In vitro propagation of infectious hypodermal hematopoietic necrosis virus (Penaeus stylirostris penstyldensovirus) in PmLyO-Sf9 cells.

Infectious hypodermal hematopoietic necrosis virus (IHHNV/PstDVI) was isolated and propagated in the hybrid shrimp-insect cell line PmLyO-Sf9. A few hours after inoculation with an infected tissue extract or virus suspension, cytopathic changes could be observed in the cell line, including clustering, enlargement, syncytium formation, granulation, vacuole formation, tapering, irregularities in the plasma membrane with extended tails, detachment, cell death, and accumulation of cellular debris. Expression of viral genes, the presence of virions, and cytological changes observed using transmission electron microscopy suggested replication of the virus in these cells. The virus was purified by ultracentrifugation, negatively stained, and examined using an electron microscope, and the purified virus was found to be infectious both in vitro and in vivo. This development opens avenues for the study of the basic molecular mechanism of IHHNV infection, pathogenesis, and replication, which is much needed for developing an antiviral strategy in aquaculture.

A Novel Approach of Transducing Recombinant Baculovirus into Primary Lymphoid Cells of Penaeus monodon for Developing Continuous Cell Line.

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30 years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5 mM sodium butyrate to the culture medium.

Cytotoxic impacts of treated electroplating industrial effluent and the comparative effect of their metal components (Zn, Hg, and Zn+Hg) on Danio rerio gill (DrG) cell line.

Effluent produced during the electroplating process can contain high concentrations of heavy metals that can enter the environment and induce toxicity to aquatic organisms. Relatively high concentrations of zinc (Zn) and mercury (Hg) have been detected in treated electroplating industrial effluent (TEPIE), though the cytotoxic potential of these compounds has not been well assessed in fish gills. A novel cell line, Danio rerio gill (DrG), were exposed to TEPIE and concentrations of Zn, Hg, and Zn + Hg previously measured in treated effluent to evaluate the use of the DrG cell line following exposure to environmental pollutants. Several cytotoxic assays were employed to assess the effect of TEPIE, Zn, and Hg on this cell line. The percent cell viability was significantly reduced in a concentration-dependent manner following exposure to TEPIE, Zn, Hg, and Zn + Hg (p < 0.05) for 24 h, with additional morphological changes observed in exposure treatments relative to controls. Additionally, there was a significant induction of DNA damage detected in all exposure treatments determined through comet assay tail length. An increase in intracellular ROS generation was also observed in cells exposed to TEPIE, Zn, Hg, and Zn + Hg, corresponding to dose-dependent increases in apoptosis. Our study confirmed that TEPIE and the metals present in it induced cytotoxicity in the DrG cell line, demonstrating its usefulness as a model to explore relationships between pollutants and fish gills.

Effects of zinc and mercury on ROS-mediated oxidative stress-induced physiological impairments and antioxidant responses in the microalga Chlorella vulgaris.

The rapid growth of industrialization and urbanization results in deterioration of freshwater systems around the world, rescinding the ecological balance. Among many factors that lead to adverse effects in aquatic ecology, metals are frequently discharged into aquatic ecosystems from natural and anthropogenic sources. Metals are highly persistent and toxic substances in trace amounts and can potentially induce severe oxidative stress in aquatic organisms. In this study, adverse effects of the two metal elements zinc (maximum concentration of 167.25 mg/L) and mercury (104.2 mg/L) were examined using Chlorella vulgaris under acute and chronic exposure period (48 h and 7 days, respectively). The metal-induced adverse effects have been analyzed through photosynthetic pigment content, total protein content, reactive oxygen species (ROS) generation, antioxidant enzymatic activities, namely catalase and superoxide dismutase (SOD) along with morphological changes in C. vulgaris. Photosynthetic pigments were gradually reduced (~32-100% reduction) in a dose-dependent manner. Protein content was initially increased during acute (~8-12%) and chronic (~57-80%) exposure and decreased (~44-56%) at higher concentration of the two metals (80%). Under the two metal exposures, 5- to 7-fold increase in ROS generation indicated the induction of oxidative stress and subsequent modulations in antioxidant activities. SOD activity was varied with an initial increase (58-129%) followed by a gradual reduction (~3.7-79%), while ~1- to 12-fold difference in CAT activity was observed in all experimental condition (~83 to 1605%). A significant difference was observed in combined toxic exposure (Zn+Hg), while comparing the toxic endpoint data of individual metal exposure (Zn and Hg alone). Through this work, lethal effects caused by single and combined toxicity of zinc and mercury were assessed, representing the significance of appropriate monitoring system to trim down the release of metal contaminants into the aquatic ecosystems.

Improved lipid production in oleaginous brackish diatom Navicula phyllepta MACC8 using two-stage cultivation approach.

A two-stage cultivation method involving the initial growth in optimized conditions for biomass production followed by those for lipid production in oleaginous brackish diatom Navicula phyllepta MACC8 resulted in a proportional increase of lipid concentration along with biomass production. The diatom was further subjected to stress conditions by altering the nutrient components such as nitrate, phosphate, silicate, and temperature. Silicon deprivation resulted in the highest lipid percentage of 28.78% of weight at the end of the 18th day of the second stage. A significant increase in lipid content was observed on the complete removal of the nutrients silicon and urea one at a time, while the biomass showed a considerable reduction. The application of multiple nutrient stress conditions had a profound influence on the increased rate of lipid production. A combination of phosphate deprivation, silicate limitation and temperature reduction resulted in a significant increase in lipid percentage of 32.13% at the cost of reduced biomass (1.1 g L-1), whereas phosphate deprivation, urea limitation, and temperature reduction resulted in lipid percentage of 27.58% with a biomass of 1.44 g L-1 at the end of the second stage. Further, the results were supported by Nile red staining, FTIR, fatty acid profile and oxidative stress marker analyses. The changes in biochemical composition and oxidative stress parameters within the various stress conditions demonstrated the profound influence of the selected stress factors on the biodiesel productivity of the diatom, besides its stress tolerance. A two-phase culturing system, with multifactor stress application, especially nitrogen limitation along with phosphate starvation and temperature stress, would be the suitable method for gaining maximum biomass productivity and lipid content in diatom Navicula phyllepta MACC8 towards biofuel production.

Optimisation of critical medium components and culture conditions for enhanced biomass and lipid production in the oleaginous diatom Navicula phyllepta: a statistical approach.

Diatoms hold great promise as potential sources of biofuel production. In the present study, the biomass and lipid production in the marine diatom Navicula phyllepta, isolated from Cochin estuary, India and identified as a potential biodiesel feedstock, were optimized using Plackett-Burman (PB) statistical experimental design followed by central composite design (CCD) and response surface methodology (RSM). The growth analyses of the isolate in different nitrogen sources, salinities and five different enriched sea water media showed the best growth in the cheapest medium with minimum components using urea as nitrogen source at salinity between 25 and 40 g kg-1. Plackett-Burman experimental analyses for screening urea, sodium metasilicate, sodium dihydrogen phosphate, ferric chloride, salinity, temperature, pH and agitation influencing lipid and biomass production showed that silicate and temperature had a positive coefficient on biomass production, and temperature had a significant positive coefficient, while urea and phosphate showed a negative coefficient on lipid content. A 24 factorial central composite design (FCCD) was used to optimize the concentration of the factors selected. The optimized media resulted in 1.62-fold increase (64%) in biomass (1.2 ± 0.08 g L-1) and 1.2-fold increase (22%) in estimated total lipid production (0.11 ± 0.003 g L-1) compared to original media within 12 days of culturing. A significantly higher biomass and lipid production in the optimized medium demands further development of a two-stage strategy of biomass production followed by induction of high lipid production under nutrient limitation or varying culture conditions for large-scale production of biodiesel from the marine diatom.