Development and characterization of a new gill cell line from the striped catfish, Pangasianodon hypophthalmus (Sauvage, 1878).

Cell lines as an in vitro model developed from different target organs of fish find their use in virus susceptibility, cytotoxicity, gene expression studies. The striped catfish, Pangasianodon hypophthalmus, is one of the main species in aquaculture, especially in Southeast Asian countries like Thailand, Indonesia, China, India, Bangladesh, and Vietnam. The present study reports the development of a new permanent cell line from the gills of P. hypophthalmus designated as PHG and its application in toxicological research. Leibovitz's L-15 cell culture medium supplemented with 15% fetal bovine serum (FBS) was used to maintain cell line PHG. The morphology of the PHG cell line was observed fibroblastic-like. PHG cells grew well at varying temperatures ranging from 24 to 30 °C with an optimum temperature of 28 °C. The PHG cell line was characterized using a sequence of mitochondrial cytochrome C oxidase subunit I, which authenticated the species of origin of the cell line. The cell line was transfected with a pEGFP-C1 plasmid, and the transfection reporter gene was successfully expressed 48 h post-transfection with 9% transfection efficiency. The toxicity assessment of two organophosphate pesticides, chlorpyrifos, and malathion using the PHG cell line revealed that the two organophosphate pesticides were cytotoxic to the cell line at varying concentrations.

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.

Teleost Nonapeptides, Isotocin and Vasotocin Administration Released the Milt by Abdominal Massage in Male Catfish, Clarias magur.

In the present work the nonapeptides i.e., isotocin and vasotocin alone or in a combination were tested in C. magur to evaluate their effect on stripping by abdominal massage. Also, we used chitosan-carbon nanotube nanocomposites to conjugate the nonapetides isotocin (abbreviated as COOH-SWCNTCSPeP) and isotocin and vasotocin (COOH-SWCNTCSPePs) with the aim of sustaining the effect for a longer duration. The conjugation of nonapeptides with nanocomposites was confirmed by Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). Two experiments were conducted to study the effect of naked (without nanoparticles) and conjugated nonapeptides on the milt release by stripping. Both the experiments consisted of eight treatments which included four naked groups two nanoconjugated groups and two controls. Both naked and nonconjugated formulations were successful in stripping the male catfish. The mRNA expression of selected reproductive genes was analysed to decipher the effect of nanopeptides at the molecular level. Nonapeptide treatment either naked or nanoconjugated, resulted in the upregulation of the transcript level of genes. Histological analysis revealed the concentration of spermatozoa was more in peptide injected groups than in the controls. The synergistic effects of nonapeptides and Ovatide had a positive impact on GSI. Thus, the present formulations were successful in stripping the male catfish to obtain the milt with significant reproductive success. Even though the naked groups perform better but the number of males required to fertilize the eggs in nanoconjuagted groups was smaller making it worth using for the delivery of nonapeptides.

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.