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.

Probing Biothiols Using a Red-Emitting Pyridoxal Derivative by Adopting Copper(II) Displacement Approach and Cell Imaging.

An aggregation-induced emission (AIE) active Schiff base L was obtained by reacting pyridoxal and 2-hydroxy-1-naphthaldehyde with p-phenylenediamine in two simple steps. The colorimetric, UV/VIS and fluorescence studies of L revealed that the yellow emissive L (λem =540 nm, λex =450 nm) in pure DMSO turned to a red-emissive L, when the poor solvent fraction (HEPES buffer, 10 mM, pH 7.4) was increased above 50 % in DMSO. The SEM and DLS results indicated the formation of self-aggregates of L that restricted the intramolecular motion and promoted the excited state intramolecular proton transfer (ESIPT) process. The cations sensing ability of the AIEgen L was explored in HEPES buffer (5 % DMSO, 10 mM, pH 7.4), where Cu2+ selectively quenched the fluorescence at 608 nm due to the chelation-enhanced fluorescence quenching (CHEQ) effect with an estimated sensitivity limit of 0.9 µM. Subsequently, the in situ formed AIEgen L-Cu2+ complex was applied for the cascade detection of glutathione (GSH), cysteine (Cys) and homocysteine (Hcy). The decomplexation of Cu2+ from the AIEgen L-Cu2+ by GSH, Cys and Hcy restored the quenched fluorescence emission of AIEgen L at 608 nm. With this Cu2+ displacement approach, the concentration of Cys, Hcy and GSH can be detected down to 2.8 µM, 3.12 µM and 2.0 µM, respectively. The practical utility of AIEgen L and AIEgen L-Cu2+ was examined by monitoring the selective analytes in real environmental and biological samples, and also applied successfully for the cell imaging applications.

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.

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.

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.

In silico studies on the interaction of phage displayed biorecognition element (TFQAFDLSPFPS) with the structural protein VP28 of white spot syndrome virus.

White spot disease caused by the white spot syndrome virus (WSSV) incurs a huge loss to the shrimp farming industry. Since no effective therapeutic measures are available, early detection and prevention of the disease are indispensable. Towards this goal, we previously identified a 12-mer phage displayed peptide (designated as pep28) with high affinity for VP28, the structural protein of the white spot syndrome virus (WSSV). The peptide pep28 was successfully used as a biorecognition probe in the lateral flow assay developed for rapid, on-site detection of WSSV. To unravel the structural determinants for the selective binding between VP28 and pep28, we used bioinformatics, structural modeling, protein-protein docking, and binding-free energy studies. We performed atomistic molecular dynamics simulations of pep28-pIII model totaling 300 ns timescale. The most representative pep28-pIII structure from the simulation was used for docking with the crystal structure of VP28. Our results reveal that pep28 binds in a surface groove of the monomeric VP28 ß-barrel and makes several hydrogen bonds and non-polar interactions. Ensemble-based binding-free energy studies reveal that the binding is dominated by non-polar interactions. Our studies provide molecular level insights into the binding mechanism of pep28 with VP28, which explain why the peptide is selective and can assist in modifying pep28 for its practical use, both as a biorecognition probe and a therapeutic.

A comparative synthesis of transcriptomic analyses reveals major differences between WSSV-susceptible Litopenaeus vannamei and WSSV-refractory Macrobrachium rosenbergii.

Since the 1990s White Spot Syndrome Virus (WSSV) has severely affected shrimp aquaculture worldwide causing a global pandemic of White Spot Disease (WSD) in penaeid culture. However, not all decapod species that can be infected by WSSV show the same susceptibility to the virus, thus raising interesting questions regarding the potential genetic traits that might confer resistance to WSSV. In order to shed light into the genetic markers of WSSV resistance, we employed a dual approach: i) we initially analysed the transcriptomes derived from the hepatopancreas of two species, the susceptible white shrimp Litopenaeus vannamei and the refractory fresh water prawn Macrobrachium rosenbergii, both infected with WSSV. We found a large number of differentially expressed genes (DEGs) belonging to the immune system (mostly anti-microbial peptides and haemolymph clotting components) that were generally up-regulated in M. rosenbergii and down-regulated in L. vannamei. Further, in both species we identified many up-regulated DEGs that were related to metabolism (suggesting a metabolic shift during the infection) and, interestingly, in L. vannamei only, we found several DEGs that were related to moult and suggested an inhibition of the moult cycle in this species following WSSV infection. ii) we then identified a limited number of genetic markers putatively linked with WSD tolerance by employing an ecological genomics approach in which we compared published reports with our own RNA-seq datasets for different decapod species infected with WSSV. Using this second comparative approach, we found nine candidate genes which are consistently down-regulated in susceptible species and up-regulated in refractory species and which have a role in immune response. Together our data offer novel insights into gene expression differences that can be found in susceptible and refractory decapod species infected with WSSV and provide a valuable resource towards our understanding of the potential genetic basis of tolerance to WSSV.

Global distribution of white spot syndrome virus genotypes determined using a novel genotyping assay.

White spot disease, caused by infection with white spot syndrome virus (WSSV), is a serious panzootic affecting prawn aquaculture. The disease has spread rapidly around the prawn-culturing regions of the world through a number of previously identified mechanisms. The ability to distinguish and trace strains of WSSV is of great benefit to identify, and then limit, the translocation routes of the disease. Here, we describe a novel genotyping method using 34 short tandem repeat regions of the viral genome concurrently. This technique is highly sensitive to strain differences when compared to previous methods. The efficacy of the described method is demonstrated by testing WSSV isolates from around the globe, showing regional genotypic differences. The differences in the genotypes were used to create a global minimum spanning network, and in most cases the observed relationships were substantiated with verification of transboundary movement. This novel panel of STR markers will provide a valuable epidemiological tool for white spot disease. We have applied this to an outbreak of the disease in Queensland, Australia, that occurred in 2016. While the results indicate that the source of this outbreak currently remains cryptic, the analyses have provided valuable insights with which to further study the origins of the strains involved.

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.

ICTV Virus Taxonomy Profile: Nodaviridae.

The family Nodaviridae includes two genera, Alphanodavirus and Betanodavirus. The family name derives from the Japanese village of Nodamura where Nodamura virus was first isolated from Culex tritaeniorhynchus mosquitoes. Virions are non-enveloped and spherical in shape with icosahedral symmetry (T=3) and diameters ranging from 25 to 33 nm. The genome consists of two molecules of single-stranded positive-sense RNA: RNA1 and RNA2. The virion capsid consists of 180 protein subunits arranged on a T=3 surface lattice. Alphanodaviruses infect insects, whereas betanodaviruses are pathogens of fish. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Nodaviridae, which is available at www.ictv.global/report/nodaviridae.

ICTV virus taxonomy profile: Sarthroviridae.

The family Sarthroviridae includes a single genus, Macronovirus, which in turn includes a single species, Macrobrachium satellite virus 1. Members of this species, named extra small virus, are satellite viruses of Macrobrachium rosenbergii nodavirus, an unclassified virus related to members of the family Nodaviridae. Both viruses have isometric, spherical virions, infect giant freshwater prawns and together cause white tail disease, which is responsible for mass mortalities and severe economic losses in hatcheries and farms. Infection is caused by both vertical and horizontal transmission of virus. Aquatic insects act as a carrier to transmit the disease in prawns. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Sarthroviridae, which is available at www.ictv.global/report/sarthroviridae.

In vitro screening of selected antiviral drugs against betanodavirus.

The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1 mM, 1 mM, 10 mM, 100 mM and 500 mM) and chlorpromazine hydrochloride (1 µM, 10 µM, 100 µM, 200 µM and 500 µM) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1 mM, 1 mM and 10 mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1 µM, 10 µM and 100 µM were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1 mM and 10 mM, and chlorpromazine hydrochloride of 10 µM and 100 µM could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.

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.

Studies on the occurrence of infectious myonecrosis virus in pond-reared Litopenaeus vannamei (Boone, 1931) in India.

Whiteleg shrimp, Litopenaeus vannamei, with clinical sign of muscle opaqueness with reddish colour at the distal abdominal segments were observed in farms located in West Bengal State, India. The mortality of shrimp in all disease outbreak ponds ranged from 20% to 50%, and mortality increased gradually. The RT-PCR assay of these samples using primer sets specific to infectious myonecrosis virus (IMNV) revealed its presence in the disease outbreak ponds. The IMNV infection was reproduced in healthy shrimp by intramuscular injection to satisfy River's postulates. The virus caused mortality in intramuscularly challenged shrimp, but failed to cause mortality by oral route. Tissue distribution of IMNV in infected shrimp by RT-PCR assay revealed the presence of this virus in haemolymph, gill, hepatopancreas and muscle. This study confirms that the disease outbreak which occurred in the shrimp farms located at Purba Medinipur District, West Bengal, India, was due to IMNV.

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.