Aparna Chaudhari

DNA constructs expressing long-hairpin RNA (lhRNA) protect Penaeus monodon against White Spot Syndrome Virus

The use of stable and cost-effective DNA vaccines for silencing genes of White Spot Syndrome Virus (WSSV) in Penaeus monodon through RNA interference has been tested. DNA constructs that express long-hairpin RNA (lhRNA) constitutively in vivo against two WSSV envelop genes vp19 and vp28 (pCMV-VP19-LH and pCMV-VP28-LH) were used to confer protection against the pathogen. A construct expressing lhRNA directed against gfp gene (pCMV-GFP-LH) was used to estimate the non-specific protection and to show that on intramuscular administration the plasmid spreads all across the shrimp body and persists for at least 30 days. Shrimp injected with 10 microg lhRNA expressing constructs were challenged with purified WSSV after 24h and pCMV-VP28-LH provided best protection as evidenced by higher survival, lower viral load and silencing of the target viral gene. At an increased dose (35 microg), this construct showed better protection with 75% survival and 81% silencing of the target vp28 mRNA. The construct could actually reduce the viral copy number from the initial 10(3) to 10(5) copies to 10-100 copies. The non-specific pCMV-GFP-LH also showed improved survival and silencing of viral genes, although its effect was less than one third of pCMV-VP28-LH. Silencing constructs can be a good prophylactic approach in commercial shrimp hatcheries to protect high value brooders and to check the vertical transmission of virus, which is a major route of viral entry. Silencing by lhRNA expressed in vivo, confirms the presence of a functional microRNA pathway in shrimp.

A GFP-based bacterial biosensor with chromosomally integrated sensing cassette for quantitative detection of Hg(II) in environment.

A mercury biosensor was constructed by integrating biosensor genetic elements into E. coli JM109 chromosome in a single copy number, using the attP/attB recombination mechanism of lambda phage. The genetic elements used include a regulatory protein gene (merR) along with operator/promoter (O/P) derived from the mercury resistance operon from pDU1358 plasmid of Serratia marcescens. The expression of reporter gene gfp is also controlled by merR/O/P. Integration of the construct into the chromosome was done to increase the stability and precision of the biosensor. This biosensor could detect Hg(II) ions in the concentration range of 100-1700 nmol/L, and manifest the result as the expression of GFP. The GFP expression was significantly different (P < or = 0.05) for each concentration of inducing Hg(II) ions in the detection range, which reduces the chances of misinterpretation of results. A model using regression method was also derived for the quantification of the concentration of Hg(II) in water samples.

Genetic Diversity in Metapenaeus dobsoni using RAPD Analysis

IntroductionMaintenance of both genetic diversity and population distinctiveness is a major goalin the conservation management plans of overexploited species (Meffe 1994).Genetic data and phylogeographic structure throughout the species’ range constitutethe basic information required to understand their evolution and biogeographichistory. A widespread decline in the shrimp population in Indian waters has led to apressing need to assess genetic variability in the wild population for properconservation.Metapenaeus dobsoni (Miers, 1878) is a valuable shrimp distributed in thecoastal waters of India, Sri Lanka, Thailand, Indonesia, Malaysia, and thePhilippines (Fischer and Bianchi 1984). It contributes significantly to shrimpcatches from the east and west coasts of India and Sri Lanka. Overexploitation ofjuveniles and the use of destructive fishing methods in the estuarine environmentcould be responsible for the dwindling of this resource. Knowledge of thepopulation genetic structure of this species can help to determine the exploitationpattern and, hence, conservation.Randomly amplified polymorphic DNA (RAPD) is a simple and easy molecularmethod to determine genetic diversity, taxonomic identity, and systemic details ofvarious organisms (Welsh and McClelland 1990; Williams et al. 1990). Thistechnique has been used to estimate genetic diversity in penaeid shrimp, fish, andshellfish (Bardakci and Skibinski 1994; Garcia et al. 1994; Garcia and Benzie 1995;

Molecular phylogeny of elasmobranchs inferred from mitochondrial and nuclear markers

The elasmobranchs (sharks, rays and skates) being the extant survivors of one of the earliest offshoots of the vertebrate evolutionary tree are good model organisms to study the primitive vertebrate conditions. They play a significant role in maintaining the ecological balance and have high economic value. Due to over-exploitation and illegal fishing worldwide, the elasmobranch stocks are being decimated at an alarming rate. Appropriate management measures are necessary for restoring depleted elasmobranch stocks. One approach for restoring stocks is implementation of conservation measures and these measures can be formulated effectively by knowing the evolutionary relationship among the elasmobranchs. In this study, a total of 30 species were chosen for molecular phylogeny studies using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA gene and nuclear Internal Transcribed Spacer 2. Among different genes, the combined dataset of COI and 12S rRNA resulted in a well resolved tree topology with significant bootstrap/posterior probabilities values. The results supported the reciprocal monophyly of sharks and batoids. Within Galeomorphii, Heterodontiformes (bullhead sharks) formed as a sister group to Lamniformes (mackerel sharks): Orectolobiformes (carpet sharks) and to Carcharhiniformes (ground sharks). Within batoids, the Myliobatiformes formed a monophyly group while Pristiformes (sawfishes) and Rhinobatiformes (guitar fishes) formed a sister group to all other batoids.

In silico analysis and expression studies of kisspeptin gene in C. catla

We report the characterization of kisspeptin gene which is considered to be essential for successful animal reproduction. The full-length cDNA sequence of kiss2 was 583 bp, consisted of 11 bp 5′-UTR (untranslated region) and 194 bp 3′-UTR, respectively. Open reading frame of 378 bp encoding a putative protein of 125 amino acids. The Catla catla kiss2 protein was having a molecular weight of 14.51 kDa and isoelectric point (pI) of 8.46. There were four serine (Ser), four threonine (Thr) and two tyrosine (Tyr) phosphorylation sites and no N-glycosylation sites on the predicted protein. The amino acids on positions 8, 11, 24, 80 and 114 were detected to be ligand binding sites. The signal peptide analysis predicted that C. catla kiss2 is a secretory protein. Kiss2 protein is localized in nuclear region (49.7%) and the extracellular region (38.3%) of the cell. Analysis of tissue distribution revealed that, kiss2 transcripts were predominantly expressed in the brain and gonads, with expression levels in female higher than those of male. Ontogenetic analysis of kiss2 demonstrated that expression level was low during early phase of development stages and more expression was observed during mature stage. Overall present results lay a strong basis for understanding the role of kisspeptin in the neuroendocrine system in teleosts.

Identification, ontogeny and expression analysis of a novel laboratory of genetics and physiology 2 (LGP2) transcript in Asian seabass, Lates calcarifer

ABSTRACT LGP2 (laboratory of genetics and physiology 2) is an important member of the retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs), which plays a significant role in antiviral innate immunity. In this study, we have cloned the full‐length cDNA sequence of LGP2 from Asian seabass, Lates calcarifer (AsLGP2). The complete AsLGP2 cDNA sequence consisted of 2586 nucleotides encoding a putative protein of 681 amino acids with a molecular mass of 77.6 kDa. From the AsLGP2 protein, four different conserved domains were predicted: a DExDc (DEAD/DEAH box helicase domain), a bacterial type III restriction enzyme domain (RES III), a HELICc (Helicase superfamily c‐terminal domain and a RIG‐I_C‐RD (RIG‐I C‐terminal regulatory domain). The transcript of AsLGP2 could be detected in all the 11 tissues tested in healthy animals with high expression noticed in tissues facing external environment such as gill, hindgut and skin. The ontogenic expression profile of AsLGP2 implies a possible maternal transfer of this gene as it has been detected in all early embryonic developmental stages along with unfertilized eggs. Viral analogue, poly I:C, injection resulted in rapid up‐regulated expression in different tissues with the highest modulation of expression observed in kidney followed by liver and gill. A rapid response of AsLGP2 expression was also observed in the different tissues of Vibrio alginolyticus‐injected L. calcarifer, while significant change in expression was noticed following Staphylococcus aureus infection. Similarly, exposure to different pathogen‐mimicking microbial analogues such as poly I:C, LPS and PGN resulted in enhanced expression of AsLGP2 in SISK cell‐line. Taking together, these observations suggest that AsLGP2 can act as both antiviral and antibacterial cytosolic receptor and may play a significant role in embryonic and larval development in marine euryhaline teleosts like Asian seabass. HighlightsA novel LGP2 transcript identified, cloned and characterized from Asian seabass, Lates calcarifer (AsLGP2).AsLGP2 constitutively expressed in embryonic and larval developmental stages.Poly I:C positively modulates the AsLGP2 expression in vivo and in vitro.AsLGP2 responds towards bacterial infection and different bacterial ligands.AsLGP2 may act as a positive regulatory molecule with both antiviral and antibacterial functions in marine euryhaline teleost.

Report of leucine-rich repeats (LRRs) from Scylla serrata: Ontogeny, molecular cloning, characterization and expression analysis following ligand stimulation, and upon bacterial and viral infections.

Leucine-rich repeat (LRR) proteins are present in all living organisms, and their participation in signal transduction and defense mechanisms has been elucidated in humans and mosquitoes. LRRs possibly involve in protein-protein interactions also and show differential expression pattern upon challenge with pathogens. In the present study, a new LRR gene was identified in mud crab, Scylla serrata. LRR gene mRNA levels in different developmental stages and various tissues of S. serrata were analysed. Further, the response of the gene against different ligands, Gram-negative bacterium, and white spot syndrome virus (WSSV) was investigated in vitro and in vivo. Full-length cDNA sequence of S. serrata LRR (SsLRR) was found to be 2290 nucleotide long with an open reading frame of 1893bp. SsLRR encodes for a protein containing 630 deduced amino acids with 17 conserved LRR domains and exhibits significant similarity with crustacean LRRs so that these could be clustered into a branch in the phylogenetic tree. SsLRR mRNA transcripts were detected in all the developmental stages (egg, Zoea1-5, megalopa and crab instar), haemocytes and various tissues such as, stomach, gill, muscle, hepatopancreas, hematopoietic organ, heart, epithelial layer and testis by reverse-transcriptase PCR. SsLRR transcripts in cultured haemocytes showed a 2-fold increase in expression at 1.5 and 12h upon Poly I:C induction. WSSV challenge resulted in significant early up-regulation at 3h in-vitro and late up-regulation at 72h in-vivo. Peptidoglycan (PGN)-induction resulted in marginal up-regulation of SsLRR at timepoints, 6, 12 and 24h (fold change below 1.5) and no significant change in the expression at early timepoints. LPS-stimulation, on the other hand, showed either down-regulation or normal level of expression at all timepoints. However, a delayed 5-fold up-regulation was observed in vivo against Vibrio parahaemolyticus infection at 72hpi. The constitutive expression of the LRR gene in all the early life-stages, and its response to various ligands and to viral challenge suggest the possible role of the LRR in immune defense in mud crab. The result provides additional information which would help in future studies in understanding the innate immune pathways in crustaceans.

DNA barcoding of elasmobranchs from Indian coast and its reliability in delineating geographically widespread specimens.

Abstract Identification of elasmobranchs by conventional taxonomy is difficult due to similarities in morphological characters. Species-specific molecular markers are good choice for identifying species irrespective of its life stage. Recently, mitochondrial cytochrome c oxidase subunit I (COI) gene got global recognition as a barcode gene to discriminate all animals up-to species level. In this study, mitochondrial COI partial gene was used to develop DNA barcodes for 18 species of elasmobranchs (10 species of sharks and 8 species of rays). The COI barcodes clearly distinguished all the species with high interspecific distance values than intraspecific values. The average interspecific and intraspecific distance values are 8.6% and 0.3% for sharks, respectively and 12.4% and 0.63% for rays, respectively using K2P method. The Neighbor-Joining tree showed distinct clusters shared by the species of same genera. The COI barcodes were also used to estimate allopatric divergences for selected species across broad geographical locations and found that Sphyrna lewini, Aetobatus narinari and Neotrygon kuhlii have cryptic diversity.

Molecular characterisation, ontogeny and expression analysis of melanoma differentiation-associated factor 5 (MDA5) from Asian seabass, Lates calcarifer

ABSTRACT MDA5 is the pivotal member of the retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs) and is reported to play a crucial role in type I IFN‐mediated responses against pathogen‐associated molecular patterns (PAMPs), especially nucleic acids. In this study, we have identified and cloned the full‐length cDNA sequence of MDA5, which comprises 3398 nucleotides and encodes for a putative protein of 978 AA length, in Asian seabass, Lates calcarifer. From the putative amino acid sequence of AsMDA5, four different conserved domains could be predicted: two N‐terminal CARD domains, a DExDc domain, a HELICc domain and a C‐terminal RIG‐1_C‐RD domain. The mRNA transcript of AsMDA5 could be detected in all the 11 tissues tested in healthy animals with the highest expression in heart followed by gill and skin. The ontogenetic expression profile showed constitutive expression in developmental stages starting from unfertilized eggs, which implies the possibility of maternally acquired immunity of RLRs in offspring. The viral analogue poly I:C could modulate the AsMDA5 expression both in vivo and in vitro. In all the tissues, AsMDA5 expression was found to be highly regulated following injection with poly I:C with the highest expression observed in kidney. The expression level of AsMDA5 was found to be modulated at different time‐points following challenge with Gram‐negative bacterium, Vibrio alginolyticus, and Gram‐positive bacterium, Staphylococcus aureus. Similarly, noticeable change in AsMDA5 expression was detected in SISK cell line induced with either LPS or PGN. The observations made in this study suggest that in euryhaline marine teleosts like Asian seabass, MDA5 gene serves as one of the pivotal receptor for the detection of viral and bacterial PAMP, and might play an important antimicrobial role during early embryonic development. HighlightsFull‐length cDNA sequence of MDA5 was identified, cloned and characterized from Asian seabass, Lates calcarifer (AsMDA5).AsMDA5 constitutively expressed in embryonic and larval developmental stages.Viral analogue, poly I:C positively modulates the AsMDA5 expression in vivo and in vitro.AsMDA5 responds towards bacterial pathogens and different bacterial ligands.AsMDA5 may be the pivotal RLR molecule with both antiviral and antibacterial functions in marine euryhaline teleost.

Toll-like receptor (TLR) 22, a non-mammalian TLR in Asian seabass, Lates calcarifer: Characterisation, ontogeny and inductive expression upon exposure with bacteria and ligands

&NA; Toll‐like receptor (TLR) 22 is a non‐mammalian TLR found mostly in teleosts and characterized initially as a cell surface surveillance receptor for detecting extracellular long dsRNA. In the current study, the full‐length cDNA sequence consisting of 3312 nucleotides encoding for 960 amino acids in Asian seabass (Lates calcarifer) TLR22 (AsTLR22) was identified. From the putative protein sequence, signature TLR domains such as 18 LRR domains, two transmembrane domains, a single LRR_CT domain and an intracellular TIR domain could be predicted. Phylogenetic analysis showed that AsTLR22 is clustered with other teleost TLR22 and is distinctly different from the other TLR groups. The transcript of AsTLR22 was ubiquitously expressed in all the tissues tested of healthy juveniles with the highest expression in gill followed by hindgut, spleen and skin. The AsTLR22 mRNA transcript was also detected in all the developmental stages as early as unfertilized eggs with higher expression in later stages such as neurula and early embryo. The dsRNA viral analogue, poly (I:C) and Gram‐negative bacterium, Vibrio alginolyticus, were found to modulate the AsTLR22 expression in different tissues with the highest expression in kidney and liver. Gram‐positive bacterium, Staphylococcus aureus, was also found to regulate the AsTLR22 expression at certain time‐points with the highest expression in gill. Similarly, noticeable change in AsTLR22 expression was detected in SISK cell line induced with different ligands such as poly (I:C), LPS and PGN. The findings indicate that AsTLR22 responds in transcript level towards bacteria‐borne PAMPs and extracellular dsRNA in the euryhaline teleost Asian seabass. Further, this might act as an important pathogen surveillance receptor during early developmental stages. HighlightsThe TLR22 transcript was identified and characterized in Asian seabass, Lates calcarifer (AsTLR22).AsTLR22 constitutively expressed in embryonic and larval developmental stages.Viral analogue, poly I:C positively modulates the AsTLR22 expression in vivo and in vitro.AsTLR22 responds towards bacterial challenge and different bacterial ligands.