Studies on expression pattern of toll-like receptor 5 (TLR5) in Edwardsiella tarda infected Pangasianodon hypophthalmus.

TLR5 is one of the important PRR (pathogen recognition receptors) and plays a fundamental role in pathogen recognition and activation of innate immune responses. It recognizes bacterial flagellin and stimulates the production of proinflammatory cytokines, through signalling via the adaptor protein MyD88. In this study, we characterized partial TLR5 (soluble form) gene from Pangasianodon hypophthalmus and analysed its expression profile upon challenge by Edwardsiella tarda. Bioinformatic analysis of gene sequence revealed a putative protein of 266 amino acids with four Leucine rich repeats. Quantitative expression analysis of TLR 5S showed its wide distribution in various organs and tissues. However, significant expression of TLR5S was observed in liver and spleen at 12 h (∼207.8 fold, p < 0.05). Significant upregulation was observed in kidney at 72 h.p.i. (50 folds, p < 0.05) indicating that the kidney provides longer protection almost till the activation of the adaptive immune system. This study enriches the knowledge of TLR5S in boosting the innate immunity against bacterial invasion in fish.

Expression Profiling of WSSV ORF 199 and Shrimp Ubiquitin Conjugating Enzyme in WSSV Infected Penaeus monodon.

White spot syndrome virus (WSSV) is one of the major viral pathogens affecting shrimp aquaculture. Four proteins, WSSV199, WSSV 222, WSSV 249 and WSSV 403, from WSSV are predicted to encode a RING-H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimp can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, a time course expression profile analysis of WSSV Open Reading Frame (ORF) 199 and Penaeus monodon ubiquitin conjugating enzyme (PmUbc) was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge by semi-quantitative RT-PCR as well as Real Time PCR. EF1α was used as reference control to normalize the expression levels. A significant increase in PmUbc expression at 24 h post infection (h.p.i) was observed followed by a decline till 72 h.p.i. Expression of WSSV199 was observed at 24 h.p.i in WSSV infected P. monodon. Since the up-regulation of PmUbc was observed at 24 h.p.i where WSSV199 expression was detected, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. However, further studies need to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this chaos in the shrimp culture industry.

Stock structure analysis of the endemic fish, Barbodes carnaticus (Jerdon 1849), for conservation in a biodiversity hotspot.

The population structure of Barbodes carnaticus species was studied using conventional (based on body morphometrics and meristic) and image-based analysis (truss network system) methods. The study was carried out with four stocks, namely Karnataka (KA) and Tamil Nadu (TN) stocks from the River Cauvery, Kerala (KE) stock from the River Chalakudy and farm-reared stock (CI) from Central Institute of Freshwater Aquaculture, Bangalore. A total of 27 morphometric, 9 meristic and 30 truss measurements were used in the study for the stock structure. Fifteen landmarks were used to generate 30 truss distance measurements. The principal component analysis (PCA), factor analysis (FA), discriminant function analysis (DFA) and cluster analysis (CA) were deployed to determine the variation using both the conventional and truss variables. Variations (86.9%) among the morphometric characters were explained by five principal components, while four principal components explain 96.01% of the variation among the truss distances. DFA using conventional method correctly classified 100% of the original grouped classes of the KA, KE and CI and 93.8% of TN stocks. The DFA employed with truss distance was classified into the stocks CI, KA, KE and TN, and the values are 100, 89.1, 8.6 and 6.1%, respectively. Factor analysis based on truss morphometry showed that factor one is related to body shape and factor two is related to head shape. Two clusters were identified in both the conventional and the truss distance analysis. Truss distance-based cluster showed that the KE and CI stocks are similar compared to the TN stock. In contrary, morphometry-based cluster showed the KE and TN stocks are similar compared to CI stock. The multivariate analysis showed that the farm-reared stock (CI) is different from the wild stocks (KA, KE and TN). This study explained that the combination of the conventional and image-based truss network analysis helps to discriminate various stocks of B. carnaticus. Based on the PCA, bilinear data models were generated using R 3.5.3 software for predicting the stock of each individual. Stock discrimination of this species was mainly due to the geographic isolation, river ecology and temperature variations. The stocks of B. carnaticus are highly exploited from the studied rivers, and the species is an important candidate for species diversification to enhance aquaculture production. Within stock variations are found to be minimum in the present morphometric study, hence the gene pool identification and marker study are required for better understanding of the stocks. This stock structure study may help to develop conservation programmes for this endemic species through a more scientific approach.

Expression Profile of Penaeus monodon Ubiquitin Conjugating Enzyme (PmUbc) at Protein Level in White spot syndrome virus Challenged Shrimp.

White spot syndrome virus (WSSV) is one of the major pathogens in shrimp aquaculture. Four proteins of WSSV are predicted to encode a RING H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimps can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, expression profile of Penaeus monodon Ubiquitin conjugating enzyme (PmUbc) was studied at protein level in WSSV challenged shrimp. A time point analysis of the expression of PmUbc was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge in P. monodon. Recombinant PmUbc (rPmUbc) was produced in prokaryotic expression vector, BL21 (DE3) pLys S. The PmUbc expression pattern was studied by ELISA with rPmUbc antibodies raised in rabbit. A significant increase in PmUbc expression at 24 h post infection (hpi) was observed followed by a decline till 72 hpi. Since the up-regulation and a tremendous decline of PmUbc protein expression was observed at 24 and in 72 hpi respectively in ELISA, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. Many findings have shown that viral infection can up-regulate expression of ubiquitin and that the ubiquitin system plays a key role in the course of viral infection. The present study reveals the expression patterns of PmUbc at protein level in WSSV infected P. monodon. However, further studies are to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this major culprit in shrimp culture industry.