DNA barcoding detected improper labelling and supersession of crab food served by restaurants in India.

BACKGROUND: Detection of improper labelling of raw and processed seafood is of global importance for reducing commercial fraud and enhancing food safety. Crabs are crustaceans with intricate morphological as well as genetic divergence among species and are popular as seafood in restaurants. Owing to the high number of crab species available, it can be difficult to identify those included in particular food dishes, thus increasing the chance of supersession. DNA barcoding is an advanced technology for detecting improper food labelling and has been used successfully to authenticate seafood. RESULTS: This study identified 11 edible crab species from India by classical taxonomy and developed molecular barcodes with the cytochrome c oxidase I (COI) gene. These barcodes were used as reference barcodes for detecting any improper labelling of 50 restaurant crab samples. Neighbour-joining tree analysis with COI barcodes showed distinct clusters of restaurant samples with respective reference species. The study demonstrated 100% improper labelling of restaurant samples to cover up acts of inferior crab supersession. CONCLUSION: DNA barcoding successfully identified 11 edible crabs in accordance with classical taxonomy and discerned improper crab food labelling in restaurants of India.

In vitro cytotoxicity assessment of two heavy metal salts in a fish cell line (RF).

Labeo rohita (rohu) is one of most important of Indian major carps, which is highly ranked among the important freshwater aquaculture species in the world. Heavy metals are major risk factors for aquatic health, which are biomagnified through the food chain. The present study was carried out to investigate the effect of different divalent salts of two heavy metals, such as zinc (ZnCl(2), ZnSO(4) and ZnNO(3)) and cadmium (CdCl(2) and CdSO(4)), in an established fish cell line, RF developed from fin tissue of L. rohita. The RF cell line was used for assessment of heavy metal cytotoxicity through various endpoint assays, including maximum tolerated dose (MTD) determination, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, neutral red (NR) uptake assay, and Coomassie brilliant blue (CBB) assay. Results revealed that these heavy metal salts were cytotoxic to the RF cell line at varied concentrations. MTD values were found to be 1.563, 3.125, 6.25, 12.5 and 25 mg/L for CdCl(2), CdSO(4), ZnCl(2), Zn(NO(3))(2) and ZnSO(4), respectively. The half-maximal inhibitory concentration values calculated by MTT, NR and CB assay were 53.83 ± 7.02, 58.03 ± 9.12 and 79.20 ± 15.27 for ZnSO(4), 26.44 ± 7.01, 36.60 ± 7.82 and 155.6 ± 14.75 for Zn(NO(3))(2), 20.26 ± 17.95, 16.94 ± 7.05 and 87.54 ± 7.58 for ZnCl(2), 5.166 ± 0.57, 15 ± 1 and 41.80 ± 8.38 for CdSO(4) and 4.966 ± 0.56, 9.56 ± 1.73 and 20.93 ± 4.47 for CdCl(2). This study establishes the RF cell line as an in vitro tool for assessment and monitoring of heavy metal concentration in the aquatic environment.

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 LC(50) of profenofos was found to be 2.68 µgL(-1). On the basis of the obtained LC(50) 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.

Development of cell culture system from the giant freshwater prawn Macrobrachium rosenbergii (de Man).

A new cell culture system (MRH) was developed for the first time from 2 months old freshwater prawn, Macrobrachium rosenbergii. Primary cultures were developed from heart tissues by explant culture technique. Cell outgrowth was obtained from the heart explant after 14 days of explant culture. The culture medium used was Leibovitz-15 supplemented with 20% Fetal Bovine Serum along with 1% prawn hemolymph serum, 0.1% glucose, 0.5% NaCl and antibiotics (Penicillin 10,000 Units ml(-1), Streptomycin 10,000 microg ml(-1), Amphotericin B 500 mg ml(-1)) with a final osmomolality of 470-550 mmol kg(-1). The pH of the growth medium found suitable for the growth of the cells was 7.20. The viability of cells was found to be 60% when revived after a month of storage in liquid nitrogen.

Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn).

Two new cell lines (CCF and CCH) were established from fin and heart tissues of common carp, Cyprinus carpio. The cells were optimally maintained in Leibovitz-15 medium supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml of basic fibroblastic growth factor (bFGF). The effects of temperature, concentration of FBS and bFGF on the growth of CCF and CCH cells were examined. The temperature ranged from 24 to 32°C for good growth of the cells. The growth rate of cells was higher in medium containing 10% FBS and the addition of bFGF to the medium significantly increased the growth rate. The CCF cells were found to be epithelial, while the CCH cells were fibroblastic in nature. The cytogenetic analysis of the cell lines revealed a diploid number of 100 chromosomes in C. carpio. The viability of CCF and CCH cell lines were 70 and 72%, respectively, after six months of storage in liquid nitrogen (-196° C). Molecular characterization of the cell lines using 16S rRNA and Cytochrome Oxidase Subunit I (COI) revealed the origin of the cell lines. These new cell lines will be useful for isolation of fish viruses and other in vitro biotechnological studies.

Molecular identification and phylogenetic relationships of seven Indian Sciaenids (Pisces: Perciformes, Sciaenidae) based on 16S rRNA and cytochrome c oxidase subunit I mitochondrial genes.

The partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) mitochondrial genes were analyzed for species identification and phylogenetic relationships among the commercially important Indian sciaenids (Otolithes cuvieri, Otolithes ruber, Johnius dussumieri, Johnius elongatus, Johnieops vogleri, Otolithoides biauritus and Protonibea diacanthus). Sequence analysis of both genes revealed that the seven species fell into three distinct groups, which were genetically distant from each other and exhibited identical phylogenetic resolution. Partial sequences of both the genes provided sufficient phylogenetic information to distinguish the seven sciaenids indicating the usefulness of mtDNA-based approach in species identification.

Genetic relatedness and phylogenetics of five Indian pufferfishes.

The taxonomy and phylogeny of the pufferfishes belonging to the family Tetraodontidae found in India are poorly understood. We investigated five species of freshwater and marine pufferfishes using partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) of mitochondrial genes. The sequence alignment of 16S rRNA yielded 573 bp, whereas COI gene sequence alignment yielded 614 bp. The sequence analysis of the genes revealed two distinct groups of freshwater and marine origin, which are genetically distinct from each other and exhibit identical phylogenetic resolution. The partial sequences of both the genes provided sufficient phylogenetic resolution to distinguish all the five species of pufferfishes. The COI sequences could be used as DNA barcodes for identification of the pufferfishes.

Genetic differentiation of Ompok bimaculatus (Teleostei: Siluridae) population based on mtDNA cytochrome b gene.

Ompok bimaculatus commonly called as Indian butter catfish is a commercially important food fish and distributed throughout India and Southeast Asia. Genetic differentiation of O. bimaculatus was investigated using cytochrome b (cyt b) gene of three different riverine populations. A total of 37 sequences of cyt b gene (1118 bp) were generated from O. bimaculatus. The mitochondrial DNA analysis revealed that O. bimaculatus has relatively high genetic diversity. The estimated haplotype and nucleotide diversity were 0.063 and 0.005, respectively. The analysis of molecular variance indicated 73.53% genetic variability between the populations and 26.47% genetic variation within population. The understanding of genetic differentiation of O. bimaculatus populations will play a key role in conservation and management of the important endangered fish species.

Permanent genetic resources added to molecular ecology resources database 1 December 2012-31 January 2013.

This article documents the addition of 268 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alburnoides bipunctatus, Chamaerops humilis, Chlidonias hybrida, Cyperus papyrus, Fusarium graminearum, Loxigilla barbadensis, Macrobrachium rosenbergii, Odontesthes bonariensis, Pelteobagrus vachelli, Posidonia oceanica, Potamotrygon motoro, Rhamdia quelen, Sarotherodon melanotheron heudelotii, Sibiraea angustata, Takifugu rubripes, Tarentola mauritanica, Trimmatostroma sp. and Wallago attu. These loci were cross-tested on the following species: Alburnoides fasciatus, Alburnoides kubanicus, Alburnoides maculatus, Alburnoides ohridanus, Alburnoides prespensis, Alburnoides rossicus, Alburnoides strymonicus, Alburnoides thessalicus, Alburnoides tzanevi, Carassius carassius, Fusarium asiaticum, Leucaspius delineatus, Loxigilla noctis dominica, Pelecus cultratus, Phoenix canariensis, Potamotrygon falkneri, Trachycarpus fortune and Vimba vimba.

Molecular identification of three Ompok species using mitochondrial COI gene.

A DNA-based barcode identification system that is applicable to all animal species will provide a simple, universal tool for the identification of fish species. The barcode system is based on sequence diversity in subunit 1 cytochrome c oxidase (COI) gene. Identification and characterization of fish species based on morphological characters are sometimes found to be erroneous and environmentally affected. There are no studies on the genus Ompok in India at molecular level and species identification of the Ompok is usually carried out through morphological features. A total of 106 samples from three species Ompok pabda, O. pabo and O. bimaculatus were collected from eight sampling sites of seven Indian rivers. One hundred and six sequences were generated from COI region of three Ompok species and 21 haplotypes were observed. The sequence analysis of COI gene revealed three genetically distinct Ompok species and exhibited identical phylogenetic resolution among them. The partial COI gene sequence can be used as a diagnostic molecular marker for identification and resolution of taxonomic ambiguity of Ompok species.

Morphological taxonomy and molecular divergence of four balitorid species (subfamily: Nemachelinae) from Central Himalaya, India.

The fishes in this study belong to the difficult group. It is difficult to identify about more than 50 nemacheilid loach species in India, mainly due to the poor quality of the original descriptions, the lack of good reviews, and the similarity of a number of the banded species. In view of this, a morphometric and genetic study was conducted on four species that have been documented in Central Himalayan region of India. Using comparative methods, a contribution to the knowledge about the variability of four Himalayan hillstream loaches, a molted loach Acanthocobitis botia (Hamilton, 1822), Nemacheilus corica (Hamilton, 1822), a creek loach Schistura beavani (Gunther, 1868), and Schistura montana (McClelland, 1838), was accomplished. Morphometric data were analyzed by univariate (CV and analysis of variance) and multivariate analyses (discriminant function analysis and cluster analysis). Based on the results of these statistical analyses, generally all four species grouped into their own species cluster with minimal overlap between two species of genus Schistura. Mitochondrial DNA cytochrome b (Cyt b) gene analysis revealed 7.7-17.6% genetic distances among the four species. Molecular phylogenetic relationship among these species and other Balitoridae species was investigated using published mitochondrial Cyt b sequences. Dendrograms obtained by the maximum likelihood and maximum parsimony method analyses exhibited the same topology. According to this topology, all the four species represented distinct species group and A. botia form the most distinct species while S. beavani and S. montana are clustered together with Nemacheilus species to form a single group. This work may build the base for the revision of taxonomic identity of fishes of the family Balitoridae. The results may further help to enhance the knowledge of the ichthyologists in understanding the fish fauna of India and assist them in planning conservation and management strategies for the propagation of these less studied small indigenous species along their natural range of distribution.

Development and characterization of two cell lines PDF and PDH from Puntius denisonii (Day 1865).

The Puntius denisonii colloquially and more popularly referred to as Miss Kerala is a subtropical fish belonging to the genus Puntius (Barb) and family Cyprinidae. Two cell lines PDF and PDH were developed from the caudal fin and heart of P. denisonii, respectively. The cell lines were optimally maintained at 26°C in Leibovitz-15 medium supplemented with 10% fetal bovine serum. A diploid count of 50 chromosomes at passage 50 was observed in both the cell lines. The high growth potential of the cell lines was reflected from the cell doubling time of 28 and 30 h of PDF and PDH cell lines, respectively. The viability of the PDF and PDH cell lines was 70% and 76%, respectively, after 4 mo of storage in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 653 bp fragments of cytochrome oxidase subunit I of mitochondrial DNA genes.

Development and characterization of three new diploid cell lines from Labeo rohita (Ham.).

Development of cell lines from fish for identifying the pathogenesis of viral diseases and for vaccine production against viral and bacterial diseases is imperative where they are of commercial importance. Three new diploid fish cell lines (RF, RH, and RSB) were developed from fin, heart, and swim bladder of an Indian major carp, Labeo rohita, commonly called Rohu. All the cell lines were optimally maintained at 28 degrees C in Leibovitz-15 medium supplemented with 10% FBS. The propagation of RH and RSB cells was serum dependent, with a low plating efficiency (<16%), whereas RF cells showed 20% efficiency. The cytogenetic analysis revealed a diploid count of 50 chromosomes. The cells of RF and RSB were found to be epithelial, where as the cells of RH were mostly fibroblastic. The viability of the RF, RH, and RSB cell lines was 75, 70 and 72%, respectively after 6 months of storage in liquid nitrogen. The origin of the cell lines was confirmed by the amplification of 496 and 655 bp fragments of 16S rRNA and Cytochrome Oxidase Subunit I (COI) of mtDNA. The new cell lines would facilitate viral disease diagnosis and genomic studies.

Development and characterization of a new epithelial cell line PSF from caudal fin of Green chromide, Etroplus suratensis (Bloch, 1790).

A new cell line [pearlspot fin (PSF)] has been developed from caudal fin of Etroplus suratensis, a brackish/freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10% fetal bovine serum (FBS). The PSF cell line consisted predominantly of epithelial-like cells. The cells were able to grow at temperatures between 25 degrees C and 32 degrees C with optimum temperature of 28 degrees C. The growth rate of PSF cells increased as the FBS proportion increased from 2% to 20% at 28 degrees C with optimum growth at the concentration of 10% FBS. One marine fish virus (fish nodavirus) was tested on this cell line and found not susceptible. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed epithelial-like morphology and reached confluency on the third d after subculture. Polymerase chain reaction amplification of mitochondrial 16S rRNA and COI indicated identity of this cell line with those reported from this fish species, confirming that the cell line was of pearlspot origin. The cells were successfully cryopreserved and revived at the tenth, 25th, and 35th passages. The bacterial extracellular products from Vibrio cholerae MTCC 3904 were found to be toxic to PSF. Karyotyping analysis indicated that the modal chromosome number was 48.

Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn)

Two new cell lines (CCF and CCH) were established from fin and heart tissues of common carp, Cyprinus carpio. The cells were optimally maintained in Leibovitz-15 medium supplemented with 10 percent fetal bovine serum (FBS) and 10 ng/ml of basic fibroblastic growth factor (bFGF). The effects of temperature, concentration of FBS and bFGF on the growth of CCF and CCH cells were examined. The temperature ranged from 24 to 32 °C for good growth of the cells. The growth rate of cells was higher in medium containing 10 percent FBS and the addition of bFGF to the medium significantly increased the growth rate. The CCF cells were found to be epithelial, while the CCH cells were fibroblastic in nature. The cytogenetic analysis of the cell lines revealed a diploid number of 100 chromosomes in C. carpio. The viability of CCF and CCH cell lines were 70 and 72 percent, respectively, after six months of storage in liquid nitrogen (-196 ° C). Molecular characterization of the cell lines using 16S rRNA and Cytochrome Oxidase Subunit I (COI) revealed the origin of the cell lines. These new cell lines will be useful for isolation of fish viruses and other in vitro biotechnological studies.