N. S. Nagpure

Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis.

Chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridylphosphorothioate) is one of the organophosphate pesticides widely used in agricultural practices throughout world and irreversible inhibitor of cholinesterase in all animal species. Limited efforts have been made to study acute genotoxic effects of chlorpyrifos (CPF) in different tissues of fish using genotoxic biomarkers. Therefore, the present investigation was aimed to study the induction of DNA damage by CPF in freshwater teleost fish Channapunctatus using micronucleus assay (MN assay) and alkaline single-cell gel electrophoresis (comet assay). The value of LC(50) - 96 h of CPF was determined as 811.98 microgl(-1) for C. punctatus, in a semi-static system and on the basis of LC(50) value three acute concentrations viz., 203, 406 and 609 microgl(-1) were determined. The fishes were exposed to the different concentrations of CPF for 96 h and samplings were done at regular intervals for assessment of the MN frequencies and DNA damage. In general, significant effects (P<0.01) from both concentrations and time of exposure were observed in exposed fishes. It was found that the micronucleus induction was highest on 96 h at all concentrations in the peripheral blood. Similar trend was observed for the DNA damage measured in terms of the percentage of tail DNA in the lymphocyte and gill cells. This study explored the combined use of micronucleus assay and comet assay for in vivo laboratory studies using fresh water fish for screening the genotoxic potential of xenobiotics.

Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Chlorpyrifos (CPF) is the single largest selling agrochemical that has been widely detected in surface waters in India. The studies on long-term genotoxic effects of CPF in different tissues of fish using genotoxic biomarkers are limited. Therefore, in the present study DNA damage by CPF in freshwater fish Channapunctatus using micronucleus (MN) and comet assays was investigated. The LC(50) - 96 h of CPF was estimated for the fish in a semi-static system. On this basis of LC(50) value sublethal and nonlethal concentrations were determined. The DNA damage was measured in lymphocytes and gill cells as the percentage of DNA in comet tails and micronuclei were scored in erythrocytes of fishes exposed to above concentrations of CPF. In general, significant effects for both the concentrations and time of exposure were observed in treated fish. It was found that MN induction in the blood was highest on day 14 at 203.0 microg/l of CPF. The highest DNA damage was observed on day 5, followed by a gradual non-linear decline in the lymphocytes and gill cells. The study indicated MN and comet assays to be sensitive and rapid methods to detect mutagenicity and genotoxicity of CPF and other pollutants in fishes.

Investigation of the Genotoxicity of Malathion to Freshwater Teleost Fish Channa punctatus (Bloch) Using the Micronucleus Test and Comet Assay

Malathion [S-(1,2-dicarboethoxyethyl) O, O-dimethyl phosphorodithioate] is a widely used organophosphorus insecticide throughout the world. However, limited efforts have made to study its genotoxic effect in different fish tissues. The present investigation was aimed to assess the genotoxic potential of the pesticide to the freshwater teleost fish Channa punctatus at sublethal concentrations using the micronucleus test and comet assay. Initially, the 96-h LC50 value of commercial-grade malathion (50% EC) was determined as 5.93xa0ppm in a semistatic system. Based on LC50, three test concentrations (viz. sublethal I, sublethal II, and sublethal III) were determined to be 1.48, 0.74, and 0.59xa0ppm, respectively, and the fish specimens were exposed to these concentrations. Tissue samplings were done on days 0, 1, 3, 7, 15, 22 and 29 of malathion exposure for assessment of the induction of micronuclei (MN) frequency and DNA damage. The MN formation in the peripheral blood cells was found to be significantly higher (pxa0<xa00.05) in the treated specimens at all sampling intervals compared to the control. The MN frequency reached maximum on days 3 and 7 at sublethal I and II concentrations, respectively, followed by a nonlinear decline with the progression of the experiment. Similarly, significant effects (pxa0<xa00.05) of both concentration and time of exposure were observed on DNA damage in the gill, kidney, and lymphocytes. All of the tissues exhibited a concentration-dependent increase in DNA damage up to day 3, followed by a nonlinear decrease with the duration of exposure. A comparison of the extent of DNA damage among the tissues showed the sensitivity of gill tissue to malathion.

Chromosomal localization of 18S and 5S rDNA using FISH in the genus Tor (Pisces, Cyprinidae)

Dual color fluorescence in situ hybridization (FISH) was performed to study the simultaneous chromosomal localization of 18S and 5S ribosomal genes in the genus Tor for the first time. The 18S and 5S rDNAs in four Tor species were amplified, sequenced and mapped on the metaphase chromosomes. The number and distribution of 18S and 5S rDNA clusters were examined on metaphase chromosome spreads using FISH. The specimens of T. chelynoides, T. putitora and T. progeneius showed six bright fluorescent signals of 18S rDNA and T. tor exhibited ten such signals. The 5S rDNA signals were present only on one pair of chromosomes in all the four Tor species. Ag-NORs were observed on two pairs of chromosomes in T. chelynoides, T.putitora, T. progeneius and four pairs in T. tor. Comparison of the observed 18S rDNA FISH signals and Ag-NORs strongly suggested a possible inactivation of NORs localized at the telomeres of a subtelocentric and telocentric chromosome pairs in all four species. The 5S rDNA contained an identical 120xa0bp long coding region and 81xa0bp long highly divergent non-transcribed spacers in all species examined. 18S and 5S rDNA sequencing and chromosomal localization can be a useful genetic marker in species identification as well as phylogenetic and evolutionary studies.

Population distribution of 45S and 5S rDNA in golden mahseer, Tor putitora: population-specific FISH marker

Chromosomal locations of major 45S and minor 5S ribosomal DNAs (rDNAs) and organization of 5S rRNA genes were analysed in five different populations of golden mahseers (Tor putitora) using fluorescence in situ hybridization (FISH) and Southern blot hybridization. All five populations of T. putitora (2n = 100) showed a similar type of macro-karyotype composed of 12 metacentric, 22 submetacentric, 14 subtelocentric and 52 telocentric chromosomes. Analysis of active nucleolar organizer regions (NORs) by silver staining did not show any differences in number and chromosomal position in different populations. But FISH data showed significant difference between the populations, four of the five populations showed six 18S (three pairs) and two 5S (one pair) signals with positional polymorphism, while one population showed eight 18S and four 5S signals, respectively. Southern blot data confirms that 5S rDNA clusters present on two different chromosome pairs in Kosi river population contain non-transcribed spacers (NTS) of same length. In the present study, simultaneous localization of 45S and 5S rDNA by in situ hybridization helped us to develop the discrete population-specific markers in different geographically isolated populations of T. putitora.

Nucleotide variation and physical mapping of ribosomal genes using FISH in genus Tor (Pisces, Cyprinidae)

Molecular cytogenetic studies were carried out for localization of 18S and 5S ribosomal DNAs on chromosomes of three cyprinid fish species viz., T.khudree, T. mussullah and T. mosal mahanadicus using two color fluorescence in situ hybridization (FISH). All the species typically possessed 100 diploid chromosomes with minor variation in karyo-morphology. The 18S rDNA signals were observed on two pair of chromosomes in T. khudree and T. mussullah, and three pairs in T. mosal mahanadicus. The location of 18S signals also showed affinity to silver nitrate and chromomycin A3 staining. Similarly, variation in localization of 5S rDNA among the three species has been detected with the presence of FISH signals on one pair of chromosome in T. khudree and T. mussullah, and on two pairs in T.xa0mosal mahanadicus. These molecular markers could be used as species specific markers for taxonomic identification and can further add in understanding the dynamics of genome organization and karyotypic evolution of these species. The 18S rDNA region was sequenced that generated 1811, 1810 and 1776xa0bp long 18S sequence in T. khudree, T. mussullah and T. mosal mahanadicus, respectively. The 18S rDNA sequence showed 95–98% identity among the subject species. Similarly, 5S sequencing generated 203 bp long fragments in these species with 100% identity in coding and 9.63% variability in non-transcribed spacer regions. The nucleotide sequence variations could be used for understanding the genetic diversity and will add new informative characters in comparative genomics. These results, in general, would enhance the value and interpretation of ecological assessment data for conservation of Tor species.

Investigation on acute toxicity and behavioral changes in Channa punctatus (Bloch) due to organophosphate pesticide profenofos

Acute toxicity of an organophosphate pesticide profenofos (O-4-bromo-2- chlorophenyl-O-ethyl S-propyl phosphorothioate) to freshwater fish, Channa punctatus (Bloch), was studied in a static bioassay. Estimated 96-hour LC50 of profenofos was found to be 2.68 μgL−1. On the basis of the obtained LC50 values for 96-hour exposure intervals, profenofos can be rated as highly toxic to C. punctatus. Fish exposed to profenofos showed hyper excitability, discoloration, erratic swimming, and secretion of excess amounts of mucus on the body and gills with eventual exhaustion and death.

Molecular characterization of major and minor rDNA repeats and genetic variability assessment in different species of mahseer found in North India

Relationship among the mahseer species (Family: Cyprinidae) has long been debated in fish systematics. Present study concentrates on the nature of the phylogenetic relationship among the five mahseer species using the sequence of major ribosomal DNA (45S rDNA). We have covered rDNA sequence of approximately 5.2 kb per individual, 26.0 kb per species and 130.0 kb as a whole. We also characterized the 45S and 5S rDNA regions with respect to their nucleotide composition. For phylogenetic analyses, nucleotide sequences were divided into four datasets. First and second datasets contained 18S rDNA and ITS1 sequence, whereas third and fourth datasets consisted of ITS2 and complete 18S-ITS1-5.8S-ITS2-28S, respectively. The NJ tree was constructed for all the datasets. The mahseer species under study formed a monophyletic group well separated from the outgroup species. Similarly, the individuals of Neolissochilus hexagonolepis form monophyletic group with Tor species, indicating Neolissochilus as a sister genus of Tor. The findings from the present study provide greater insights into taxonomic status of mahseer, and set the stage for future investigations dealing with phylo-geography, taxonomy, conservation and co-evolution within this interesting and important group of fish.

Characterization of two freshwater silurid catfish using conventional and molecular cytogenetic techniques.

Catfish family Siluridae includes nearly 100 extant species. Ompok is an important genus of this family that retains four freshwater fish species in India namely: O. bimaculatus (Indian butter catfish), widely distributed in India and other countries of Southeast Asia; O. malabaricus, found in Western Ghats of Kerala, Goa and Maharashtra; O. pabda (pabdah catfish), distribution confined to Indus and Brahmaputra drainages; and O. pabo, mainly found in Ganga and Brahmaputra River systems in North Bengal and Assam, respectively. These species differ among themselves mostly in colour pattern, in addition to minor variation in size of anal fin, barbels and shape of caudal fin lobes. O. bimaculatus and O. pabda are highly priced and preferable fishes in North India, after Hilsa, due to flesh quality and taste. Cytogenetic mapping and molecular organization of ribosomal and other repetitive DNA sequences have provided important aid in characterization of biodiversity and evolution of the ichthyofauna. Classical cytogenetic studies have determined the diploid chromosome number, constitutive heterochromatin (CH) distribution pattern, and location of nucleolar organizer regions (NORs) by silver nitrate and chromomycin A3 (CMA3) staining. In higher eukaryotes, the genes coding major 45S ribosomal RNA occur in tandem arrays at one or several specific regions on chromosomes. Each repeat of ribosomal RNA coding gene (rDNA) unit contains a transcriptional unit, i.e. 18S, 5.8S, 28S, two internal transcribed spacers (ITS 1 and 2) to separate them and two external transcribed spacers (5′ ETS and 3′ ETS) surrounds them. Each transcriptional unit is separated by highly variable nontranscribed spacer (NTS) region. The genes coding for 18S, 5.8S and 28S rRNA are highly conserved among

Investigation of Cadmium-Induced Genotoxicity and Oxidative Stress Response in Indian Major Carp, Labeo rohita

ABSTRACT We investigated genotoxicity and oxidative stress in the gills of Labeo rohita exposed to 33.6, 67.1, and 100.6 mg L–1of cadmium chloride at 96 h. Genotoxicity was assessed using single cell gel electrophoresis whereas oxidative stress was monitored through lipid peroxidation induction and antioxidant response parameters, namely reduced glutathione (GSH), glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, and catalase (CAT) activities. Significant (p < .05) effect of both concentration and time of exposure was observed on the extent of DNA damage in treated fish. Similarly, malondialdehyde content, level of GSH, and activities of antioxidant enzymes were significantly elevated in treated groups, except CAT. The increased DNA damage and lipid peroxidation (LPO) content along with fluctuation in antioxidant defense system in fish indicated the interaction of cadmium (Cd) with DNA repair processes and production of reactive oxygen species. Thus, Cd is liable for induction of LPO, alteration of antioxidant defenses, and DNA damage in gills of L. rohita.