Indigo dyes: Toxicity, teratogenicity, and genotoxicity studies in zebrafish embryos.

Wastewater released by textile dyeing industries is a major source of pollution. Untreated wastewater released from indigo dyeing operations affects aquatic ecosystems and threatens their biodiversity. We have assessed the toxicity of natural and synthetic indigo dye in zebrafish embryos, using the endpoints of teratogenicity, genotoxicity, and histopathology. The zebrafish embryo toxicity test (ZFET) was conducted, exposing embryos to ten concentrations of natural and synthetic indigo dyes; the 96-hour LC50 values were approximately 350 and 300 mg/L, respectively. Both dyes were teratogenic, causing egg coagulation, tail detachment, yolk sac edema, pericardial edema, and tail bend, with no significant difference in effects between the natural and synthetic dyes. Both dyes were genotoxic (using comet assay for DNA damage). Real-time RT-PCR studies showed upregulation of the DNA-repair genes FEN1 and ERCC1. Severe histological changes were seen in zebrafish larvae following exposure to the dyes. Our results show that indigo dyes may be teratogenic and genotoxic to aquatic organisms, underscoring the need for development of sustainable practices and policies for mitigating the environmental impacts of textile dyeing.

Assessment of toxicity potential of neglected Mithi River water from Mumbai megacity, India, in zebrafish using embryotoxicity, teratogenicity, and genotoxicity biomarkers.

The Mithi River begins at Vihar Lake and flows through the industrial hub of the city of Mumbai, India, and merges with the Arabian Sea at Mahim Creek. The current study was carried out to assess the ecotoxicological effects of the Mithi River surface water in zebrafish (Danio rerio) embryos. Water samples were collected from ten sampling sites (S1 to S10) located along the course of the Mithi River. The toxicity of water samples was assessed using a zebrafish embryo toxicity test (ZFET). Water samples were diluted from all sites at 1:0, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128 times. The lowest and highest LDil 20 values for 96 h were estimated as 9.16 and 74.18 respectively for the S2 and S5 sites. The results of embryotoxicity and teratogenicity assays indicated a significant difference (p < 0.0001) between embryos exposed to control and sampling sites (except S1) for various endpoints such as mortality, egg coagulation, pericardial edema, yolk sac edema, tail bend, and skeletal deformities. The histopathological analysis revealed various lesions, ascertaining the toxic effects of water samples. The comet assay revealed significantly higher DNA damage (except S1) in embryos exposed to sites S5 and S6 with OTM values of 4.46 and 2.48 respectively. The results indicated that the Mithi River is polluted with maximum pollution load at the middle stretches. The study further indicated that the pollutants in the Mithi River (except S1) could potentially be hazardous to the aquatic organisms; therefore, continuous biomonitoring of the river is needed for its revival.

Gonadotropin inhibitory hormone receptors (GnIHRs): Molecular characterization and synergistic effect of different drugs in Indian major carp, Labeo catla.

After the discovery of Gonadotropin-inhibitory hormone (GnIH) in birds in 2000, it showed different roles in different vertebrate classes and even in different species of same classes. In birds and mammals, GnIH inhibits the expression of gonadotropins during reproduction, while in fishes it exerts both inhibitory and stimulatory effect on reproduction. The current study evaluates the role of GnIH during reproduction in Labeo catla. The partial cDNA sequence of GnIHR1 and GnIHR3 receptor genes was identified by degenerate PCR. The mRNA expression analysis of GnIHRs during different reproductive phases showed that the expression of all three GnIH receptor genes is highest during spawning phase. The expression of GnIH receptors is detected in both brain and gonads except for GnIHR3 which only expressed in gonads. The in vivo experiments with GnIH antagonist, RF313 drastically reduced the expression level of reproduction related genes like LH, FSH, and GnRH at 1 h post-injection. In another experiment the surge induced by cGnIH-III peptide on gonadotropins gene expression is further increased when co-injected with LHRHa. However, co-injection of melatonin along with cGnIH-III peptide had opposite effects. These results showed that the GnIH/GnIHRs system has positive effect on reproduction in L. catla.

Characterization of the complete mitochondrial genome of Neolissochilus hexastichus (McClelland, 1839).

The complete mitochondrial genome of the near threatened mahseer fish, Neolissochilus hexastichus, was characterized for the first-time using Ion Torrent NGS platform. The total length of the mitogenome was 16,538 bp including a standard set of 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), 13 protein-coding genes and a non-coding control region. Twenty-eight of the 37 genes are located on the light strand and, the remaining nine genes are situated on the heavy strand. Phylogenetic analysis showed the sister relationship between N. hexastichus and N. hexagonolepis. The mitogenome could be useful for phylogenetics, population genetics, and conservation of the mahseers.

Characterization, Docking and Molecular Dynamics Simulation of Gonadotropin-Inhibitory Hormone Receptor (GnIHR2) in Labeo Catla.

BACKGROUND/AIMS: GnIH receptors (GnIHRs) belong to the family of G-protein coupled receptors (GPCRs) and play a key role in the regulation of reproduction from fishes to mammals, either by inhibiting or stimulating the expression of gonadotropins. The aim of this study was to characterize GnIH receptor (GnIHR2) from Indian Major Carp, Labeo catla and its docking and simulation with GnIH antagonist RF313. METHODS: The full length sequence of GnIHR2 was obtained with RACE PCR. The docking analysis of RF313 with GnIHR2 receptor was performed with AutoDock v. 4.2.6 and molecular dynamics (MD) simulation with GROMACS 5.0. RESULTS: In the present study, we cloned full-length cDNA (1733 bp) of GnIHR2 from the brain of L. catla. The phylogenetic analysis showed clustering of catla GnIHR2 with goldfish and zebrafish in the GPR147 group. L. catla GnIHR2 receptor comprised seven transmembrane domains and the 3D-structure was predicted by I-TASSER tool. The docking analysis revealed high binding affinity (-11.6 kcal/mol) of GnIH antagonist, RF313 towards GnIHR2 receptor. The primary bonds involved were alkyl and hydrogen bonds while the amino acids participated were proline 43, 210, 339, cysteine 214, leucine 211, serine 213 and phenylalanine 338. The MD simulation analysis of docked complex for 100 nano-seconds (ns) in the lipid membrane environment showed the stability of the complex with time. CONCLUSION: Our study showed that GnIH antagonist, RF313 interact tightly with the GnIH receptor, GnIHR2 of L. catla. To the best of our knowledge, this is the first report on computational modelling and MD simulation of GnIH receptor in fishes. This will help in functional characterization studies of GnIH/GnIHR system in vertebrates.

Ontogenetic and tissue-specific expression of gonadotropin-inhibitory hormone (GnIH) and its receptors in Catla catla.

Gonadotropin-inhibitory hormone (GnIH) is an RFamide peptide, and its role in reproduction is well studied from fish to mammals, but very few reports are available about the function of GnIH during larval development. In this study, we examined the GnIH and GnIH receptors (GnIHRs) expression from embryogenesis to adult stage and tissue-specific expression in adult Catla catla using quantitative real-time (qRT) PCR. The qRT PCR analysis of GnIH mRNA during ontogenetic development showed the increasing trend from early developmental stages to the adult stage with the highest expression in 24 months fish. However, the expression of two GnIH receptors, GnIHR1 and GnIHR2 also increased from larval stages to the adults with a peak at 17 days post-hatching, while GnIHR3 showed the higher mRNA expression during embryogenesis and then decreasing gradually. Tissue distribution analysis of GnIH showed the highest mRNA expression of GnIH in the brain, followed by gonads of both the sexes. GnIHR1 and GnIHR2 were also highly expressed in the brain and gonads of both the sexes, while GnIHR3 showed the highest expression in gonads of both the sexes without any expression in the brain. These results suggest that the brain is the primary site of action for GnIH, GnIHR1 and GnIHR2, while gonads for GnIHR3.

Molecular characterization of gonadotropin-inhibitory hormone (GnIH) gene and effect of intramuscular injection of GnIH peptide on the reproductive axis in Catla catla.

Gonadotropin-inhibitory hormone (GnIH) plays an important role in reproduction by inhibiting the expression of gonadotropins in birds and mammals, but in fishes, it is ambiguous. In this study, we cloned 606 bp long cDNA of GnIH from Catla catla brain (cGnIH). The encoded preproGnIH peptide generated three putative peptides (cGnIH-I, -II, -III) of different size. Phylogenetic analysis of GnIH showed clustering of different peptide sequence with its orthologs in separate clades. The real-time PCR analysis showed the expression of cGnIH in brain, gonads, intestine, stomach, heart, gill and liver with the highest expression in the brain and gonads of both sexes. The basal GnIH mRNA expression was higher in spawning and spent phase of the male brain and spawning phase of the female brain. In testis, the expression was highest in spent phase, while in ovary the expression did not change significantly during reproductive phases. The in vivo experiment of cGnIH-III peptide exhibited the higher expression of HPG axis genes, lhb, fshb, cgnrh, kiss2 and kiss1r and serum hormone level of LH and FSH as soon as 3 h after the intramuscular delivery. These results suggest that the GnIH is positively involved in regulation of reproduction in HPG axis of C. catla.

In Silico Mining of Conserved miRNAs of Indian Catfish Clarias batrachus (Linnaeus, 1758) from the Contigs, ESTs, and BAC End Sequences.

MicroRNAs are small non-coding RNAs which play significant role in RNA interference. The present work deals with mining of the conserved miRNA and their target genes from the contigs, ESTs, and BAC end sequences of commercially important catfish, Clarias batrachus, from India. A total of 138, 1 and 1 conserved pre-miRNA sequences, were mined from the contigs, ESTs, and BAC end sequences, respectively. The analysis of families of the conserved pre-miRNA revealed conservation of the fish-specific family mir-430 and other important families, such as mir-455, let-7, mir-133, and mir-137. The mir-455 is involved in hypoxia signaling, let-7 family represents potential anti-tumor molecules involved in human cancer therapy, whereas mir-133 and mir-137 have high therapeutic potentials. Using an alternate computational in silico approach, mining of mature miRNAs resulted in identification of 210 mature miRNAs from contigs, 1 from EST, and 2 each from forward as well as reverse BAC end sequences. Target prediction of these putative miRNAs resulted in the identification of 66,758 and 18,747 target genes in C. batrachus and Danio rerio, respectively. Functional annotation of these miRNAs indicated their involvement in diverse biological functions. The findings of the present study can serve as a valuable resource for further functional genomics studies in C. batrachus.

Development and characterization of genic SSR markers from low depth genome sequence of Clarias batrachus (magur).

Indian magur (Clarias batrachus) is an important freshwater catfish, which is listed as endangered under A3cde+4acde ver. 3.1 categories by the IUCN (2015) due to decreasing population trend. Microsatellites or short sequence repeats (SSRs) tagged to genes have been utilized as gene marker. In the present study, 31,814 SSRs of C. batrachus (magur) were identified using microsatellite identification tool programme from the next generation sequencing data generated on Roche 454 and Ion Torrent platforms. A bioinformatics pipeline, with stringent criteria resulted in selection of 1672 microsatellite loci falling in the genic region. Initially, a total of 30 loci were selected for primer development; and of these 14 were successfully amplified and five were found to be polymorphic in 30 individuals of C. batrachus (magur). The observed as well as expected heterozygosity ranged from 0.038 to 0.526 and 0.434 to 0.784, respectively, and the number of observed alleles ranged from three to five. The study reported the application of next generation sequencing technologies for rapid development of microsatellite loci in Indian catfish species, C. batrachus (magur).

Low-depth shotgun sequencing resolves complete mitochondrial genome sequence of Labeo rohita.

Labeo rohita, popularly known as rohu, is a widely cultured species in whole Indian subcontinent. In the present study, we used in-silico approach to resolve complete mitochondrial genome of rohu. Low-depth shotgun sequencing using Roche 454 GS FLX (Branford, Connecticut, USA) followed by de novo assembly in CLC Genomics Workbench version 7.0.4 (Aarhus, Denmark) revealed the complete mitogenome of L. rohita to be 16 606 bp long (accession No. KR185963). It comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNAs and 1 putative control region. The gene order and organization are similar to most vertebrates. The mitogenome in the present investigation has 99% similarity with that of previously reported mitogenomes of rohu and this is also evident from the phylogenetic study using maximum-likelihood (ML) tree method. This study was done to determine the feasibility, accuracy and reliability of low-depth sequence data obtained from NGS platform as compared to the Sanger sequencing. Thus, NGS technology has proven to be competent and a rapid in-silico alternative to resolve the complete mitochondrial genome sequence, thereby reducing labors and time.

Authentication of five Barilius species from Indian waters using DNA barcoding.

Authentic identification of fish species is essential for conserving them as a valuable genetic resource in our environment. DNA barcoding of living beings has become an important and ultimate tool for establishing their molecular identity. Among cyprinids, Barilius is an important genus having nearly 23 species in Indian region whose morphological identification is often difficult due to minute differences in their features. Five species collected from Indian waters and primarily identified as Opsarius bakeri (syn. Barilius bakeri), B. gatensis, B. vagra, B. bendelisis and B. ngawa were authenticated by their DNA barcoding based on mitochondrial COI gene sequences. Five individuals of each species were taken for barcode preparation by COI gene sequencing which yielded one barcode for B. ngawa, two barcodes each for O. bakeri, B. gatensis, B. bendelisis and three barcodes for B. vagra. The order of inter and intra-specific variation was estimated to know a preliminary status of variation prevailing in these cold stream fish species significant for evolution and conservation of these valued species of our ichthyofauna. Average variation within genera was found to be 13.6% with intra-specific variation ranging from 0.0% (B. ngawa) to 0.6% (B. gatensis). These distance data are in the same order found by various researchers globally using COI barcode sequences in different fish species. Phylogenetic relatedness among Barilius species and some other cyprinids validate their status of individual species as established by conventional taxonomy.

Assessment of pollution of river Ganges by tannery effluents using genotoxicity biomarkers in murrel fish, Channa punctatus (Bloch).

River pollution due to rapid industrialization and anthropogenic activities adversely affects the aquatic organisms, especially fish. Here, we assessed the genotoxicity, mutagenicity and bioaccumulative aspects of tannery effluents in freshwater murrel, Channa punctatus, an inhabitant of river Ganges. Test specimens were collected from three different polluted sites of the river within and nearby Kanpur area during different seasons and blood samples of these specimens were processed for comet assay and micronucleus test as genotoxicity biomarkers. A significantly (P < 0.05) higher micronuclei induction, nuclear abnormalities and % tail DNA was observed in the specimens collected from the polluted sites. Bioaccumulation studies in the muscle (1.202 µg/g) and gill tissues (< 0.300 µg/g) of the specimens revealed the concentration of chromium (core component of tanning industry) above the maximum permissible limits as prescribed by World Health Organization (WHO). The findings of the present analysis indicated contamination of river Ganges with tannery effluents which induce genotoxicity in fish with seasonal variation.

Assembly and variation analyses of Clarias batrachus mitogenome retrieved from WGS data and its phylogenetic relationship with other catfishes.

Whole genome sequencing (WGS) using next generation sequencing technologies paves the way to sequence the mitochondrial genomes with greater ease and lesser time. Here, we used the WGS data of Clarias batrachus, generated from Roche 454 and Ion Torrent sequencing platforms, to assemble the complete mitogenome using both de novo and reference based approaches. Both the methods yielded almost similar results and the best assembled mitogenome was of 16,510 bp size (GenBank Acc. No. KM259918). The mitogenome annotation resulted in 13 coding genes, 22 tRNA genes, 2 rRNA genes and one control region, and the gene order was found to be identical with other catfishes. Variation analyses between assembled and the reference (GenBank Acc. No. NC_023923) mitogenome revealed 51 variations. The phylogenetic analysis of coding DNA sequences and tRNA supports the monophyly of catfishes. Two SSRs were identified in C. batrachus mitogenome, out of which one was unique to this species. Based on the relative rate of gene evolution, protein coding mitochondrial genes were found to evolve at a much faster pace than the d-loop, which in turn are followed by the rRNAs; the tRNAs showed wide variability in the rate of sequence evolution, and on average evolve the slowest. Among the coding genes, ND2 evolves most rapidly. The variations present in the coding regions of the mitogenome and their comparative analyses with other catfish species may be useful in species conservation and management programs.

ATPase 8/6 GENE BASED GENETIC DIVERSITY ASSESSMENT OF SNAKEHEAD MURREL, Channa striata (Perciformes, Channidae).

The mitochondrial DNA (mtDNA) ATPase 8/6 gene has been used in phylogenetic as well as in phylogeographic studies along with other mtDNA markers. In this study, ATPase gene sequences were used to assess the genetic structuring and phylogeographic patterns in Channa striata. Out of 884 nucleotide positions generated in ATPase 8/6 genes, 76 were polymorphic. The study suggested 23 unique haplotypes from 67 individuals of nine populations collected from different riverine systems of India. The ATPase 8/6 sequence revealed highest haplotype as well as nucleotide diversities in Imphal River population and lowest diversities in Tapti River population. The pattern of genetic diversity and haplotype network indicated distinct mitochondrial lineages for Chaliyar population, whereas mismatch distribution strongly suggested a population expansion in mid pleistocene epoch (0.4 Mya) with distinct genetic structuring in C. striata. The baseline information on genetic variation and the population sub-structuring would facilitate conservation and management of this important snakehead murrel.

DNA damage and oxidative stress modulatory effects of glyphosate-based herbicide in freshwater fish, Channa punctatus.

The present study was undertaken to evaluate the genotoxic and oxidative stress modulatory effects of commercial formulation of glyphosate-based herbicide (Roundup(®)) in freshwater fish Channa punctatus. Three sublethal test concentrations of the herbicide viz., SL-I (1/10th of LC50=∼3.25mgL(-1)), SL-II (1/8th of LC50=∼4.07mgL(-1)) and SL-III (1/5th of LC50=∼6.51mgL(-1)) were calculated using 96-LC50 value and the test specimens were exposed to these concentrations. Blood and gill cells of the exposed specimens were sampled on day 1, 7, 14, 21, 28 and 35 to examine the DNA damage using comet assay and to assess the alteration in lipid peroxidation and antioxidant enzymes activities. The highest DNA damage was observed on day 14 at all test concentrations followed by gradual non-linear decline. Induction of oxidative stress in the blood and gill cells were evidenced by increased lipid peroxidation level, while antioxidants namely superoxide dismutase, catalase and glutathione reductase responded in a concentration-dependent manner. The results supported the integrated use of comet and antioxidant assays in determining the toxicity of water pollutants which could be used as part of monitoring programs.

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.

A SRCF cell line from snowtrout, Schizothorax richardsonii: development and characterization.

Schizothorax richardsonii, commonly called snowtrout, is an important indigenous coldwater fish of the Himalayas, India with high commercial values as food and game fish. A cell line named as SRCF was developed from the caudal fin tissue of S. richardsonii. The cell line has been maintained in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS) at 24°C. The cells showed fibroblastic morphology, high plating efficiency and cell doubling time of 48h. Chromosomal analysis of SRCF cells revealed a diploid count of 98 chromosomes. The origin of the cell line was confirmed by the amplification of 655 and 578bp of cytochrome oxidase subunit I (COI) and 16S rRNA of mitochondrial DNA (mt-DNA) genes, respectively. Transfection of SRCF cells with pEGFP-C1 plasmid resulted in bright fluorescent signals, suggesting the application of cell line in transgenic and genetic improvement programme. In addition, genotoxicity assessment illustrated the utility of the cell line as an in vitro model for aquatic toxicological studies.

Interaction between shrimp and white spot syndrome virus through PmRab7-VP28 complex: an insight using simulation and docking studies.

White spot disease is a devastating disease of shrimp Penaeus monodon in which the shrimp receptor protein PmRab7 interacts with viral envelop protein VP28 to form PmRab7-VP28 complex, which causes initiation of the disease. The molecular mechanism implicated in the disease, the dynamic behavior of proteins as well as interaction between both the biological counterparts that crafts a micro-environment feasible for entry of virus into the shrimp is still unknown. In the present study, we applied molecular modeling (MM), molecular dynamics (MD) and docking to compute surface mapping of infective amino acid residues between interacting proteins. Our result showed that α-helix of PmRab7 (encompassing Ser74, Ile143, Thr184, Arg53, Asn144, Thr184, Arg53, Arg79) interacts with ß-sheets of VP28 (containing Ser74, Ile143, Thr184, Arg53, Asn144, Thr184, Arg53, Arg79) and Arg69-Ser74, Val75-Ile143, Leu73-Ile143, Arg79-Asn144, Ala198-Ala182 bonds contributed in the formation of PmRab7-VP28 complex. Further studies on the amino acid residues and bonds may open new possibilities for preventing PmRab7-VP28 complex formation, thus reducing chances of WSD. The quantitative predictions provide a scope for experimental testing in future as well as endow with a straightforward evidence to comprehend cellular mechanisms underlying the disease.

Genetic divergence and molecular phylogenetics of Puntius spp. based on the mitochondrial cytochrome b gene.

Puntius is an important genus of freshwater food and ornamental fish belonging to the family Cyprinidae. A total of 60 samples from twelve species of the genus Puntius were collected from eight sampling sites of eight Indian Rivers. Twelve species of Puntius (P. chola, P. sophore, P. filamentosus, P. fasciatus, P. vittatus, P. chelynoides, P. gonionotus, P. denisonii, P. ticto, P. gelius, P. conchonius and P. sarana) were investigated using 60 partial sequences of the mitochondrial cytochrome b (Cyt b, 1096 bp) gene to estimate genetic divergence and to establish the phylogenetic relationship. The average intraspecies diversity was estimated as 0.002, whereas the average interspecies diversity was estimated as 0.177. The sequence analysis of the Cyt b gene revealed four distinct groups, which are genetically distinct species and exhibited identical phylogenetic relationship. The present study validated the utility of the Cyt b gene in genetic diversity and phylogenetic studies.

Genetic characterization of two hill stream fish species Barilius bendelisis (Ham.1807) and Barilius barna (Ham.1822) using RAPD markers.

Genetic structure of four wild populations of two hill stream fishes Barilius bendelisis (Ham.1807) and B. barna (Ham. 1822) from Uttarakhand, India, was studied using RAPD markers. Eight selective primers provided distinct and consistent RAPD profiles in both the species, producing a total of 47 and 35 scorable bands in B. bendelisis and B. barna respectively. The bands in the range 666-4,830 bp were scored for consistent results. The RAPD profiles generated by all the eight primers revealed varying degrees of polymorphism (25.00-50.00 %). The average genetic diversity (h) was estimated as 0.1661 and 0.1606 among the four populations of B. bendelisis and B. barna respectively.