Flow cytometry analysis of apoptotic progression and expression analysis of four apoptosis-related genes in Penaeus vannamei in response to white spot syndrome virus infection.

Flow cytometry analysis was carried out to detect the progression of apoptosis in haemocytes of WSSV infected Penaeus vannamei at different time-points (1.5 hpi, 18 hpi and 56 hpi). Apoptosis in haemocytes was found to increase with time of infectivity from 5.06 to 69.63%. Quantitative real-time PCR (qPCR) was used for the expression analysis of four apoptosis-related genes such as Death-associated protein 1, caspase-5, translationally controlled tumor protein, and cathepsin D. The evidence of apoptosis in haemocytes of P. vannamei was established as shown by significant increase in the percentage of late apoptotic cells due to WSSV infection in shrimp. The present study gives an insight to the apoptosis rate in a WSSV infected shrimp during the course of infection and the role of apoptosis related genes.

Abundance, community structure and diversity of nitrifying bacterial enrichments from low and high saline brackishwater environments.

The study reports diversity in nitrifying microbial enrichments from low (0·5-5‰) and high (18-35‰) saline ecosystems. Microbial community profiling of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) enrichments was analysed by sequencing 16S rRNA and was processed using Mothur pipeline. The α-diversity indices showed the richness of nitrifying bacterial consortia from the high saline environment and were clustering based on the source of the sample. AOB and NOB enrichments from both the environments showed diverse lineages of phyla distributed in both groups with 38 and 34 phyla from low saline and 53 and 40 phyla in high saline sources, respectively. At class level, α- and γ-proteobacteria were found to be more dominant in both the enrichments. AOBs and NOBs in enrichments from low saline environments were dominated by Nitrosomonadaceae, Gallionellaceae (Nitrotoga sp.) and Ectothiorhodospiraceae and Nitrospira, respectively. Though Chromatiaceae were present in both AOB and NOB enrichments, Nitrosoglobus and Nitrosococcus dominated the AOBs while NOBs were dominated by uncultured genera, whereas Rhizobiales were found in both the enrichments. AOBs and NOBs in enrichments from high saline environments were dominated by Nitrospira-like AOBs, Nitrosomonas and Nitrosococcus genera, whereas ammonia-oxidizing archaea (AOA) group included Nitrosopumilus and Nitrososphaera genera comprising and Nitrospirae, respectively. The majority of the genera obtained in both the salinities were found to be either uncultured or unclassified groups. Results of the study suggest that the AOB and NOB consortia have unique and diverse microbes in each of the enrichments, capable of functioning in aquaculture systems practised at different salinities (0-60 ppt).

A comparative synthesis of transcriptomic analyses reveals major differences between WSSV-susceptible Litopenaeus vannamei and WSSV-refractory Macrobrachium rosenbergii.

Since the 1990s White Spot Syndrome Virus (WSSV) has severely affected shrimp aquaculture worldwide causing a global pandemic of White Spot Disease (WSD) in penaeid culture. However, not all decapod species that can be infected by WSSV show the same susceptibility to the virus, thus raising interesting questions regarding the potential genetic traits that might confer resistance to WSSV. In order to shed light into the genetic markers of WSSV resistance, we employed a dual approach: i) we initially analysed the transcriptomes derived from the hepatopancreas of two species, the susceptible white shrimp Litopenaeus vannamei and the refractory fresh water prawn Macrobrachium rosenbergii, both infected with WSSV. We found a large number of differentially expressed genes (DEGs) belonging to the immune system (mostly anti-microbial peptides and haemolymph clotting components) that were generally up-regulated in M. rosenbergii and down-regulated in L. vannamei. Further, in both species we identified many up-regulated DEGs that were related to metabolism (suggesting a metabolic shift during the infection) and, interestingly, in L. vannamei only, we found several DEGs that were related to moult and suggested an inhibition of the moult cycle in this species following WSSV infection. ii) we then identified a limited number of genetic markers putatively linked with WSD tolerance by employing an ecological genomics approach in which we compared published reports with our own RNA-seq datasets for different decapod species infected with WSSV. Using this second comparative approach, we found nine candidate genes which are consistently down-regulated in susceptible species and up-regulated in refractory species and which have a role in immune response. Together our data offer novel insights into gene expression differences that can be found in susceptible and refractory decapod species infected with WSSV and provide a valuable resource towards our understanding of the potential genetic basis of tolerance to WSSV.

Temporal changes in transcriptome profile provide insights of White Spot Syndrome Virus infection in Litopenaeus vannamei.

Shrimp aquaculture is severely affected by WSSV. Despite an increasing effort to understand host/virus interaction by characterizing changes in gene expression (GE) following WSSV infection, the majority of published studies have focussed on a single time-point, providing limited insight on the development of host-pathogen interaction over the infection cycle. Using RNA-seq, we contrasted GE in gills of Litopenaeus vannamei at 1.5, 18 and 56 hours-post-infection (hpi), between WSSV-challenged and control shrimps. Time course analysis revealed 5097 differentially expressed genes: 63 DEGs were viral genes and their expression in WSSV group either peaked at 18 hpi (and decreased at 56 hpi) or increased linearly up to 56 hpi, suggesting a different role played by these genes during the course of infection. The remaining DEGs showed that WSSV altered the expression of metabolic, immune, apoptotic and cytoskeletal genes and was able to inhibit NF-κB and JAK/STAT pathways. Interestingly, GE changes were not consistent through the course of infection but were dynamic with time, suggesting the complexity of host-pathogen interaction. These data offer novel insights into the cellular functions that are affected during the course of infection and ultimately provide a valuable resource towards our understanding of the host-pathogen dynamics and its variation with time.

Comparative analysis of shrimp (Penaeus vannamei) miRNAs expression profiles during WSSV infection under experimental conditions and in pond culture.

In recent years, the importance of viral and host microRNAs (miRNAs) in mediating viral replication and control of host cellular machinery, has been realised and increasing efforts have been taken in order to understand the interactions of miRNAs from host and pathogen during infection. However, all existing studies has thus far been conducted in controlled experimental conditions and the veracity of these data for field conditions are yet to be established. In this framework, small RNA sequencing was performed to identify the miRNAs involved in shrimp (Penaeus vannamei) immune responses under two different WSSV infection conditions of natural infection and experimentally challenged conditions. The expression profiles of miRNAs of shrimp infected with WSSV under two contrasting conditions were compared and as a result, 23365 known miRNAs and 481 novel miRNAs were identified. Amongst the most abundantly expressed miRNAs, the hypoxia related miR-210 and immune pathway related miR-29b were expressed only in infected shrimps of both conditions. miR-8-5p, having a functional role in modulation of chitin biosynthesis was exclusively represented in higher numbers in the WSSV -infected shrimps under natural conditions whilst four of the miRNAs (mja-miR-6493-5p, mja-miR-6492, mmu-miR-3968, tcf-miR-9b-5p) identified from shrimps collected from pond culture targeted chitinase, an important enzyme involved in growth and moulting in shrimps, indicating an interaction between WSSV infection and moult cycle under culture conditions. Some of the miRNAs (tca-miR-87b-3p, cte-miR-277a) and miRNAs belonging to class miR-9, miR-981 that were identified only in WSSV infected shrimps under experimental conditions, are known to respond against WSSV infection in shrimps. Moreover, the miRNA target prediction revealed several immune-related gene targets such as cathepsin, c-type lectin, haemocyanin and ubiquitin protein ligase were commonly identified under both the conditions. However, the miRNAs identified from challenge experiment had wide number of gene targets as compared to the miRNAs of natural infection. The shrimp miRNA mja-miR-6489-3p, was also found to target early virus gene wsv001 of WSSV. Our study, therefore, provides the comparative analysis of miRNA expression from shrimp during WSSV infection in two different conditions.

Genome size estimation of brackishwater fishes and penaeid shrimps by flow cytometry.

Flow cytometry was used for estimating the genome size of five brackishwater finfish and four shrimp species. The genome size for Lutjanus argentimaculatus was 0.95 ± 0.10 and 0.79 ± 0.01 pg for Scatophagus argus. The genome sizes for Chanos chanos (0.72 ± 0.01 pg), Etroplus suratensis (1.71 ± 0.16 pg) and Liza macrolepis (0.87 ± 0.02 pg) which are important aquaculture species are reported for the first time in this study. The phylogenetic tree constructed using sixty-seven sequence accessions of cytochrome c oxidase subunit 1 (COI) gene of Lates calcarifer revealed two separate clades. The Indian Lates calcarifer species with estimated genome size of 0.44 ± 0.02 pg belonged to a clade different than that of South East Asia and Australia reported to have larger genome size. The genome size for the four major species of genus Penaeus (Penaeus monodon, Penaeus indicus, Penaeus vannamei and Penaeus japonicus) were found in similar range. The genome size of female shrimps ranged from 2.91 ± 0.03 pg (P. monodon) to 2.14 ± 0.02 pg (P. japonicus). In male shrimps, the genome size ranged from 2.86 ± 0.06 pg (P. monodon) to 2.19 ± 0.02 pg (P. indicus). Significant difference was observed in the genome size between male and female shrimp of all species except in P. monodon. The highest relative difference of 12.78% was observed in the genome size between the either sex in P. indicus. The interspecific relative difference of 30.59% in genome size was highest between the male shrimps of P. monodon and P. indicus and 35.98% between the female shrimps of P. monodon and P. japonicus. The stored gills and pleopod tissues could be successfully used up to 3 weeks to estimate the genome size in shrimps.

First Report of a Complete Genome Sequence of White spot syndrome virus from India.

White spot syndrome virus is a major pathogen of shrimp, causing economic loss to the aquaculture industry. For the first time, a complete de novo genome of an Indian isolate of this virus has been deciphered using Illumina and Nanopore sequencing technologies. The genome has 280,591 bp with 442 predicted coding genes.

Effect of immune gene silencing in WSSV infected tiger shrimp Penaeus monodon.

White spot syndrome virus, continues to cause huge economic loss to aquaculture industry. In the absence of effective therapeutics to control WSSV, it is important to understand the host pathogen interaction at the molecular level. Suppression subtractive hybridization (SSH) cDNA library was constructed which led to identification of several differentially expressed genes in response to WSSV infection in Penaeus monodon. The genes expressed in SSH cDNA library of shrimp gill and gut tissues belonged to a wide range of biological functions. The three differentially expressed genes, Single von Willebrand factor type C domain protein (pmSVC), P53 protein gene (pmP53) and ADP ribosylation factor (pmArf) were up-regulated against WSSV infection and were further characterized by gene silencing to study the role of these shrimp immune genes on WSSV multiplication. The sequence-specific knock down of pmSVC, pmP53 and pmArf using the dsRNA revealed that in pmSVC-dsRNA inoculated shrimps WSSV replication was more with increased viral copy numbers when compared with pmP53-dsRNA and pmArf -dsRNA inoculated shrimps. The varied response of immune genes to WSSV infection, indicated that host genes may either inhibit virus replication to some extent or might act as a target to facilitate viral pathogenesis.

Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

Recent insights into host-pathogen interaction in white spot syndrome virus infected penaeid shrimp.

Viral disease outbreaks are a major concern impeding the development of the shrimp aquaculture industry. The viral disease due to white spot syndrome virus (WSSV) observed in early 1990s still continues unabated affecting the shrimp farms and cause huge economic loss to the shrimp aquaculture industry. In the absence of effective therapeutics to control WSSV, it is important to understand viral pathogenesis and shrimp response to WSSV at the molecular level. Identification and molecular characterization of WSSV proteins and receptors may facilitate in designing and development of novel therapeutics and antiviral drugs that may inhibit viral replication. Investigations into host-pathogen interactions might give new insights to viral infectivity, tissue tropism and defence mechanism elicited in response to WSSV infection. However, due to the limited information on WSSV gene function and host immune response, the signalling pathways which are associated in shrimp pathogen interaction have also not been elucidated completely. In the present review, the focus is on those shrimp proteins and receptors that are potentially involved in virus infection or in the defence mechanism against WSSV. In addition, the major signalling pathways involved in the innate immune response and the role of apoptosis in host-pathogen interaction is discussed.

High salinity induced expression profiling of differentially expressed genes in shrimp (Penaeus monodon).

Four suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed salinity stress responsive genes of black tiger shrimp, Penaeus monodon exposed to high (55 ppt) salinity conditions. One each of the forward and reverse SSH cDNA libraries were developed from the gill and gut tissues of shrimp and clones having inserts larger than 300 bp were unidirectionally sequenced. Based on the sequence homology search, the identified genes were categorized for their putative functions related to a wide range of biological roles, such as nucleic acid regulation and replication, immune response, energy and metabolism, signal transduction, cellular process, structural and membrane proteins, stress and osmoregulation. Gene expression levels in response to high salinity conditions at 2 weeks post salinity stress for some of the differentially expressed genes (Na(+)/K(+)-ATPase α-subunit, glutathione peroxidase, intracellular fatty acid binding protein, elongation factor 2, 14-3-3 like protein, penaeidin, translationally controlled tumor protein, transglutaminase and serine proteinase inhibitor B3) identified from SSH cDNA libraries were analysed by real-time RT-PCR. The highest gene expression levels was observed for Na(+)/K(+)-ATPase α-subunit in gill tissues (15.23-folds) and antennal glands (12.01-folds) and intracellular fatty acid binding protein in gut tissues (14.05-folds) respectively. The differential and significant levels of gene expression indicate the functional role of these genes in shrimp salinity stress adaptive mechanisms.

Molecular cloning and characterization of acyl-CoA binding protein (ACBP) gene from shrimp Penaeus monodon exposed to salinity stress.

Acyl-CoA binding protein (ACBP), a protein present ubiquitously in wide range of organisms play significant role in transport of acyl groups for macromolecular biosynthesis involved in various functional and regulatory processes. In crustaceans, ACBP has functional role in growth, reproduction and temperature tolerance. In the present study, two suppression subtractive hybridization (SSH) cDNA libraries were performed using gut tissues of shrimp Penaeus monodon exposed to low (3 ppt) and high (55 ppt) salinity stress conditions. SSH library resulted in identification of differentially expressed genes that belonged to various functional classes such as the nucleic acid regulation and replication, defence proteins, allergen protein, signal transduction pathways, apoptosis, energy and metabolism, cell cycle regulation and hypothetical proteins. ACBP was identified as one of the differentially expressed gene in both the SSH libraries of shrimp P. monodon subjected to low and high salinity stress. The full-length cDNA of P. monodon ACBP gene was isolated and the sequence revealed 273 bp open reading frame encoding 90 amino acids with molecular mass of 10 kDa and pI 6.8. The ORF showed presence of four phosphorylation sites, with absence of signal peptide sequence and glycosylation sites. The deduced amino acid sequence of ACBP exhibited high sequence identity (92%) with ACBP class of protein identified from Fenneropenaeus chinensis. Real time PCR analysis of shrimps subjected to 3 ppt salinity conditions after 2 weeks revealed an increase in expression of ACBP transcripts, in the gut (28.08-folds), gills (11.71-folds) and in the muscle tissues (1.70-folds). Whereas, shrimps exposed to 55 ppt salinity conditions after 2 weeks exhibited increased ACBP transcript levels in the gut (11.95-folds), gills (1.052-folds) and muscle tissues (7.35-folds). The significant increase in expression levels of ACBP in various tissues of shrimps suggests a functional role of this gene in salinity stress tolerance and adaptation.

Identification and expression analysis of differentially expressed genes from shrimp (Penaeus monodon) in response to low salinity stress.

Four suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed salinity stress responsive genes of black tiger shrimp, Penaeus monodon exposed to low (3 ppt) salinity conditions. Forward and reverse SSH cDNA libraries were developed from the gill and gut tissues of shrimp and clones having inserts larger than 300 bp were unidirectionally sequenced. Based on the sequence homology search, the identified genes were categorized for their putative functions related to a wide range of biological roles, such as nucleic acid regulation and replication, immune response, energy and metabolism, cell signaling, cellular process, cytoskeleton and membrane structure, stress and osmoregulation. Gene expression levels in response to low salinity conditions at 2 weeks post salinity stress of thirteen selected differentially expressed genes identified from SSH cDNA libraries (14-3-3 like protein, crust in, lysozyme, arginine kinase, Naþ/Kþ-ATPase a-subunit, intracellular fatty acid binding protein, cathepsin B, anti-lipopolysaccharide factor, ferritin, ubiquitin conjugating enzyme E2, calreticulin, innexin 2 and heat shock protein 21) were analyzed by RT-PCR. The highest gene expression levels were observed for Naþ/Kþ-ATPase a-subunit (34.28-folds) in gill tissues, intracellular fatty acid binding protein (13.30-folds) in gut tissues and innexin 2 (14.43-folds) in muscle tissues respectively. The differential and significant levels of gene expression indicate the functional role of these genes in shrimp salinity stress adaptive mechanisms.

Identification, cloning and expression analysis of Catechol-O-methyltransferase (COMT) gene from shrimp, Penaeus monodon and its relevance to salinity stress.

O-methyltransferase (OMT), a protein present ubiquitously in wide range of organisms plays significant role in methylation of small macro molecules for various functional and regulatory purposes. In crustaceans, OMT has functional role in growth, reproduction, ovarian development and molting. In the present study, suppression subtractive hybridization (SSH) performed using gill tissues of low (3ppt) and high (55ppt) salinity stressed shrimp Penaeus monodon resulted in identification of differentially expressed genes involved in signal transduction pathways, metabolism, defense proteins, DNA repair and synthesis, apoptosis, cell cycle regulation along with unknown and hypothetical proteins. Catechol-O-methyltransferase (COMT) a type of OMT was identified by SSH as one of the differentially expressed genes of shrimp P. monodon subjected to low and high salinity stress. The full length cDNA of COMT was cloned from the gills of P. monodon which consisted an open reading frame of 666 bp, encoding 221 amino acids. The ORF revealed one each of N-glycosylation and O-glycosylation sites and nine phosphorylation sites. The deduced amino acid sequence of COMT exhibited high sequence identity (92%) with COMT class of protein from Fenneropenaeus chinensis. Real time PCR analysis of the shrimp samples exposed to low salinity conditions at 3ppt revealed significant increase in expression of COMT transcripts in the guts at 24 h, 48 h, 1 week and 2 weeks, gills at 24 h and in the muscle tissues at 48 h, with maximum expression of the COMT levels by 5 fold in guts (1 week), 1 fold in gills (24 h) and 1.5 fold in muscle (48 h) respectively. The increased expression level of COMT at different time intervals in different tissues suggests a possible role of this gene in salinity stress tolerance in shrimps under low salinity conditions.

Transcript Analysis of White spot syndrome virus Latency and Phagocytosis Activating Protein Genes in Infected Shrimp (Penaeus monodon).

Viral latency has been recently observed to be associated with White spot syndrome virus (WSSV) infection in shrimp. In the present study, shrimp samples (Penaeus monodon) surviving WSSV infection were examined for presence of WSSV in latent phase. Virus latency was observed in shrimp which were either experimentally challenged with WSSV and survived the infection or those which survived the natural infection. Three viral transcripts (ORFs 427, 151, 366) associated with latency were analyzed by real-time PCR. The shrimp surviving the natural WSSV infection on estimation with RT-PCR were found to have low grade of WSSV infection (less than 56 copies of WSSV). All the shrimp samples were RT-PCR negative for structural protein genes of WSSV, VP24 and VP28, indicating that these samples were harboring latent phase virus. RT-PCR of all the shrimp samples which survived WSSV infection revealed amplification of phagocytosis activating protein (PAP) gene (435 bp) with higher gene expression levels in experimentally challenged shrimp when compared to naturally infected shrimp. The expression of PAP in WSSV infected shrimp samples indicates its possible role in host response for resistance against WSSV infection. PAP was cloned and expressed as recombinant protein for protection studies. Shrimp were injected with three doses (5, 15 and 20 µg g(-1) body weight) of recombinant PAP. Relative percent survival of 10 % was observed in shrimp immunized with the dose of 15 µg g(-1) body weight of recombinant PAP. The expression of both WSSV latency associated and PAP genes obtained from shrimp surviving the WSSV infection, indicates the possible role of these genes in host-pathogen interaction.

Molecular biological characterization and biostimulation of ammonia-oxidizing bacteria in brackishwater aquaculture.

In the present study, molecular methods based on sequencing of clone libraries have been used to provide sequence and the phylogenetic information of ammonia oxidizing bacteria (AOB). Ammonia monooxygenase (amoA) gene, which catalyzed the oxidation of ammonia to hydroxyl amine in the initial rate-determining step of nitrification was targeted for detection and characterization of AOB using gene-specific primers. The amoA genes obtained through the clone library construction are closely affiliated with Nitrosomonas sp. and other uncultured beta proteobacteria. The levels of nucleotide similarity and amino acid similarity ranged from 85-99% and 83-88%, respectively. The level of conservation of the amino acid sequences is 73%. The use of a matrix prepared from abundantly available lignocellulosic agrowaste-bagasse has successfully been demonstrated for biostimulation of AOB in aquaculture environment by supplementing nutritional requirement facilitating the biofilm mode of growth of the autotrophic consortia. Present study is useful in predictability and reliability of the treatment of ammonia in brackishwater aquaculture.

Viral nerve necrosis in hatchery-produced fry of Asian seabass Lates calcarifer: sequential microscopic analysis of histopathology.

We studied the natural progression of viral nerve necrosis (VNN) in larvae of Asian seabass Lates calcarifer Bloch from 0 to 40 days post-hatch (dph). The hatchlings were reared in the vicinity of a confirmed nodavirus-affected older batch. Using light and electron microscopy (EM), we made a sequential analysis of histopathological manifestations in nerve tissue and other organs. There were no changes from the day of hatching until 4 dph. Larvae at 4 dph had viral particles in the intramuscular spaces underlying the skin, but the nerve cells of the brain were normal. The first signs of necrosis of the brain cells were observed at 6 dph. EM observations revealed characteristic membrane-bound viral particles measuring 30 nm in the cytoplasm of nerve cells of the brain, spinal cord and retina. Histological samples of fry examined when group mortalities reached 20 to 35% revealed highly vacuolated brains, empty nerve cell cytoplasm and viral particles in the intercellular spaces. Viral particles occurred extensively in the intramuscular spaces and the epidermal layers. These observations were corroborated by positive immunostaining of the virus-rich intramuscular spaces. EM studies also revealed progressive necrotic changes in the cells harboring the virus. Results emphasize the need to maintain hygiene in the hatchery environment and to develop strategies for prevention of disease spread among cohabiting seabass and other susceptible fish larvae. Intramuscular localization of the nodavirus in both preclinical healthy-looking and post-clinical moribund larvae suggests that virus neutralization strategies during larval development could be effective in controlling VNN-associated mortalities.

White spot syndrome virus isolates of tiger shrimp Penaeus monodon (Fabricious) in India are similar to exotic isolates as revealed by polymerase chain reaction and electron microscopy.

Microbiological analysis of samples collected from cases of white spot disease outbreaks in cultured shrimp in different farms located in three regions along East Coast of India viz. Chidambram (Tamil Nadu), Nellore (Andhra Pradesh) and Balasore (Orissa), revealed presence of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas spp. but experimental infection trials in Penaeus monodon with these isolates did not induce any acute mortality or formation of white spots on carapace. Infection trials using filtered tissue extracts by oral and injection method induced mortality in healthy P. monodon with all samples and 100% mortality was noted by the end of 7 day post-inoculation. Histopathological analysis demonstrated degenerated cells characterized by hypertrophied nuclei in gills, hepatopancreas and lymphoid organ with presence of intranuclear basophilic or eosino-basophilic bodies in tubular cells and intercellular spaces. Analysis of samples using 3 different primer sets as used by other for detection of white spot syndrome virus (WSSV) generated 643, 1447 and 520bp amplified DNA products in all samples except in one instance. Variable size virions with mean size in the range of 110 x 320 +/- 20 nm were observed under electron microscope. It could be concluded that the viral isolates in India involved with white spot syndrome in cultured shrimp are similar to RV-PJ and SEMBV in Japan, WSBV in Taiwan and WSSV in Malaysia, Indonesia, Thailand, China and Japan.

Nodavirus infection causes mortalities in hatchery produced larvae of Lates calcarifer: first report from India.

Larvae (15 to 21 d post hatch, dph) of the Asian sea bass Lates calcarifer (Bloch) suffered heavy mortalities (60 to 90%) during the hatchery-rearing phase. Darkened and moribund larvae showed no evidence of bacterial or parasitic infections. Tissue sections of brain and spinal cord showed clear necrotic vacuolation. Electron microscopy revealed membrane-bound viral particles in the cytoplasm of the nerve cells. The viral particles measured 28 to 30 nm in diameter. Primer sets, designed for the amplification of the RNA2 segment of the piscine nodavirus coat protein gene, were used in the RT-PCR analysis of moribund larvae of 20 and 21 dph which produced the amplified product of 430 bp. The clinical manifestations, pathology and electron microscopy observations supported by the RT-PCR analysis suggest that the nerve necrosis was due to nodavirus infection in the larvae. This is the first report of piscine nodavirus infection from the Indian sub-continent.