Gain of function studies on predicted host receptors for white spot virus.

Three decades after its first outbreak, the shrimp white spot virus (WSV) is still a global cause of concern due to considerable losses and lack of effective control measures. Several candidate host receptor proteins have been identified, but the pathogenesis is not clearly understood, although the key role of the WSV envelope protein VP28 in virus internalization is established. Here, protein-protein docking is applied to evaluate the interaction of VP28 trimeric extracellular region with four host (Penaeus monodon) receptors reported earlier, Rab7 GTPase (PmRab7), glucose transporter 1 (PmGLUT1), C-type lectin (PmCTL) and calreticulin (PmCRT). The stability of predicted complexes evaluated in terms of binding energy per unit buried surface area ranged from -8.46 to -11.82 cal mol-1/Å2, which is not sufficient for functional interaction. Nevertheless, each of these host proteins was tested by a gain-of-function approach by observing their ability to make a fish cell line permissive to the shrimp WSV. Full-length expression constructs of the four receptors were transfected into SSN1 snakehead fish cells that are non-permissive to WSV. Transfected SSN1 cells and WSV permissive insect Sf9 cells were challenged with purified WSV. After 24 h, the presence of receptor transcripts was confirmed in the treated SSN1 cells, and not in the non-transfected SSN1 cells. Further, vp28 transcript was detected in Sf9 cells, but not in any of the treated SSN1 cells, indicating that none of the receptors were singly sufficient to make SSN1 cells permissive to WSV, even though PmRab7 was a strong candidate that alone showed >85% protection in virus neutralization experiments. For the other 3 candidates, previous reports predicted the involvement of co-receptors, which is confirmed here by their inability to act singly.

Transcriptome analysis of Clarias magur brain and gonads suggests neuro-endocrine inhibition of milt release from captive GnRH-induced males.

Transcriptome analysis of Clarias magur brain and gonads at preparatory, mature, 6 and 16 h post-GnRH injection (hpi) stages yielded 9.5 GB data with 39,738 contigs. Sequences of 45 reproductive genes were identified for the first time in C. magur along with unique and differentially expressed genes. The expression of 20 genes was validated by qRT-PCR. Upregulation of Cyp11A1, Cyp17A1 and FTZF1 genes in the 16hpi testis accompanied by the 17ß-HSD3 expression indicates testosterone (T) synthesis in response to LH surge, while reduced expression of CYP11B1 suggests a high T: 11-KT ratio. It is evident by the gene expression analysis that the inhibitory neurotransmitter GABA, altered T: 11-KT, increased testicular bile acids, and oxytocin-like neuropeptide in the male brain, appear to be involved in arresting the pulsatile motion of testicular smooth muscles. The work generates important leads for an effective induced breeding strategy for silurid catfish.

Molecular characterization and expression analysis of secretory immunoglobulin M (IgM) heavy chain gene in rohu, Labeo rohita.

Immunoglobulin M (IgM) is the major isotype among teleost immunoglobulins. The present study was aimed to explore IgM heavy chain gene and its expression profile in rohu. Full-length IgM heavy chain cDNA of rohu consisted of 1994 bp encoding a polypeptide of 576 amino acid residues including a leader peptide, variable (VH) and constant (CH1-CH2-CH3-CH4) domains confirming the secretory form of IgM. The sequence carries conserved residues such as cysteine, tryptophan and amino acid motifs like 'YYCAR' and 'FDYWGKGT-VTV-S'. The predicted 3 D model confirmed various domains of rohu IgM heavy chain. Phylogenetic tree analysis revealed that IgM heavy chain gene of rohu shared the same cluster with that of other cyprinid fishes. Tissue distribution analysis showed the predominant level of IgM heavy chain gene expression in kidney, spleen and intestine. IgM heavy chain gene expression in rohu kidney was found to be up-regulated and reached a maximum at 7 days post-challenge with Aeromonas hydrophila. These findings demonstrate the first report of full-length secretory IgM heavy chain gene in rohu. Besides, IgM heavy chain gene was highly expressed in major lymphoid tissues and bacterial challenge influenced its expression which further confirmed its role in the adaptive humoral immune response.

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.

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.

Molecular characterization of kisspeptin gene and effect of nano-encapsulted kisspeptin-10 on reproductive maturation in Catla catla.

Kisspeptin, a member of the RF-amide-related peptide family, has emerged recently as an essential gatekeeper of various reproductive processes via its ability to activate kisspeptin receptors at puberty. In this study, the kiss1 gene and its receptor kiss1rb were cloned and characterized from the brain of Catla catla. Further, the effects of kissppetin-10 (K-10) and chitosan-encapsulated K-10 nanoparticles (CK-10) on gene expression were assessed. The full-length complementary DNA sequence of kiss1 is 754 bp with an open reading frame of 351 bp that encodes a putative protein of 116 amino acids. The kiss1rb complementary DNA is 1,280 bp long and contains a 5'-untranslated region of 30 bp, 3'-untranslated region of 149 bp, and an open reading frame (open reading frame) of 1,101 bp. The expression patterns of kiss1 and kiss1rb messenger RNA (mRNA) in basal tissues revealed that they are mainly expressed in the brain, pituitary gland, and gonads. CK-10 nanoparticles with a particle size of 125 nm and a zeta potential of 36.45 mV were synthesized and compared with K-10. Chitosan nanoparticles showed 60% entrapment efficiency for K-10. The mRNA expression of reproductive genes (GnRH, LH, and FSH) in fish injected with K-10 declined after 6 h, whereas those injected with CK-10 showed controlled and a sustained surge of mRNA expression of these genes with a peak at 12 h. Histologic examination of ovaries indicated a pronounced effect of CK-10 on maturation and gonadal development. The study reports that this sustained release delivery system will help in increasing the half-life of K-10 and other therapeutic protein drugs in the biological system. Besides, the nanoformulation developed in the present study may be useful for developing therapies against various reproductive dysfunctions in vertebrates.

Expression studies on NA+/K(+)-ATPase in gills of Penaeus monodon (Fabricius) acclimated to different salinities.

The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K(+)-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35 per thousand salinities and maintained for 20 days to observe long-term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5 per thousand compared to 25 per thousand (isosmotic salinity) and 0.48 folds lower at 35 per thousand. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5 per thousand the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25 per thousand and 35 per thousand NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25 per thousand. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5 per thousand measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.