Vertical transmission and prevalence of white spot syndrome virus (WSSV) in the wild spawning population of the Indian white shrimp, Penaeus indicus.

White spot disease, caused by white spot syndrome virus (WSSV), has historically been the most devastating disease in shrimp aquaculture industry across the world. The mode of virus transmission is the most crucial stage in the dynamics and management of virus infection. This study explored the mechanism of vertical transmission of WSSV in Indian white shrimp, Penaeus indicus, potential native species for domestication and genetic improvement, using quantitative real time PCR (q RT PCR), light and electron microscopy, and in situ hybridization. Wild brooders of P. indicus (n = 2576) were sampled along the South east coast of India, during 2016 to 2021. Of these âˆ¼ 58 % of the brooders were positive for WSSV, and almost 50 % of infected wild brooders were at the various stages of reproductive maturation. WSSV-PCR positive brooders (n = 200) were analysed for vertical WSSV transmission. The q RT PCR studies of reproductive tissues revealed that 61 % (n = 13) of spermatophore, 54 % (n = 28) of immature ovaries and 48 % (n = 27) of ripe ovaries were infected with WSSV. The lowest level of infection was recorded in females with ripe ovaries (6.84 × 101 ± 9.79 × 100 ng genomic DNA) followed by fertilized eggs (1.59 × 102 ± 3.69 × 101 ng genomic DNA), and larvae (nauplius and zoea). The histology of gravid females with high WSSV copies showed pyknotic and karyorrhectic germinal vesicle with degenerated cortical rods. Conversely, the gravid females with low WSSV copies showed fully developed ovary without characteristic signs of WSSV infection. Transmission electron microscopic studies clearly established the presence of WSSV particles in both ovaries and spermatophores. When subjected to in situ hybridization, WSSV-specific signals were observed in connective tissues of spermatophore, although gravid ovary and fertilized eggs were failed to produce WSSV specific signals. The present study provides the first molecular and histological evidence for trans-ovarian vertical transmission of WSSV. Development of disease-free base population being the cornerstone and first step in establishing the breeding program, the present findings could be a basis for development of such programs.

Evaluation of in vitro and in vivo potential of Bacillus subtilis MBTDCMFRI Ba37 as a candidate probiont in fish health management.

Bacillus subtilis MBTDCMFRI Ba37 (B. subtilis Ba37), an antibacterial strain isolated from the tropical estuarine habitats of Cochin, was evaluated for in vitro and in vivo potential, and its application as a candidate probiont in fish health management. B. subtilis Ba37 was characterized using their morphological and biochemical properties. It exhibited exoenzymatic activities, tolerance to various physiological conditions and a wide spectrum of antibacterial activity against aquaculture pathogens such as Vibrio and Aeromonas. In co-culture assay, B. subtilis Ba37 inhibited Vibrio anguillarum O1 (V. anguillarum O1) even with the initial cell count of 104 CFUmL-1. Cytotoxicity assay performed using the cell free supernatant (CFS) of B. subtilis Ba37 revealed its non toxic nature. A twenty one days of feeding trial was conducted in juveniles of Etroplus suratensis (E.suratensis) by administrating B. subtilis Ba37 to evaluate its efficacy on growth, immune parameters and antioxidant enzyme activities. Overall the supplementation of B. subtilis Ba37 enhanced significantly (P < 0.05) the survival rate, weight gain, specific growth (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and feed efficiency (FE) of the fed animals as compared with the control. The immune parameters and antioxidant activities such as total protein, alkaline phosphatase (ALP), superoxide dismutase (SOD) and catalase were also improved significantly (P < 0.05) while serum alanine aminotransferase (SGOT) and serum aspartate aminotransferase (SGPT) activities were decreased slightly than the control. After fifteen days of challenge test, the fish fed with B. subtilis Ba37 showed higher relative percentage survival (RPS) than the control. Thus the study indicated the advantages of B. subtilis Ba37 to be used as a candidate probiont, which could be effectively utilized in managing diseases in aquaculture systems and to improve the health of the host.

Isolation and characterization of a novel antimicrobial oxatetracyclo ketone from Bacillus stercoris MBTDCMFRI Ba37 isolated from the tropical estuarine habitats of Cochin.

Antimicrobial compounds from the safest source have gained greater relevance because of their wide spectrum of possible applications, especially in aquaculture industry, where pathogenic threat and antibacterial resistance are serious concerns. Bacillus stercoris MBTDCMFRI Ba37 isolated from mangrove environment of tropical estuarine habitats of Cochin exhibited a wide spectrum of antibacterial activity against major aquaculture pathogens belonging to genus Vibrio and Aeromonas. The structural characterization of the antibacterial compound from this strain may help in identifying their role as a biocontrol agent in aquaculture and allied sectors. The highest antibacterial activity was detected in 3rd day culture, grown in a modified Bacillus medium containing 1% of glycerol and 0.5% of glutamic acid at 30 °C, pH 8.0 and 15 ppt saline conditions. The inhibitory activity of the cell free supernatant was evident even at 20% v/v dilution. Preliminary studies on the nature of antibacterial action indicated that the bioactive principle is stable at temperatures up to 70 °C, between pH 6-9 and instable to lyzozyme and proteolytic enzymes. Bioassay guided purification followed by spectroscopic characterization of active fractions of B. stercoris MBTDCMFRI Ba37 revealed that the compound 1-(1-Hydroxyethyl)-1,7,10,12,13,15,17 heptamethyl-16-oxatetracyclo[8.7.0.02,3.012,13]heptadecan-5-one, is responsible for its major antibacterial activity. This is the first report on isolation and characterization of an antibacterial compound from the species B. stercoris. The results of this study indicated that B. stercoris MBTDCMFRI Ba37 has beneficial antibacterial properties which could be useful in developing novel antimicrobial therapeutics against a variety of aquaculture and other pathogens.

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).

Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP).

Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.

Prospective impact of Corona virus disease (COVID-19) related lockdown on shrimp aquaculture sector in India - a sectoral assessment.

The lockdown on account of the Coronavirus disease 2019 (COVID-19) adversely impacted the food production sector including aquaculture, globally. Unfortunately, it coincided with the major shrimp farming season in India which contributes 60% of the national annual shrimp production hence the impact was substantial. An on-line survey was carried out among the stakeholders of the shrimp farming sector to evaluate the prospective impact of COVID-19 related lockdown across the shrimp supply chain. The study estimated an economic loss of 1.50 billion USD to the shrimp aquaculture sector during the current year. It is expected that shrimp production and its export performance may be declining by 40% in the current season. The Garret ranking and Rank Based Quotient analyses projected severe constraints in shrimp seed production and supply, disruptions in the supply chain, logistics, farming, processing, marketing and loss of employment and income for the workers due to the pandemic. To mitigate the impact, the Government of India declared fisheries and aquaculture as an essential activity, facilitated the movement of inputs and services. Further, a major Fisheries Development Scheme(PMMSY) with a financial outlay of 267 million USD has been announced to usher in a blue revolution by strengthening the value chain, doubling the fisher/farmer income, employment generation, economic and social security for fishers/fish farmers adhering to the sustainability principles. Short and medium-term technical and policy measures are suggested to tide over the impact of COVID-19 related lockdown and related restrictions.

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.

Perkinsus olseni in the short neck yellow clam, Paphia malabarica (Chemnitz, 1782) from the southwest coast of India.

Parasites of the genus Perkinsus predominantly infect bivalves, and two species among them, P. olseni and P. marinus, are notifiable to OIE. P. olseni infections are known to cause extensive damage to wild as well as farmed bivalves globally with enormous implications to its fishery. Consequent to the initiation of a surveillance programme for aquatic animal diseases in India, Perkinsus infections were observed in many species of bivalves. The present paper describes P. olseni infections in the short neck yellow clam, Paphia malabarica from the southwest coast of India. Diagnosis of the parasite was carried out using Ray's Fluid Thioglycollate Medium culture, histology, in-situ hybridisation and molecular taxonomy. Pathology of infection and development of zoospores is also described. This forms the first report of a P. olseni infection in P. malabarica. High prevalence and intensity of infection of Perkinsus in clams raises concerns, as clam reserves in this geographical area sustain fisheries and the livelihoods of local fishing communities.

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.

Molecular approach for the rapid detection of Bacillus and Pseudomonas genera--dominant antagonistic groups--from diverse ecological niches using colony multiplex PCR.

Bacillus and Pseudomonas are the dominant groups of bacteria known for their antagonistic potential against many plant and animal pathogens. Presently, exploration of these genera with antagonistic property for disease management of aquaculture system is gaining more importance to overcome the use of antibiotics and related resistance issues. Rapid screening and identification of these genera from diverse bacterial populations by conventional methods is laborious, cost-intensive, and time-consuming. To overcome these limiting factors, in the present study, a colony multiplex PCR (cmPCR) method was developed and evaluated for the rapid detection of Bacillus and Pseudomonas. The technique amplifies the partial 16S rRNA gene of Bacillus and Pseudomonas with a product size of ~1,100 and ~375 bp, respectively, using single forward (BSF2) and two reverse primers (PAGSR and BK1R). Reliability of the cmPCR method was confirmed by screening 472 isolates obtained from ten different eco-stations, of which 133 isolates belonged to Bacillus and 32 to Pseudomonas. The cmPCR method also helped to identify six different Pseudomonas spp. and 14 different Bacillus spp. from environmental samples. Of the total 472 isolates studied, 46 showed antagonistic activity, among which 63 % were Bacillus and 17.4 % were Pseudomonas. Thus, the newly developed molecular approach provides a quick, sensitive, and potential screening tool to detect novel, antagonistically important Bacillus and Pseudomonas genera for their use in aquaculture. Further, it can also act as a taxonomic tool to understand the distribution of these genera from wide ecological niches and their exploitation for diverse biotechnological applications.

Molecular cloning, characterization and expression analysis of F-type lectin from pearl oyster Pinctada fucata.

F-type lectin is an important type of pattern recognition receptor that can recognize and bind carbohydrate moieties on the surface of potential pathogens through its carbohydrate recognition domains (CRDs). This paper reports the cloning of an F-type lectin (designated as pfF-type lectin) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this pfF-type lectin contains an open reading frame (ORF) of 588 bp coding for196 amino acids. A signal peptide at the N-terminus of the deduced polypeptide was predicted by the signal P program and the cleavage site is located between the positions of Gly(19)and Tyr(20). Conserved domain search at NCBI revealed the pfF-type lectin domain extends from Lys(55)to Val(192). Semi-quantitative analysis in adult tissues showed that the pfF-type lectin mRNA was abundantly expressed in haemocytes and gill and rarely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfF-type lectin mRNA in haemocytes was increased, reaching the highest level at 4 h, then dropping to basal levels at 36 h. These results suggest that F-type lectin play a critical role in the innate immune system of the pearl oyster P. fucata.

Molecular cloning, characterization and expression analysis of cytoplasmic Cu/Zn-superoxide dismutase (SOD) from pearl oyster Pinctada fucata.

Because of its capacity to rapidly convert superoxide to hydrogen peroxide, superoxide dismutase (SOD) is crucial in both intracellular signalling and regulation of oxidative stress. In this paper we report the cloning of a Cu/Zn SOD (designated as pfSOD) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this Cu/Zn SOD contains an open reading frame (ORF) of 471 bp coding for 156 amino acids. No signal peptide was identified at the N-terminal amino acid sequence of Cu/Zn SOD indicating that this pfSOD encodes a cytoplasmic Cu/Zn SOD. This is supported by the presence of conserved amino acids required for binding copper and zinc. Semi-quantitative analysis in adult tissues showed that the pfSOD mRNA was abundantly expressed in haemocytes and gill and scarcely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfSOD mRNA in haemocytes was increased, reaching the highest level at 8 h, then dropping to basal levels at 36 h. These results suggest that Cu/Zn SOD might be used as a bioindicator of the aquatic environmental pollution and cellular stress in pearl oyster.

First report of Perkinsus beihaiensis in Crassostrea madrasensis from the Indian subcontinent.

Protozoan parasites of the genus Perkinsus are considered important pathogens responsible for mass mortalities in many wild and farmed bivalve populations. The present study was initiated to screen populations of the Indian edible oyster Crassostrea madrasensis, a promising candidate for aquaculture along the Indian coasts, for the presence of Perkinsus spp. The study reports the presence of P. beihaiensis for the first time in C. madrasensis populations from the Indian subcontinent and south Asia. Samples collected from the east and west coasts of India were subjected to Ray's fluid thioglycollate medium (RFTM) culture and histology which indicated the presence of Perkinsus spp. PCR screening of the tissues using specific primers amplified the product specific to the genus Perkinsus. The taxonomic affinities of the parasites were determined by sequencing both internal transcribed spacer (ITS) and actin genes followed by basic local alignment search tool (BLAST) analysis. Analysis based on the ITS sequences showed 98 to 100% identity to Perkinsus spp. (P. beihaiensis and Brazilian Perkinsus sp.). The pairwise genetic distance values and phylogenetic analysis confirmed that 2 of the present samples belonged to the P. beihaiensis clade while the other 4 showed close affinities with the Brazilian Perkinsus sp. clade. The genetic divergence data, close affinity with the Brazilian Perkinsus sp., and co-existence with P. beihaiensis in the same host species in the same habitat show that the remaining 4 samples exhibit some degree of variation from P. beihaiensis. As expected, the sequencing of actin genes did not show any divergence among the samples studied. They probably could be intraspecific variants of P. beihaiensis having a separate lineage in the process of evolution.

Diversity and characterization of antagonistic bacteria from tropical estuarine habitats of Cochin, India for fish health management.

Mortalities due to pathogenic bacteria are a major problem in aquaculture, especially in larval rearing systems. Use of antibiotics to overcome this problem is not an option any more due to the increasing antibiotic resistance among pathogens. The present study aims to understand the diversity of bacteria with antagonistic properties in the tropical estuarine habitats of Cochin, located along the southwest coast of India, and to use them as an alternative to antibiotics in aquaculture. Among the 4,870 isolates screened, approximately 1 % showed significant antibacterial activity against six common aquaculture pathogens belonging to the genera Aeromonas and Vibrio. The antagonistic bacteria were identified as Bacillus (81 %) and Pseudomonas (19 %) using biochemical and 16S rRNA gene sequence homology. The isolates showing stable and higher levels of antibacterial activity were subjected to enzymatic expression profile, antibiotic resistance pattern and abiotic stress tolerance assays. As a result, five Pseudomonas spp. and four Bacillus spp., were identified as promising antagonistic isolates that could be exploited as probionts or microbial products (MP's), to control bacterial diseases in aquaculture rearing systems.

Mitochondrial DNA based diversity studies reveal distinct and substructured populations of pearlspot, Etroplus suratensis (Bloch, 1790) in Indian waters.

Pearlspot (Etroplus suratensis) is one of the most commercially important brackish water fish species widely found along the coastal regions of peninsular India and Sri Lanka. Pearlspot is known for its tender flesh, delectable taste, culinary tourism and highyielding market value. Information on the genetic makeup of stocks/populations is extremely vital as it forms the basis for future genetic studies. For this, we utilized ATPase6/8 genes of mtDNA of pearlspot populations collected from nine different locations ranging from Ratnagiri in Maharashtra state on the west coast to Chilika in Odisha on the east coast. Sequence analyses of these genes revealed 33 polymorphic sites, which include 17 singleton and 16 parsimony informative sites. Pair-wise genetic differentiation study (FST = 0.75) indicated significant (P<0.001) differences among all the pairs of stocks except those from Chilika and Nagayalanka. The spatial analysis of molecular variance (SAMOVA) significantly delineated the population into four groups (FCT = 0.69, P = 0.0001), namely northwest (Ratnagiri and Goa); southwest (Mangalore and lakes at Vembanad, Ashtamudi and Vellayani in Kerala); southeast (Pulicat in Tamil Nadu) and northeast (Chilika in Odisha and Nagayalanka in Andhra Pradesh). The above delineation is supported by clades of the phylogenetic tree and also the clusters of median joining haplotype network. The high haplotype diversity (0.84), low nucleotide diversity (0.003), and negative values of Tajima's D (-1.47) and Fu's Fs statistic (-14.89) are characteristic of populations having recently undergone demographic expansion. Mantel test revealed significant isolation by distance. The study identifies highly delineated structured populations with restricted gene flow. If such a stock is overfished, it is highly unlikely that it would recover through migration. For any future breeding programme in this species, it would be desirable to form a base population which incorporates the genetic material from all the locations so that we get a wide gene pool to select from.

Early Post-Vaccination Gene Signatures Correlate With the Magnitude and Function of Vaccine-Induced HIV Envelope-Specific Plasma Antibodies in Infant Rhesus Macaques.

A better understanding of the impact of early innate immune responses after vaccine priming on vaccine-elicited adaptive immune responses could inform rational design for effective HIV vaccines. The current study compared the whole blood molecular immune signatures of a 3M-052-SE adjuvanted HIV Env protein vaccine to a regimen combining the adjuvanted Env protein with simultaneous administration of a modified Vaccinia Ankara vector expressing HIV Env in infant rhesus macaques at days 0, 1, and 3 post vaccine prime. Both vaccines induced a rapid innate response, evident by elevated inflammatory plasma cytokines and altered gene expression. We identified 25 differentially-expressed genes (DEG) on day 1 compared to day 0 in the HIV protein vaccine group. In contrast, in the group that received both the Env protein and the MVA-Env vaccine only two DEG were identified, implying that the MVA-Env modified the innate response to the adjuvanted protein vaccine. By day 3, only three DEG maintained altered expression, indicative of the transient nature of the innate response. The DEG represented immune pathways associated with complement activation, type I interferon and interleukin signaling, pathogen sensing, and induction of adaptive immunity. DEG expression on day 1 was correlated to Env-specific antibody responses, in particular antibody-dependent cytotoxicity responses at week 34, and Env-specific follicular T helper cells. Results from network analysis supported the interaction of DEG and their proteins in B cell activation. These results emphasize that vaccine-induced HIV-specific antibody responses can be optimized through the modulation of the innate response to the vaccine prime.

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.

Non-lethal screening of Asian seabass (Lates calcarifer) by monoclonal antibody based indirect enzyme linked immunosorbent assay for viral nervous necrosis.

Viral nervous necrosis (VNN) is a serious viral disease of several species of farmed and wild fishes. Adult fish are asymptomatic and become carriers of the virus when infected with nervous necrosis virus (NNV) and they transmit the virus to the offspring through eggs. ELISA is ideal for non-lethal screening of adult fish for VNN. Asian seabass (Lates calcarifer) IgM was purified using Protein A affinity column and hybridoma clones secreting monoclonal antibodies (MAb) specific to the heavy chain of IgM was developed. An Indirect ELISA using anti-seabass IgM MAb was developed by optimizing all the reagents. The assay was used to screen adult Asian seabass from grow-out farms in comparison to RT-PCR. The assay was also used to assess the immune response in Asian seabass immunized with inactivated Red-spotted grouper NNV (RGNNV). Seabass IgM on SDS-PAGE analysis revealed three heavy chain bands of size 96, 82 and 76 kDa and a single light chain of size 25 kDa. Out of 18 positive hybridoma clones, two selected clones reacted specifically with the 76 kDa heavy chain band. Out of 28 serum samples of Asian seabass from grow-out farms 26 were positive for NNV antibodies while 22 were positive by RT-PCR. Fish immunized with inactivated RGNNV showed immune response by one week post-immunization, and the peak immune response was observed four weeks post-immunization. The assay developed can be used for non-lethal screening of adult Asian seabass for VNN and to assess the immune response after vaccination.