Antibacterial activity of haemolymph reduced to 46-67 percent on the 45th day of the experiment. The results of the study suggest that, the criteria used for the selection of putative probiotic strains in the present study, such as predominant growth on primary isolation media, ability to produce extracellular enzymes and siderophores, did not bring about the desired effect in vivo and improve the immune system in shrimp. Hence, new protocols have to be evolved for selection of microbe(s) as putative probiotics and that, detailed understanding of proven probiotics, employed presently on empirical basis may provide a clue on the selection procedure.

Messenger RNA degradation in Saccharomyces cerevisiae.

The analysis of 17 functional mRNAs and two recombinant mRNAs in the yeast Saccharomyces cerevisiae suggests that the length of an mRNA influences its half-life in this organism. The mRNAs are clearly divisible into two populations when their lengths and half-lives are compared. Differences in ribosome loading amongst the mRNAs cannot account for this division into relatively stable and unstable populations. Also, specific mRNAs seem to be destabilized to differing extents when their translation is disrupted by N-terminus-proximal stop codons. The analysis of a mutant mRNA, generated by the fusion of the yeast PYK1 and URA3 genes, suggests that a destabilizing element exists within the URA3 sequence. The presence of such elements within relatively unstable mRNAs might account for the division between the yeast mRNA populations. On the basis of these, and other previously published observations, a model is proposed for a general pathway of mRNA degradation in yeast. This model may be relevant to other eukaryotic systems. Also, only a minor extension to the model is required to explain how the stability of some eukaryotic mRNAs might be regulated.

Messenger RNA stability in Saccharomyces cerevisiae: the influence of translation and poly(A) tail length.

A comparison between the half-lives of 10 specific yeast mRNAs and their distribution within polysomes (fractionated on sucrose density gradients) was used to test the relationship between mRNA translation and degradation in the eukaryote Saccharomyces cerevisiae. Although the mRNAs vary in their distribution across the same polysome gradients, there is no obvious correlation between the stability of an mRNA and the number of ribosomes it carries in vivo. This suggests that ribosomal protection against nucleolytic attack is not a major factor in determining the stability of an mRNA in yeast. The relative lengths of the poly(A) tails of 9 yeast mRNAs were analysed using thermal elution from poly(U)-Sepharose. No dramatic differences in poly(A) tail length were observed amongst the mRNAs which could account for their wide ranging half-lives. Minor differences were consistent with shortening of the poly(A) tail as an mRNA ages.

The efficiency of folding of some proteins is increased by controlled rates of translation in vivo. A hypothesis.

We propose that the way in which some proteins fold is affected by the rates at which regions of their polypeptide chains are translated in vivo. Furthermore, we suggest that their gene sequences have evolved to control the rate of translational elongation such that the synthesis of defined portions of their polypeptide chains is separated temporally. We stress that many proteins are capable of folding efficiently into their native conformations without the help of differential translation rates. For these proteins the amino acid sequence does indeed contain all the information needed for the polypeptide chain to fold correctly (even in vitro, after denaturation). However, other proteins clearly do not fold efficiently into their native conformation in vitro. We argue that the efficiency of folding of these problematic proteins in vivo may be improved by controlled synthesis of the nascent polypeptide.

The relationship between mRNA stability and length in Saccharomyces cerevisiae.

A rapid and convenient procedure has been developed for the measurement of mRNA half-life in S.cerevisiae using the transcriptional inhibitor, 1,10-phenanthroline. A range of half-lives from 6.6 +/- 0.67 minutes to over 100 minutes, relative to the stability of the 18S rRNA control, has been obtained for fifteen mRNAs. They include the pyruvate kinase and actin mRNAs, as well as 13 randomly picked mRNAs of unknown function. The mRNAs clearly fall into two populations when their lengths and half-lives are analysed; one population is considerably more stable than the other when mRNAs of similar length are compared. Also, within each population, there is an inverse relationship between mRNA length and half-life. These results suggest that mRNA length and at least one additional factor strongly influence mRNA stability in yeast.