Development and characterization of brain cell line from Trachinotus blochii and its application in virological and gene expression studies.

A permanent cell line, SPB (Snubnose pompano brain) was established from Trachinotus blochii by the explant culture method. It has been sub-cultured more than 75 passages and showed optimal growth at 28°C using L-15 medium supplemented with 15% to 20% FBS. The SPB cells were cryopreserved at different passage levels for various applications. SPB cells were composed of fibroblastic and epithelial-like cells. The SPB cells were tested for mycoplasma contamination which was found to be negative. The origin of the SPB cell line from T. blochii was confirmed by amplification of the mitochondrial cytochrome oxidase I (COI) gene. The transfection efficiency of SPB cell line is 15% assessed by expression of green fluorescent protein using pEGFP-N1 plasmid. In addition, two CMV promotor plasmids pFNCPE42-DNA and pcDNAVP28 were transfected to SPB cells and it shows high expression levels of FNCP of fish nodavirus and VP28 protein of white spot syndrome virus by immunostaining. The SPB cells showed susceptibility to SJNNV and the infection was confirmed by RT-PCR, Western blot, ELISA, TCID50 and RT-qPCR. Experimental infection was carried out in T. blochii using SJNNV propagated in SPB cell line and found 100% mortality with clinical signs. The infection was confirmed by RT-PCR. The SPB cell line can be used for propagation of fish viral pathogens and production of the recombinant proteins.

An Indigenous, Field-Deployable, Lateral Flow Immunochromatographic Assay Rapidly Detects Infectious Myonecrosis in Shrimp, Litopenaeus vannamei.

Shrimp farming is an important socioeconomic activity worldwide. Infectious myonecrosis virus (IMNV) is an important shrimp virus responsible for significant mortality (up to 70%) in Litopenaeus vannamei. We produced recombinant capsid protein (r-IMNV31) and obtained a highly specific antibody, anti-r-IMNV31, which was used in WOAH-approved ELISA and Western blot to detect IMNV. Further, anti-r-IMNV31 was employed in an indigenously developed lateral flow immunoassay (LFA) with gold nanoparticles as a visual label. Using LFA, IMNV could be detected rapidly (20 min) from tissue homogenate with high specificity, reproducibility, and sensitivity (LOD = 103 viral particles). LFA was validated with "gold standard" qRT-PCR using 60 samples with high sensitivity (100%), specificity (86%). A Cohen's kappa coefficient of 0.86 suggested "good agreement" between LFA and qRT-PCR. With a shelf-life of ~ 1 year at ambient temperature, the use of LFA in the on-site detection of IMNV by shrimp farmers will be a reality.

Toxicological assessment of functional polymer with single-walled carbon nanotubes in zebrafish embryos and its gill cell line.

Carbon nanotubes (CNTs) have been widely used in developing polymer hybrid coatings for anticorrosive application. In the present study, poly [(3,5-dimethyl-lH-pyrazole-1-yl) methyl methacrylate-co-glycidyl methacrylate] (PyM) was prepared by solution polymerization. Single-wall carbon nanotubes (SWCNT) were incorporated in the PyM by solution blending technique at different proportions. The PyM and its SWCNT (PyM-SWCNT) nanocomposites were characterized by FT-IR spectroscopy, X-Ray Diffraction, FE-SEM and HR-TEM. Different concentrations of PyM or PyM-SWCNT prepared in the present study were assessed separately for their toxicity by in vivo and in vitro assays using zebrafish embryos and gill cell line of zebrafish (DrG), respectively. The nanocomposites at the concentration of 400 µg ml-1 of PyM in 1.0% of SWCNT was found to be non-toxic and recommended for anticorrosive application whereas the nanocomposites with above 1% of SWCNT was found to be toxic. The nanocomposites with 1.5% of SWCNT delayed the hatching rate of eggs, decreased survival rate and heart beat in zebrafish embryos, and induced the morphological changes in DrG cells. Gene expression studies revealed that PyM-SWCNT with high concentration of SWCNT induced oxidative stress by activating ROS generations in zebrafish embryos and DrG cells. The immersion study of uncoated and coated with recommended concentration of PyM-SWCNT on mild steel (MS) in sea water was studied using FE-SEM and EDS, and the results showed effective corrosion protection without leaching behaviour. The nanocomposites with novel polymer in the present study may be used in the industry for anticorrosive purpose.

In vitro propagation of infectious myonecrosis virus in C6/36 mosquito cell line.

Infectious myonecrosis (IMN) is an important shrimp viral disease caused by infectious myonecrosis virus (IMNV). Based on previous reports, an attempt was made to propagate IMNV in apparently healthy C6/36 subclone of Aedes albopictus cell line. The confirmatory assays such as RT-PCR, real-time PCR and bioassay revealed that C6/36 cells were found to be susceptible to IMNV and these cells could be used easily for isolation and propagation of IMNV. The results of real-time PCR assay showed that a lower CT value of 22.25 in IMNV-infected cells was obtained on 10 day post-infection (d p.i.), whereas the higher CT value of 35.21 was obtained in IMNV-infected cells on 2 d p.i. There is no significant difference between CT values of IMNV production in vitro using C6/36 cell line and in vivo using shrimp. The IMNV propagated in C6/36 cells is capable of infecting shrimp and caused 100% mortality in shrimp. Clinical signs observed in shrimp injected with IMNV propagated in C6/36 cell line were found to be similar to naturally infected shrimp.

Silencing of prophenoloxidase (proPO) gene in freshwater prawn, Macrobrachium rosenbergii, makes them susceptible to white spot syndrome virus (WSSV).

Prophenoloxidase (proPO) is very important to protect the invertebrates from microbial infections. Our previous studies revealed that proPO was up-regulated in WSSV-injected Macrobrachium rosenbergii and is responsible for protecting M. rosenbergii from WSSV. In order to prove this mechanism, an attempt was made in the present study to silence the proPO gene in freshwater prawn by injection of dsRNA-proPO followed by WSSV challenge. Two partial fragments of proPO with the size of 251 and 331 bp were used to synthesize dsRNA using LITMUS38i vector and E. coli. The bacterially synthesized dsRNA-proPO was used to silence proPO gene to determine its involvement in developing resistance in prawn against WSSV. In proPO gene-silenced prawn, 100% mortality was observed after WSSV challenge whereas no mortality was observed in prawn injected with WSSV alone. The WSSV infection in gene-silenced prawn was confirmed by PCR, and its propagation was quantified by ELISA and real-time PCR at different time intervals. Real-time PCR assay revealed a significant reduction in the expression of proPO gene in WSSV-challenged proPO-silenced prawn when compared to normal prawn. Level of proPO was reduced significantly in the haemolymph of proPO-silenced prawn when compared to prawn injected with PBS.

Multiple infections caused by white spot syndrome virus and Enterocytozoon hepatopenaei in pond-reared Penaeus vannamei in India and multiplex PCR for their simultaneous detection.

White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow-out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV-positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m-PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m-PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.

A new strain of white spot syndrome virus affecting Litopenaeus vannamei in Indian shrimp farms.

White spot syndrome virus (WSSV)-infected shrimp samples collected from grow-out ponds located at Nellore, Andhra Pradesh, India, showed WSSV negative and positive by PCR using primer sets specific to ORF119 and VP28 gene of WSSV, respectively. This indicated the deletion of genetic fragments in the genome of WSSV. The WSSV isolate along with lab strain of WSSV was subjected to next-generation sequencing. The sequence analysis revealed a deletion of 13,170 bp at five positions in the genome of WSSV-NS (new strain) relative to WSSV-TH and WSSV-LS (lab strain). The PCR analysis using the ORF's specific primer sets revealed the complete deletion of 10 ORFs in the genome of WSSV-NS strain. The primer set was designed based on sequence covering ORF161/162/163 to amplify a product of 2,748 bp for WSSV-LS and 402 bp for WSSV-NS. Our surveillance programme carried out since 2002 revealed the replacement of WSSV-LS by WSSV-NS in Indian shrimp culture system.

Delivery of viral recombinant VP28 protein using chitosan tripolyphosphate nanoparticles to protect the whiteleg shrimp, Litopenaeus vannamei from white spot syndrome virus infection.

The VP28 gene of white spot syndrome virus was amplified by PCR using gene specific primer set and cloned into pRSET B vector to produce recombinant VP28 (r-VP28) in E. coli GJ1158. The chitosan tripolyphosphate nanoparticles (CS/TPP) were prepared by ionic gelation process and characterized. The purified r-VP28 protein was encapsulated by CS/TPP nanoparticles. The encapsulation efficiency of CS/TPP nanoparticles was found to be 84.8% for r-VP28 protein binding with CS/TPP nanoparticles. The in vitro release profile of encapsulated r-VP28 was determined after treating with protease and chitosanase. The different types of feed were formulated and named as normal feed with PBS, Feed A coated with crude r-VP28, Feed B with purified r-VP28 and Feed C with CS/TPP encapsulated r-VP28 (Purified). Tissue distribution and clearance of r-VP28 at different time intervals were examined in shrimp fed with different types of feed by ELISA and the results showed the presence of r-VP28 protein in different organs. Various immunological parameters were assessed in experimental shrimp. The mRNA expression of five immune-related genes was analysed by qPCR in order to investigate their response to all types of feed in shrimp. A cumulative percentage mortality was also recorded in treated shrimp challenged with WSSV.

Ontogenetic changes in the expression of immune related genes in response to immunostimulants and resistance against white spot syndrome virus in Litopenaeus vannamei.

In recent years, researchers have focused on viral and plant immunostimulants which could have beneficial effects in disease prevention and control in shrimp culture. At present, the application of the recombinant VP28 protein (r-VP28) and herbal immunostimulant has been considered as a more effective approach to prevent white spot syndrome (WSS) by enhancing the immune response in shrimp. In the present study, expression of selected immune related genes in response to r-VP28 and herbal immunostimulant mix (HIM) were separately studied qualitatively and quantitatively by RT-PCR and real time PCR, respectively during ontogenetic development from nauplius to juvenile stage in Litopenaeus vannamei. The mRNA expression level of immune related genes such as anti-lipopolysaccharides (ALF), Lysozyme, cMnSOD, Crustin, Prophenoloxidase, Tumor necrosis factor receptor-associated factor 6 (TRAF6) and Haemocyanin were found to be up-regulated significantly in different ontogenetic development stages of shrimp fed with r-VP28 and HIM formulated diets. Relative percent survival (RPS) was determined in shrimp fed with immunostimulants formulated diets after oral challenge with WSSV. The survival of WSSV challenged shrimp was found to be higher in immunostimulants treated groups when compared to untreated group. The results of PCR, ELISA and real time PCR revealed the absence of WSSV in WSSV-challenged shrimp after 20 days of treatment with immunostimulants. Among these immunostimulants, HIM was found to be more effective when compared to r-VP28. After a survey of literature, we are of the opinion that this might be the first report on the expression of immune genes during ontogenetic development of L. vannamei in response to immunostimulants.

Application of fish cell lines for evaluating the chromium induced cytotoxicity, genotoxicity and oxidative stress.

In the present study, we hypothesize that cytotoxicity, genotoxicity and oxidative stress play a key role in chromium induced toxicity in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines after 24 h exposure. Three fish species namely Lates calcarifer, Etroplus suratensis and Catla catla were exposed to the concentrations of 0, 10, 20, 30, 40 and 50 mg/L of chromium for 96 h under static conditions for conducting acute toxicity tests. LC50 was then calculated. The percentage cell survival was assessed by multiple endpoints such as MTT, NR, AB and CB assays in the seven fish cell lines exposed to different concentrations of chromium and EC50 values of all the four endpoints were calculated. High significances were noted in the correlations between each in vitro cytotoxicity assays and in vivo mortality data. Cell shrinkage, cell detachment, vacuolations and cell swelling at the highest concentration of chromium (50 mg/L) were seen on microscopic examination of cell morphology. Comet assay and Hoechst staining were carried out to assess DNA damage and nuclear fragmentation in the seven fish lines exposed to chromium. The results of antioxidant parameters obtained indicate a significant reduction in the level of catalase, superoxide dismutase, glutathione S-transferase and Glutathione peroxidase, and increased level of lipid peroxidation in all the cell lines exposed to chromium. These results confirm that fish cell lines could be used as an alternative to whole fish for cytotoxicity, genotoxicity and oxidative stress assessment in chromium toxicity studies.

Effects of nicotine on zebrafish: A comparative response between a newly established gill cell line and whole gills.

A novel cell line, Danio rerio gill (DrG), derived from the gill tissue of zebrafish, was established and characterized. The cells were able to grow at a wide range of temperatures from 25°C to 32°C in Leibovitz's L-15 medium. The DrG cell line consists of epithelial-like cells with a diameter of 18-22µm. The cell line was characterized by mitochondrial 12S rRNA gene. Acute toxicity tests were conducted on D. rerio by exposing them to nicotine for 96h under static conditions. In vitro cytotoxicity of nicotine was assessed in DrG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Linear correlations between each in vitro cytotoxicity assay and the in vivo mortality data were highly significant. Nicotine induced intracellular reactive oxygen species generation in DrG cell line in a concentration dependent manner. DrG cell line and zebrafish exposed to nicotine significantly increased the elevation of lipid peroxidation (LPO) while depletion of reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidise(GPx1a) was observed. In nicotine treated fish and cells a negative correlation between reduced glutathione and LPO was observed. In addition, the production of ROS and the resulting oxidative stress resulted in increased expression of apoptosis related genes p53 and cas3.Collectively, our result suggests that nicotine has the potential to induce reactive oxygen species (ROS) production, oxidative stress and apoptosis in DrG cell line and zebrafish.

Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP-infected whiteleg shrimp, Litopenaeus vannamei (Boone, 1931), in India.

Stunted growth in pond-reared Litopenaeus vannamei was observed in different farms located in Tamil Nadu and Andhra Pradesh, India. No mortality was associated with stunted growth. PCR assay on these samples revealed the presence of Enterocytozoon hepatopenaei (EHP) in stunted shrimp. Tissue distribution of EHP in naturally and experimentally infected shrimp was studied by PCR and histology. Histological examination revealed the presence of EHP in hepatopancreas and gut, but not in other organs. The PCR assay revealed the presence of EHP in all the organs tested in both naturally and experimentally infected shrimp. Healthy shrimp were challenged with E. hepatopenaei by intramuscular injection and oral route, and no mortality was observed in both routes after 30 days post-challenge. Different developmental stages of the microsporidian parasite were observed in the hepatopancreatic epithelial cells. Biochemical parameters such as total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase were measured in the haemolymph of naturally and experimentally EHP-infected shrimp. All biochemical parameters mentioned were found to be significantly higher in EHP-infected shrimp when compared to normal shrimp. This is the first report relating AST and ALT levels to EHP infection in naturally and experimentally infected shrimp.

Immune responses of whiteleg shrimp, Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus.

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA-VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune-related genes were examined to estimate the effect of dsRNA-VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA-VP28 treatment, whereas MDA content did not change significantly. Among the ten immune-related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA-VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA-VP28 stimulation indicate that these immune-related genes were involved in dsRNA-VP28-induced innate immunity in shrimp.

Isolation, propagation, characterization, cryopreservation, and application of novel cardiovascular endothelial cell line from Channa striatus (Bloch, 1793).

There are only few primary endothelial cell cultures developed from fishes to date, but in this work the development of an endothelial cell line from Channa striatus is described. The vascular explants were plated into fibronectin (5 µg ml(-1)) and anti-CD31 antibody (100 ng ml(-1))-coated flask; after 60 h incubation explants were removed from the flask. The flask contained only endothelial and blood cells. Blood cells were cleared out after subsequent passages. The culture medium used was Leibovitz's L-15 supplemented with 20 % serum and antibiotics. The cultures were incubated at 28 °C in a normal atmosphere incubator. The plating efficiency was high (53.72 %). The endothelial cells were cryopreserved at different passage levels and revived successfully with 75-85 % survival. Polymerase chain reaction amplification of mitochondrial 16S rRNA using primer specific to C. striatus confirmed the origin of C. striatus cardiovascular endothelial (CSCVE) cell line from C. striatus. This cell line was further characterized for chromosome number, transfection, mycoplasma, cell cycle distribution, mitochondrial staining, and phagocytic activity. Cells were analyzed according to morphological appearance and expression of specific endothelial markers by fluorescent staining (von Willebrand Factor, anti-platelet endothelial cell adhesion molecule-1, and anti-Endoglin). The formation of tubules in the Matrigel and endothelial co-cultured with fibroblast like cells was observed. The cytotoxicity of ciprofloxacin on the CSCVE cell line was determined by MTT, AB, and R-123 cytotoxicity end points. Susceptibility of CSCVE cell line to nodavirus was confirmed by cytopathic effect and reverse transcriptase-polymerase chain reaction.

Cytotoxicity, genotoxicity and oxidative stress of malachite green on the kidney and gill cell lines of freshwater air breathing fish Channa striata.

The cytotoxicity, genotoxicity and oxidative stress of malachite green (MG) was investigated using the fish Channa striata kidney (CSK) and Channa striata gill (CSG) cell lines. Five concentrations ranging from 0.001 to 10 µg mL(-1) were tested in three independent experiments. Cytotoxicity was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Rhodamine 123 and Alamar Blue. The mitochondrial changes and apoptosis of MG-exposed cells were observed by Rhodamine 123 and acridine orange/ethidium bromide (AO/EB) staining, respectively. In vitro potential DNA damaging effect of MG was tested using comet assay. Mitochondrial damage, apoptosis and DNA fragmentation increased in a concentration-dependent manner. Additionally, DNA electrophoretic mobility experiments were carried out to study the binding effect of MG to double-stranded DNA (dsDNA) of cells. DNA shift mobility experiments showed that MG is capable of strongly binding to linear dsDNA causing its degradation. Biochemical parameters such as lipid peroxidation (MDA), catalase (CAT) activity and reduced glutathione (GSH) levels were evaluated after exposure to MG. In CSK and CSG cell lines exposed to MG for 48 h, a significant increase in lipid peroxidation, which might be associated with decreased levels of reduced glutathione and catalase activity in these cell lines (p < 0.001), was observed.

In vitro cytotoxic, genotoxic and oxidative stress of cypermethrin on five fish cell lines.

The indiscriminate use of pesticides and herbicides to enhance crop production has aroused great concern, because these products are likely to reach the aquatic environment, thereby posing a health concern for humans and aquatic species. Cypermethrin (CYP), a type II pyrethroid insecticide, is widely used in agriculture and for other purposes. Therefore a study was conducted for the assessment of cytotoxic, genotoxic and oxidative stress of CYP in IEG, CB, ICG, LRG and CSG cell lines at 24h exposure. The cytotoxic effect of CYP in IEG, CB, ICG, LRG and CSG cell lines was assessed using MTT, NR, AB and CB assays. Linear correlations between each EC50 values, of CYP resulting in 50% inhibition of cytotoxicity parameters after 24h exposure to CYP were calculated for IEG, CB, ICG, LRG and CSG cell lines using MTT, NR, AB and CB assays. Statistical analysis revealed good correlation with R(2)=0.90-0.939 for all combinations between endpoints employed. The percentage of DNA damage was assessed by comet assay in IEG, CB, ICG, LRG and CSG cells exposed to CYP. The results of antioxidant parameters obtained show a significant increase in lipid peroxidation (LPO) level and decreased level of GSH, SOD and CAT in IEG, CB, ICG, LRG and CSG cell lines after exposure to increasing CYP in a concentration-dependent manner. This work proves that fish cell lines could be used not only for cytotoxicity and genotoxicity studies but also for studying oxidative stress when exposed to environmental contaminants such as pesticides and other pollutants.

In vitro assay for the toxicity of silver nanoparticles using heart and gill cell lines of Catla catla and gill cell line of Labeo rohita.

Silver nanoparticles (Ag-NPs) are used in commercial products for their antimicrobial properties. The Ag-NPs in some of these products are likely to reach the aquatic environment, thereby posing a health concern for humans and aquatic species. The silver nanoparticles were synthesized and characterized using, UV-vis spectra, Dynamic light scattering (DLS) and Transmission electron microscopy (TEM) analysis. Acute toxicity tests on fish were conducted by exposing Catla catla and Labeo rohita for 96h to AgNO3 and Ag-NPs under static conditions. The cytotoxic effect of AgNO3 and Ag-NPs in Sahul India C. catla heart cell line (SICH), Indian C. catla gill cell line (ICG) and L. rohita gill cell line (LRG) was assessed using MTT and neutral red (NR) assay. Linear correlations between each in vitro EC50 and the in vivo LC50 data were highly significant. DNA damage and nuclear fragmentation (condensation) were assessed by comet assay and Hoechst staining, respectively in SICH, ICG and LRG cells exposed to Ag-NPs. The results of antioxidant parameter obtained show significantly increased lipid peroxidation (LPO) level and decreased level of GSH, SOD and CAT in SICH, ICG and LRG cell lines after exposure to increasing Ag-NPs in a concentration-dependent manner. This work proves that fish cell lines could be used as an alternative to whole animals using cytotoxicity tests, genotoxicity tests and oxidative stress assessment after exposure to nanoparticles.

Development and characterization of a new gill cell line from air breathing fish Channa striatus (Bloch 1793) and its application in toxicology: direct comparison to the acute fish toxicity.

A new cell line, Channa striatus gill (CSG), derived from the gill tissue of murrel, was established and characterized. The CSG cell line was maintained in Leibovitz's L-15 supplemented with 10% fetal bovine serum and has been subcultured more than 92 times. This cell line was able to grow in a range of temperatures from 22 to 32°C with optimal growth at 28°C. The plating efficiency was very high (52.21%) and doubling time was approximately 37h. The gill cell line was cryopreserved at different passage levels and revived successfully with 85% survival. Polymerase chain reaction amplification of mitochondrial 16S rRNA using primer specific to C. striatus confirmed the origin of this cell line from murrel. The cell line was further characterized by immunocytochemical analysis, chromosome number, transfection and mycoplasma detection. The cytotoxicity of endosulfan was assessed in CSG cell line using apoptosis assay, comet assay, mitochondrial alteration and five other endpoints such as Rhodamine 123 uptake, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red assay, Alamar Blue assay and Methylene Blue protein assay. Acute toxicity study on fish was conducted by exposing murrel for 96h to endosulfan under static conditions. Statistical analysis revealed good correlation with r(2)=0.972-0.997 among the five endpoints. Linear correlations between the in vivo lethal concentration 50 (LC50) and each in vitro effective concentration 50 (EC50) were highly significant. The present study highlights the development of a new gill cell line from an air breathing fish that could be used as an alternative in vitro tools for studying pesticide toxicity in fish.

Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r-MCP43) of giant freshwater prawn, M. rosenbergii (de Man) for immunological diagnostic methods.

White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6-histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD-infected post-larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection. The antiserum raised against r-MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti-rMCP43 and pure r-MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT-PCR to test the efficiency of antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV-positive coded samples as detected by RT-PCR.