Genetic stock identification in Perna viridis (Linnaeus1758) from the Indian Peninsula by using microsatellite markers.

BACKGROUND: Perna viridis (Linnaeus, 1758), the Asian green mussel, is native to the Asia-Pacific region. The species is extensively distributed in the Indian subcontinent and is a candidate species for aquaculture in the Southeast Asian region. Availability of genetic information on wild populations is essential for the effective conservation and management of Perna species. The present study assessed the genetic variation and population structure across the distribution range of this species from the Indian peninsula by using microsatellite markers to determine the genetic structuring among the species. METHODS: A total of 15 microsatellite loci with M13 labeling were used for the genetic characterization of P. viridis along Indian waters. Genotyped data were analyzed using analytical software to determine the genetic stocks and understand the genetic variability across the populations. RESULTS: We identified 15 polymorphic markers to understand the genetic stocks and variability across Perna populations. The mean value of the observed heterozygosity (Hobs: 0.741) for all populations was closer to the expected heterozygosity (Hexp: 0.75). The pairwise Fst values between the west and east coasts of India varied significantly, indicating the existence of significant genetic structure between the populations. CONCLUSIONS: Genetic stock identification using software analysis exhibited two distinct stocks, one along the west coast (Arabian Sea) and another along the east coast (Bay of Bengal). Bottleneck analysis indicated the genetic stability of species in the wild. P. viridis is a commercially vital species in Indian peninsular regions. The present study suggests the adoption of stock-specific relaying programs of the species from Indian waters in future studies.

Microsatellite marker development in Spanish mackerel Scomberomorus commerson using third generation sequencing technology.

Spanish mackerel S. commerson belonging to family Scombridae, represent a group of highly commercial marine fisheries with an ever-growing demand world over. Analysing the genetic diversity of this species is of utmost importance and necessary for conservation purposes. Microsatellites are molecular tools with advantages that are ideal for population analyses. This study provides the first multiplex panel set of species-specific microsatellite loci for S. commerson that can be applied when assessing both intra- and inter population genetic variation. Microsatellite marker panels were developed in S. commerson, using Third Generation Sequencing technology in PacBio RSII, based on Single-Molecule Real-Time (SMRT). Thirty- two microsatellite loci were isolated and characterized for S. commerson, by genotyping 20 individuals each obtained from the Kochi and Veraval in the Arabian sea and Chennai along Bay of Bengal coast (n = 3). The number of alleles per locus in S. commerson varied from 4 to 17, while the mean observed and expected heterozygosities ranged from 0.656 to 0.753. The Polymorphic Information Content (PIC) were highly informative, 85% loci with PIC value 0 > 0.75. This suite of markers provides the first species specific nuclear multiplex microsatellite marker panels (32 loci) for S. commerson and thus allows assessment of different populations structures of the species across its distribution range, with more specificity. These newly developed loci have also been validated for cross transferability in another scomberid fish Scomberomorus guttatus.

Population connectivity and genetic structure of Asian green mussel, Perna viridis along Indian waters assessed using mitochondrial markers.

Perna viridis (Linnaeus, 1758), the Asian green mussel, belonging to the family Mytilidae is widely distributed along the Indian coast. The species is majorly found in southeastern countries and is considered an ideal candidate for aquaculture due to its high nutritional value and growth rate. Obtaining their genetic information is essential for their sustainable capture-based production. In the present study, genetic variation, population structure, and demographic processes of the populations across the distribution of this species were assessed using the mitochondrial DNA ATPase6 and cytb gene. In total, we selected 170 samples from five localities across the Indian subcontinent including Andaman Sea. Sequence analysis of partial cytb (885 bp) and ATPase6 (714 bp) genes revealed 45 and 58 haplotypes, respectively. The significant coefficient of genetic differentiation (FST: 0.255 for cytb and 0.252 for ATPase6) and analyses of molecular variance indicated three varieties of stocks, namely Arabian Sea, Bay of Bengal, and Andaman Sea. All the populations showed low nucleotide diversity, suggesting severe historical bottleneck events and high haplotype diversity, indicating population expansion. The genetic variation and demographic process reported in this study will form the baseline information for framing policies, which can be adopted while planning stock specific ranching and relaying programmes in the Indian subcontinent with view to enhance and manage the fishery.

Aenigmachanna mahabali, a new species of troglophilic snakehead (Pisces: Channidae) from Kerala, India.

Aenigmachanna mahabali, a new species of troglophilic snakehead is described on the basis of a single specimen recovered from a well in Kerala, India, over 200km south of the type locality for the only known species in the genus. The new species can be distinguished from its congener in possessing fewer dorsal fin rays (53 vs 56-57), fewer total vertebrae (61 vs 64), fewer scales in lateral series (76 vs 83-85) and in the pectoral-fin rays being extended beyond the margin of the membrane into filaments.

New insights from nuclear and mitochondrial markers on the genetic diversity and structure of the Indian white shrimp Fenneropenaeus indicus among the marginal seas in the Indian Ocean.

Genetic variation in wild stocks of a major commercial shrimp, Fenneropenaeus indicus, from the marginal seas in the Indian Ocean was analysed using polymorphic microsatellite loci and mitochondrial COI gene. The average observed heterozygosity (Ho = 0.44 ±â€¯0.02) and the expected heterozygosity (He = 0.73 ±â€¯0.01) were high across loci and populations indicating high microsatellite variation. Pairwise FST and Bayesian clustering indicated the occurrence of four genetically distinct stocks out of the eight sampled populations with implications for specific management approaches. Mantel test for isolation by distance proved that genetic differentiation is not related to geographic distance between populations. Mitochondrial COI sequence analysis showed concordant differentiation pattern as well indicated the relevance of COI in population genetics of shrimps. Pairwise ɸST and phylogenetic and Bayesian analyses revealed four distinct clades, as observed with nuclear markers. Divergence time analysis revealed the origin and initial divergence of F. indicus corresponds to late Miocene and divergence to phylogroups in the Pleistocene. BSP analysis presented a long stable population size with a slight decrease in the late Pleistocene and gradually expanded to the current status. The information here will be useful in commercial shrimp breeding and selection programmes and management of natural stocks of Indian white shrimp.

Molecular and morphological evidences resolve taxonomic ambiguity between Systomus sarana sarana (Hamilton, 1822) and S. sarana subnasutus (Valenciennes) and suggest elevating them into distinct species.

Taxonomic ambiguity exists in genus Systomus and recently many new species were described under this genus. Systomus sarana subnasutus is considered a valid subspecies of S. sarana sarana although revisions have been done by some researchers. We employed a combination of morpho-meristics and molecular tools (Cytochrome c oxidase I, 16S and Cytochrome b genes of mitochondrial genome) to resolve the two species. Three morpho-meristic characters, head length/maxillary barbel length (HL/MxBL), Lateral Line Scales (LLSs) as well as two truss-based characters, had discernible variation between the two taxa. The sequence analysis (2353 nucleotides) depicted a separate clad of S. sarana subnasutus with high bootstrap support. The findings from combined use of morphology, meristics and mitogenes were concordant. The corroborative results suggest the possibility of two different species. The results suggest to adopt suitable management measures, accordingly.

Molecular based phylogenetic species recognition in the genus Pampus (Perciformes: Stromateidae) reveals hidden diversity in the Indian Ocean.

Pomfrets (Genus Pampus) are commercially important fishes in the Indo Pacific region. The systematics of this genus is complicated due to morphological similarities between species. The silver pomfret from Indian waters has long been considered to be Pampus argenteus. The objective of the study was to utilize the mitochondrial COI gene to establish the molecular identity of the silver pomfret distributed in Indian waters and to resolve the phylogenetic relationships among Pampus species in the world based on sequence data in the NCBI database. Seven valid Pampus species are identified in this study. The mean genetic divergence value calculated between clades representing these species was 7.9%. The mean genetic distance between the so-called Pampus argenteus from Indian waters and sequences attributed to P. argenteus from the South China Sea, where the neotype of this species was collected, was found to be greater than 12%, strongly supporting the likelihood of the Indian species being distinct. The Indian Pampus species show very close affinity to P. cinereus, with inter species differences less than 2%. The taxonomic identity of the silver pomfret in India is also discussed here, in light of molecular and morphological evidence.

Mitochondrial signatures for identification of grouper species from Indian waters.

Groupers are important commercial fish in many parts of the world. Accurate identification is critical for effective conservation assessment and fisheries management. Genetic barcodes provide a simple and reproducible method for the identification of species even in the absence of taxonomic expertise. The generation of reference barcodes from properly identified specimens is an important first step in this direction. Here, 36 species belonging to the subfamily Epinephelinae (Family: Serranidae) were collected from landings on the west coast of India and Port Blair, Andaman, and partial nucleotide sequence data of the mitochondrial cytochrome C oxidase subunit I (COI) gene was generated. Barcodes for 13 species were developed from Indian waters for the first time. Analysis using the COI gene produced phylogenetic trees in concurrence with other multi-gene studies. Epinephelus fasciatus and E. areolatus were found to be a species complex, as hypothesized in other studies. The DNA barcodes developed in the study can be used for identifying species within Epinehelinae, where taxonomic ambiguity still exists.

DNA barcoding reveals species composition of sharks and rays in the Indian commercial fishery.

DNA barcoding was successfully used for the accurate identification of chondrichthyans in the Indian commercial marine fishery. About 528 specimens of 111 chondrichthyan species and 34 families, collected from the Indian EEZ, were barcoded for a 655 bp region of the mitochondrial gene cytochrome c oxidase subunit 1 (COI). Generally, five specimens per species were barcoded, but numbers ranged from 2 to 13. The average Kimura 2 parameter (K2P) distance separating individuals within species was 0.32%, and the average distance separating species within genera was 6.73%. Ten species were suggested as putative new species requiring formal descriptions. Based on the morphology and molecular support, 11 elasmobranch species were confirmed first records for Indian waters. The present study confirms the ability of DNA barcoding for the accurate identification of sharks, rays, and their products from Indian waters.

Population genetic structure of Macrobrachium rosenbergii (Palaemonidae) from Indian waters using mitochondrial ATPase 6/8 gene.

Macrobrachium rosenbergii, giant freshwater prawn, is one of the most commercially important crustaceans. In the present study, primers for ATPase 6/8 region of mt-DNA were designed and successfully amplified (827 bp) in the species. The nucleotide variation in ATPase 6/8 gene revealed the population structuring in natural populations of M. rosenbergii in Indian waters. A total of 35 haplotypes were observed in 93 individuals collected from different locations. Low nucleotide diversity and high haplotype diversity were noticed for the ATPase 6/8 gene. Significant pairwise FST and, haplotype network indicated occurrence of distinct populations. Observed mismatch distribution and Tajima's D test suggested demographical stability of giant freshwater prawn. The genetic stock structure revealed in this study will be helpful for conservation and management of stocks of M. rosenbergii in Indian waters.

Emergence of carp edema virus in cultured ornamental koi carp, Cyprinus carpio koi, in India.

A disease outbreak was reported in adult koi, Cyprinus carpio koi, from a fish farm in Kerala, India, during June 2015. The clinical signs were observed only in recently introduced adult koi, and an existing population of fish did not show any clinical signs or mortality. Microscopic examination of wet mounts from the gills of affected koi revealed minor infestation of Dactylogyrus sp. in a few koi. In bacteriological studies, only opportunistic bacteria were isolated from the gills of affected fish. The histopathological examination of the affected fish revealed necrotic changes in gills and, importantly, virus particles were demonstrated in cytoplasm of gill epithelial cells in transmission electron microscopy. The tissue samples from affected koi were negative for common viruses reported from koi viz. cyprinid herpesvirus 3, spring viraemia of carp virus, koi ranavirus and red sea bream iridovirus in PCR screening. However, gill tissue from affected koi carp was positive for carp edema virus (CEV) in the first step of nested PCR, and sequencing of PCR amplicons confirmed infection with CEV. No cytopathic effect was observed in six fish cell lines following inoculation of filtered tissue homogenate prepared from gills of affected fish. In bioassay, the symptoms could be reproduced by inoculation of naive koi with filtrate from gill tissue homogenate of CEV-positive fish. Subsequently, screening of koi showing clinical signs similar to koi sleepy disease from different locations revealed that CEV infection was widespread. To our knowledge, this is the first report of infection with CEV in koi from India.

A new fish cell line derived from the caudal fin of freshwater angelfish Pterophyllum scalare: development and characterization.

In this study, a new cell line derived from the caudal fin of the freshwater angelfish Pterophyllum scalare was developed and characterized. The cell line was designated angelfish fin (AFF) and subcultured 44 times since its development. These cells grew well in Leibovitz's -15 medium supplemented with 10% foetal bovine saline (FBS) at 28° C and the modal chromosome number (2n) was 48. The AFF cell-line is mainly comprised of epithelial cells as confirmed by immunocytological technique using anti-cytokeratin antibodies, an epithelial cell marker. This cell line was tested for growth in a temperatures range from 20 to 37° C and at various FBS concentrations of 5-20% at 28° C. The cell line was cryopreserved at different passage levels and revived successfully with 80% survival rate. Polymerase chain reaction amplification and sequencing of partial mitochondrial 16s rRNA and coI genes confirmed that the AFF cell-line originated from angelfish. Mycoplasma sp. contamination was not detected in AFF cells and checked by Hoechst 33258 fluorescence staining. At the 42nd passage the cells were transfected with 2 µg of pAcGFP1-N1 expression vector. The AFF cells exhibited cytotoxic effects when exposed to the bacterial extra cellular products from Serratia marcescens and Proteus hauseri. The AFF cells and cells from kidney and brain did not show cytopathic effect when exposed to cyprinid herpes virus2 and viral nervous necrosis virus. The newly developed AFF cell line will be useful for the isolation of viruses affecting angelfishes, such as iridoviruses, in the future.

In vitro nuclear receptor activity and in vivo gene expression analysis in Murray-Darling rainbowfish (Melanotaenia fluviatilis) after short-term exposure to fluoxetine.

Fluoxetine (FLX) is one of numerous pharmaceuticals found in treated municipal wastewater discharged to the environment. In the present study, we investigated the effects of short-term (96h) waterborne FLX exposure (1µg/L or 100µg/L) on the expression of selected genes in brain, liver, and gonads of female Murray-Darling rainbowfish (Melanotaenia fluviatilis), a small-bodied teleost of ecotoxicological relevance in the Australasia region. Plasma 17ß-estradiol (E2) levels were also determined. In the brain, no significant changes in mRNA levels were observed for the selected genes. In ovaries, 100µg/L FLX caused a 10-fold downregulation of aromatase A (cyp19a1a) mRNA and a 4-fold upregulation of estrogen receptor α (esr1) mRNA levels. In liver, mRNA levels for vitellogenin A (vtga) and choriogenin L (chgl) were downregulated by 50-fold and 18-fold compared with controls, respectively, in response to 100µg/L FLX. Concentrations of E2 in plasma were significantly lower than controls in response to 100µg/L FLX. This could be attributable to a decrease in estrogen biosynthesis as a result of the observed downregulation of cyp19a1a mRNA. To establish whether the observed changes in gene expression could be explained by the modulation of selected nuclear receptors by FLX, we employed panel of reporter gene assays in agonistic and antagonistic modes. Apart from minor activation of ERα after exposure to high concentrations (5µM), FLX did not activate or inhibit the nuclear receptors tested. Further study is required to determine whether the observed downregulation of ovarian aromatase expression and liver estrogen-regulated genes also occurs at environmentally relevant FLX concentrations over longer exposure periods.

Genetic identification and phylogenetic relationships of Indian clariids based on mitochondrial COI sequences.

Mitochondrial cytochrome C Oxidase I (COI) sequence variation among the clariid fishes of India (Clarias magur, C. dussumieri and C. gariepinus) and their relationship with other representative clariids was studied in this work. Three species were sampled and together with 23 COI sequences from GenBank were used to reconstruct phylogenetic relationships in the family Clariidae. The study revealed two clades: one consisting of the African species with C. dussumieri, and the other of Asian species suggesting the prevalence of intra-continental diversification of catfishes. This study further revealed that the genus Clarias is monophyletic. For the COI gene, the interspecies genetic divergence ranged from 0.056 to 0.182. The mean genetic difference between C. dussumieri and other selected African species in this study is 12.1%. It was also observed that the morphological similarity of C. dussumieri and C. magur was not replicated in the genetic level. Clarias dussumieri was more close to African catfish C. gariepinus thus indicating the utility of COI phylogeny to identify the well-known African-Asian relationships within catfishes. The results also showed that C. magur and C. batrachus are genetically distinct from each other.

A new epithelial cell line, HBF from caudal fin of endangered yellow catfish, Horabagrus brachysoma (Gunther, 1864).

A new epithelial cell line, Horabagrus brachysoma fin (HBF), was established from the caudal fin tissue of yellow catfish, H. brachysoma and characterized. This HBF cell line was maintained in Leibovitz's-15 medium supplemented with 15 % fetal bovine serum (FBS) and subcultured more than 62 times over a period of 20 months. The HBF cell line consists predominantly of epithelial cells and is able to grow at temperatures between 20 and 35 °C with an optimum temperature of 28 °C. The growth rate of these cells increased as the proportion of FBS increased from 5 to 20 % at 28 °C with optimum growth at the concentrations of 15 % FBS. Partial amplification and sequencing of fragments of two mitochondrial genes 16S rRNA and COI confirmed that HBF cell line originated from yellow catfish. The HBF cells showed strong positive reaction to the cytokeratin marker, indicating that it was epithelial in nature. HBF cell line was inoculated with tissue homogenate from juveniles of Sea bass, Lates calcarifer infected with viral nervous necrosis virus (VNNV) and found not susceptible to VNNV. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the HBF cells. These cells were confirmed for the absence of Mycoplasma sp by PCR.

Hints for panmixia in Scomberomorus commerson in Indian waters revealed by mitochondrial ATPase 6 and 8 genes.

Scomberomorus commerson is an economically important migratory fish distributed worldwide. The genetic stock structure of S. commerson distributed along the Indian waters was identified using mitochondrial ATPase 6 and 8 genes. A total of 842 bp sequence of ATPase 6/8 genes obtained in this study revealed 23 haplotypes with mean low nucleotide diversity and high haplotype diversity. Co-efficient of genetic differentiation (FST) values obtained for pair wise populations were low and non-significant with an overall value of -0.02074. The high haplotype and low nucleotide diversity values together with mismatch distribution analysis suggested a history of genetic bottleneck events or founder effect, with subsequent population expansion in S. commerson. The findings of the present study indicated the panmixia nature of the species which can be managed as a unit stock in Indian waters.

Molecular phylogeny of commercially important lobster species from Indian coast inferred from mitochondrial and nuclear DNA sequences.

Lobsters constitute low-volume high-value crustacean fishery resource along Indian coast. For the conservation and management of this declining resource, accurate identification of species and larvae is essential. The objectives of this work were to generate species-specific molecular signatures of 11 commercially important species of lobsters of families Palinuridae and Scyllaridae and to reconstruct a phylogeny to clarify the evolutionary relationships among genera and species included in this study. Partial sequences were generated for all the candidate species from sampling sites along the Indian coast using markers like Cytochrome oxidase I (COI), 16SrRNA, 12SrRNA, and 18SrRNA genes, and analyzed. The genetic identities of widely distributed Thenus species along the Indian coast to be Thenus unimaculatus and the sub-species of Panulirus homarus to be P. homarus homarus were confirmed. Phylogeny reconstruction using the individual gene and concatenated mtDNA data set were carried out. The overall results suggested independent monophyly of Scyllaridae and Stridentes of Palinuridae. The interspecific divergence was found to be highest for the 12SrRNA compared with other genes. Significant incongruence between mtDNA and nuclear 18SrRNA gene tree topologies was observed. The results hinted an earlier origin for Palinuridae compared with Scyllaridae. The DNA sequence data generated from this study will aid in the correct identification of lobster larvae and will find application in research related to larval transport and distribution.

Weak genetic differentiation in cobia, Rachycentron canadum from Indian waters as inferred from mitochondrial DNA ATPase 6 and 8 genes.

Cobia, Rachycentron canadum, is an economically important migratory fish distributed in tropical waters worldwide and is a candidate fish species for aquaculture practices. The genetic stock structure of R. canadum distributed along the Indian waters was identified using mitochondrial ATPase 6 and 8 genes. A total of 842 bp sequence of ATPase 6/8 genes obtained in this study revealed 15 haplotypes with mean low nucleotide diversity (π = 0.001) and high haplotype diversity (h = 0.785). AMOVA indicated the genetic differentiation of 90.47% for individuals within the population. This is well supported by co-efficient of genetic differentiation (FST) values obtained for pairwise populations that were low and non-significant with an overall value of 0.002. The parsimony network tree revealed star-like phylogeny and all the haplotypes were connected with each other by single mutational event. The findings of the present study indicated the panmixia nature of the species which can be managed as a unit stock in Indian waters.

Mass mortality in ornamental fish, Cyprinus carpio koi caused by a bacterial pathogen, Proteus hauseri.

Moribund koi carp, Cyprinus carpio koi, from a farm with 50% cumulative mortality were sampled with the aim of isolating and detecting the causative agent. Three bacterial species viz., Citrobacter freundii (NSCF-1), Klebsiella pneumoniae (NSKP-1) and Proteus hauseri [genomospecies 3 of Proteus vulgaris Bio group 3] (NSPH-1) were isolated, identified and characterized on the basis of biochemical tests and sequencing of the 16S rDNA gene using universal bacterial primers. Challenge experiments with these isolates using healthy koi carp showed that P. hauseri induced identical clinical and pathological states within 3 d of intramuscular injection. The results suggest P. hauseri (NSPH-1) was the causative agent. In phylogenetic analysis, strain NSPH-1 formed a distinct cluster with other P. hauseri reference strains with ≥99% sequence similarity. P. hauseri isolates were found sensitive to Ampicillin, Cefalexin, Ciprofloxacin and Cefixime and resistant to Gentamycin, Oxytetracycline, Chloramphenicol, and Kanamycin. The affected fish recovered from the infection after ciprofloxacin treatment.