Trophic transfer patterns of arsenic in freshwater ecosystem layers in arsenic-endemic Ganges Delta and its potential human health risk.

Arsenic (As) is a toxic environmental contaminant with global public health concern. In aquatic ecosystems, the quantification of total As is restricted chiefly to the individual organisms. The present study has quantified the total As in different trophic layers (sediment-water-phytoplankton-periphyton-zooplankton-fish-gastropod-hydrophytes) of lentic freshwater ecosystems. As transfer pathways quantifying the transmission rate across trophic-level compartmental route were delineated using a novel model-based approach along with its potential contamination risk to humans. Lentic water bodies from Indo-Gangetic region, a core area of groundwater As, were selected for the present investigation. The study revealed that among the lower biota, zooplankton were the highest accumulator of total As (5554-11,564 µg kg-1) with magnification (rate = 1.129) of the metalloid, followed by phytoplankton (2579-6865 µg kg-1) and periphytic biofilm (1075 to 4382 µg kg -1). Muscle tissue of zooplanktivore Labeo catla is found to store higher As (80-115 µg kg-1 w.w.) compared to bottom-dwelling omnivore Cirrhinus mrigala (58-92 µg kg-1 w.w.). Whereas, Amblypharyngodon mola has accumulated higher As (203-319 µg kg-1 w.w.) than Puntius sophore (30-98 µg kg-1 w.w.) that raised further concern. The hepatic concentration indicated arsenic-mediated stress based on As stress index (threshold value = 1). Mrigal and Mola showed significant biomagnification among fishes while biodiminution was observed in Catla, Bata, Rohu and Punti. All the studied fishes were under the arsenic mediated stress. In the 'sediment-water-periphytic biofilm-gastropod' compartment, the direct grazing accumulation was higher (rate = 0.618) than the indirect path (rate = 0.587). Stems of edible freshwater macrophytes accumulated lesser As (32-190 µg kg-1 d.w.) than roots (292-946 µg kg-1 d.w.) and leaves (62-231 µg kg-1 d.w.). The target cancer risk (TCR) revealed a greater concern for adults consuming edible macrophyte regularly. Similarly, the varied level of target hazard quotient and TCR for adults consuming fishes from these waterbodies further speculated significant health concerns. The trophic transfer rate of environmental As in soil-water-biota level at an increasing trophic guild and consumer risk analysis have been unravelled for the first time in the Indo-Gangetic plains, which will be helpful for the strategic mitigation of As contamination.

Brewer's spent yeast replacement in carp diet leads to muscle biomass production, recycling, waste management and resource conservation.

Brewer's spent yeast (BSY) is among the most voluminous by-products generated in brewery industry that adds to the waste; however, smart utilization of BSY could lead to edible biomass production besides waste management. To utilize it for biomass production, it is being used in fish feeds; however, its effect on the fish physiology has been scantily studied. The present study investigated the proteomic changes in muscle tissues of carp Labeo rohita fed with BSY-based diet, to understand its impact on muscle physiology and biomass. Six feeds were prepared with different grades of BSY (0, 20, 30, 40, 50, 100% replacement of fishmeal with BSY) and fishes were fed for 90 days. Highest weight gain%, feed conversion efficiency, specific growth rate% were observed in 30% BSY-replaced group and this group was considered for the proteomic study. Comparative shotgun proteomic analysis was carried out by LC-MS/MS and data generated have been deposited in ProteomeXchange Consortium with dataset identifier PXD020093. A total of 62 proteins showed differential abundance; 29 increased and 33 decreased in the 30% BSY-replaced group. Pathway analysis using IPA and Panther tools revealed that the proteins tyrosine protein kinase, PDGFα, PKRCB and Collagen promote muscle growth by inducing the PI3K-AKT pathway. Conversely, the proteins Serine/threonine-protein phosphatase, Phosphatidylinositol 3,4,5-trisphosphate5-phosphatase 2A and Ras-specific guanine- nucleotide-releasing factor inhibit muscle growth indicating that 30% BSY-replaced feed promote muscle growth in a highly controlled manner. Findings suggest that BSY could be recycled for carp feed production in large scale thereby leading to resource conservation, reducing environmental effects.

Proteomic Profiling and Pathway Analysis of Acid Stress-Induced Vasorelaxation of Mesenteric Arteries In Vitro.

Although metabolic acidosis is associated with numerous pathophysiological conditions and its vasorelaxation effects have been well described in different animal and culture models, the molecular mechanisms of acidosis-induced vasorelaxation are not fully understood. Mesenteric artery models have been used extensively to examine the vascular response to various pathophysiological conditions. Our previous studies and several other reports have suggested the vascular responses of goat mesenteric arteries and human arteries to various stimuli, including acidic stress, are highly similar. In this study, to further identify the signaling molecules responsible for altered vasoreactivity in response to acidic pH, we examined the proteomic profile of acid stress-induced vasorelaxation using a goat mesenteric artery model. The vascular proteomes under acidic pH were compared using 2D-GE with 7 cm IPG strips and mini gels, LC-MS/MS, and MALDI TOF MS. The unique proteins identified by mass spectroscopy were actin, transgelin, WD repeat-containing protein 1, desmin, tropomyosin, ATP synthase ß, Hsp27, aldehyde dehydrogenase, pyruvate kinase, and vitamin K epoxide reductase complex subunit 1-like protein. Out of five protein spots identified as actin, three were upregulated > 2-fold. ATP synthase ß was also upregulated (2.14-fold) under acid stress. Other actin-associated proteins upregulated were transgelin, desmin, and WD repeat-containing protein 1. Isometric contraction studies revealed that both receptor-mediated (histamine) and non-receptor-mediated (KCl) vasocontraction were attenuated, whereas acetylcholine-induced vasorelaxation was augmented under acidosis. Overall, the altered vasoreactivity under acidosis observed in the functional studies could possibly be attributed to the increase in expression of actin and ATP synthase ß.

Arsenic Bioaccumulation and Identification of Low-Arsenic-Accumulating Food Fishes for Aquaculture in Arsenic-Contaminated Ponds and Associated Aquatic Ecosystems.

Arsenic-contaminated food including farmed fish is one of the main routes of human exposure. Fish farmed in contaminated environment accumulates arsenic in different tissues with great variability. Thus, it is utmost important to quantify the risk associated with different farmed fish species in arsenic-contaminated aquaculture systems. In the present study, arsenic content was measured in twelve fish species (Labeo rohita, L. catla, Cirrhinus mrigala, Oreochromis niloticus, O. mossambicus, Liza tade, Puntius javanicus, L. calbasu, Glossogobius giuris, Macrobrachium rosenbergii, Ctenopharyngodon idella, and Bellamya bengalensis (gastropod)) collected from arsenic-contaminated aquaculture systems. Among the studied finfishes, C. idella was found to accumulate the lowest amount of arsenic (< 0.05 ± 0.00 mg kg-1) whereas the highest accumulation was noticed in O. mossambicus (1.0 ± 0.18 mg kg-1). However, the estimated carcinogenic and non-carcinogenic risks of human were found to be low for all the studied fishes. The calculated target hazard quotient (THQ) value for adults ranged from 0.01 to 0.08 whereas for children it ranged from 0.05 to 0.27 for low-arsenic-accumulating fishes (arsenic conc. < 0.5 mg kg-1). Based on these findings, C. mrigala, C. idella, and M. rosenbergii could be recommended as the candidate species for aquaculture in the arsenic-contaminated areas as farming of the low-arsenic-accumulating food fishes would also lower the risk of human exposure through food chain.

Culture-dependent study of arsenic-reducing bacteria in deep aquatic sediments of Bengal Delta.

Biogeochemical release of soil-bound arsenic (As) governs mobilization of the toxic metalloid into the groundwater. The present study has examined AsV-reduction ability of bacteria from anoxic aquatic sediments that might contribute to arsenic mobilization in the Bengal Delta. Arsenic-reducing bacteria from deep layers of pond sediment were enriched and isolated in anaerobic environments and AsV reduction was assessed in culture medium. The pond sediment enrichments harboured AsV-reducing bacteria belonging to the phyla Firmicutes and Proteobacteria with dominance of Paraclostridium benzoelyticum and P. bifermentans. Among total 17 isolates, the respiratory reductase genes were not detected by the most common primers and only 3 strains had arsenic reductase ArsC gene suggesting involvement of resistance and some unknown mechanisms in AsV reduction. Presence of high levels of organic matter, As, and As-reducing bacteria might make deep aquatic sediments a hot spot of As mobilization and aquifer contamination.

Curcumin Has Protective Effect on the Eye Lens Against Arsenic Toxicity.

Arsenic is a highly carcinogenic environmental contaminant. Curcumin, the bioactive component of turmeric, exhibits therapeutic efficacy against several chronic inflammatory and infectious diseases. The present study was carried out to investigate the impact of arsenic on eye lens and evaluate the ameliorative potential of curcumin against arsenic toxicity. Gene expression analysis of α, ß, and γ-crystallins and fatty acid profile of lens tissues of arsenic-exposed Labeo rohita was examined and the protective effect of curcumin as diet supplement was evaluated. Curcumin-supplemented diet was prepared at 1.5% and 3% and fed to four groups of fish for 7 days prior to arsenic exposure (at 5 ppm and 15 ppm) for 15 days. Gene expression analysis showed downregulation of α and ß-crystallins in the eye lens of arsenic-exposed groups (fed basal diet), whereas the groups fed a curcumin-supplemented diet showed insignificant alterations. Similarly, fatty acid fingerprint of lens lipids arsenic-exposed group exhibited reduction in saturated fatty acid and docosahexaenoic acid (DHA) content. However, in 3% curcumin-supplemented diet-fed and arsenic exposed group group, fatty acid profile remained unchanged. Interestingly, concentration of one non-fatty acid, an antioxidant compound (phenol 2,4-bis 1,1 dimethyl; PD) that was identified in the GC-MS fingerprinting through NIST library (version 2.2, 2014), decreased in response to arsenic exposure which was restored to normal level in curcumin-supplemented groups proving the therapeutic potential of curcumin. The findings of the study suggest that curcumin has a protective effect on eye lens against arsenic toxicity.

Transcriptomic responses to pollution in natural riverine environment in Rita rita.

Aquatic pollution is one of the most common threats to the ecological health of aquatic ecosystems and its biota. Fish as lower vertebrates are excellent model to study the impact and responses of aquatic pollution. In fish, gill is the main organ indicator of whole animal health as it comes in contact with the surrounding water and absorbs many pollutants and contaminants; therefore, investigations on alterations in fish gill at transcriptome level could provide newer insights to the stress response mechanism(s) and pathways. For comprehensive evaluation of the impacts of pollutants (joint toxicity) prevalent in the riverine environment, comparative transcriptome analysis, by Next Generation Sequencing under Illumina HiSeq 2500 platform, was carried out in gill tissues of Rita rita collected from two stretches of river Ganga (Kanpur and Farakka) and results were validated by RT-qPCR. Out of 154,077 unigenes (Accession SRR548008), a total of 2024 differentially expressed genes (DEGs) including 942 up-regulated and 1082 down-regulated genes were identified by DESeq program. Further, Gene Ontology (GO) of DEGs showed that ribosomal large subunit biogenesis, mitochondrial ribosome and box H/ACA SnoRNA binding categories are highly affected by pollution. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis showed the involvement of the DEGs in energy metabolism, translational and transcriptional machinery, protein folding and degradation suggesting that these signalling pathways are highly affected by aquatic pollution. Among the DEGs, up-regulation of cytochrome c oxidase subunit (cox) 7a2 (69.47 fold), hsp70 subunit 14 (hsp70-14, 5.27 fold), muscle related coiled-coil protein (MURC, 21.55 fold), lysozyme G (40.14 fold), cox17 (29.36 fold) were the conspicuous ones which showed similar trends in expression when analysed by RT-qPCR. Based on fold change, perturbation values, correlation analysis by PCA and RT-qPCR validation, up-regulation of cox7a2, MURC and hsp70-14 appeared to be the most promising biomarker responses and could be useful in the evaluation of gill health and possibly be extended towards aquatic ecosystem health assessment.

Proteomic and transcriptomic changes in rat liver following oral feeding of formaldehyde.

Formaldehyde (FA), a ubiquitous volatile organic compound present in a wide range of resources, is a hazardous chemical and human carcinogen. Contamination of FA in food, especially perishable commodities like fish and meat, is a major source of exposure, although it is not recommended for use in food and food products owing to its carcinogenicity. Effects of oral feeding of FA have been studied by evaluating general health, haematology and clinical chemistry in rat. Recent studies have shown that FA exposure leads to detrimental cardiovascular effects. It regulates vascular tensions through nitric oxide-cGMP signalling pathway and ion channels in rats. Although FA is an established carcinogen, molecular studies on carcinogenic potential with dose dependency are meagre. In this context, the present study was undertaken to investigate the toxicogenomic and proteomic alterations in liver of rats fed FA through drinking water. By proteomic analysis, 621 proteins/protein-subunits showed differential abundance (proteome data available via ProteomeXchange with identifier PXD010534), whereas 536 differentially-expressed-genes were identified by transcriptome analysis (data available via Sequence Read Archive with identifier SRR7974113). Gene ontology analysis showed that binding, catalysis, signal transduction were affected in formaldehyde-fed rats. Pathway analysis revealed that formaldehyde-exposure activated PI3K-AKT pathway that leads to inhibition of caspase activity thereby assisting cells to survive against apoptosis. Decreased abundance/down-regulation of ANGPT, eNOS, STAT3 proteins/transcripts and increased abundance of EDN1 indicated decrease in angiogenesis and vasodilatation that restricted hepatic cells from becoming tumorigenic; thus, indicating FA could be less toxic and non-tumorigenic at low concentrations.

Impact assessment of barge trafficking on phytoplankton abundance and Chl a concentration, in River Ganga, India.

Impact of barge movement on phytoplankton abundance and biomass was assessed in the lower stretch of river Ganga, popularly known as Bhagirathi-Hooghly river, during April 2016 to March, 2017. Based on the magnitude of tide, intensity of shipping and boating activities, the stretch from Baranagar to Lalbag (278 km), located at latitude (22°38'33.41"N to 24°10'59.75"N) and longitude (88°21'21.29"E to 88°16'5.65"E) was divided into three zones viz. zone-I (Baranagar to Barrackpore), zone II (Triveni to Balagarh) and zone III (Nabadweep to Lalbag). Water samples were collected randomly from six stations covering 22 barge movements at their passage at three different time intervals viz., 30 minutes before 'barge movement', during 'barge movement' and 30 minutes after 'barge movement'. Analysis revealed the presence of 52 phytoplankton taxa belonged to 5 phylum during the study period. The abundance of phytoplankton was highest in zone-I followed by zone III and the zone II. A 44% decrease (1,997 ±1,510 ul-1) in phytoplankton abundance was observed during 'barge movement' with respect to normal condition (3,513 ± 2,239 ul-1) which could be due to propeller turbulence in the passage. Cell damage study revealed 21% damage in phytoplankton cell structure in 'during barge' followed by 'after barge' (10%) condition compared to natural state (6%). Study revealed that phytoplankton biomass (Chlorophyll a) was influenced by 'barge movement' in the sampling stretches and the impact was assessed by one way ANOVA. The effect was found significant at Barrackpore (p <0.01), Triveni (p <0.01), Balagarh (p <0.01) and Lalbag (p <0.01) where as it was insignificant at Baranagar and Nabadweep, which may be due to continuous and existing boat trafficking at Baranagar and Nabadweep. Two way ANOVA computed using 'barge movement' and sampling stations showed significant (p<0.01) effect on magnitude of Chl a concentrations in the sampling locations. Thus, the 'barge movement' influenced phytoplankton abundance and biomass, it had a detrimental effect on phytoplankton cell architecture also. The data set of this work serves as foundation information to understand the ecological implications augmented barge induced environmental disturbances in waterways. This is the first such study which depicts the impact of 'barge movement' on aquatic food chain linkages in Bhagirathi- Hooghly river.

Nutritional composition of food fishes and their importance in providing food and nutritional security.

Fish is a healthy food, rich in quality animal proteins, polyunsaturated fatty acids especially the (ω)-3 eicosapentaenoic acid and docosahexaenoic acid and micronutrients. In addition, fish are more available and affordable than other sources of animal proteins in tropical countries. Aquaculture, which is one of the fastest growing food production sectors, could play a big role in eradicating hunger, malnutrition and nutrient-deprivation globally. However, nutritional information on fish is necessary for utilization of fish in achieving nutritional security and will be helpful in prioritizing species for aquaculture. In this context, we have studied the detailed nutritional composition of selected fishes from India and developed a database (http://www.cifri.res.in/nutrifishin/index.php) with the food data generated. This review explore the implications of such nutritional information in consumer guidance, dietary counselling, food-policy planning and prioritization of species for aquaculture to fight hunger, malnutrition and micronutrient deficiency; ultimately contributing to food and nutritional security.

Heat stress-induced alterations in the expression of genes associated with gonadal integrity of the teleost Puntius sophore.

Temperature plays an important role on reproductive physiology of vertebrates including mammals, fish, and birds. It has varying effects on fish reproduction depending on the species; higher temperatures favor the spring-spawning species, while lower temperatures stimulate reproduction in autumn spawners. To evaluate the impact of high temperature on the reproductive physiology of minnow Puntius sophore, we carried out expression analysis of selected genes associated with gamete quality (hsp60, hsp70, hsp90, hsf1, vtg), pleuripotency (sox2, oct4, nanog), and sex determination (dmrt1) in gonads (ovary and testis) of P. sophore, heat stressed for different time periods (36 °C/7 days or 60 days) using real-time quantitative polymerase chain reaction (RT-qPCR). Expression of most of the hsp, vtg, and pleuripotency marker genes sox-2, oct-4, and nanog genes was downregulated in both ovary and testis of heat-stressed fish. The expression of dmrt-1 was upregulated in testis but downregulated in ovary of the heat-stressed fish which could be a male favoring effect of high temperature in P. sophore. This study suggests that the reproductive physiology and health of the nutrient dense P. sophore would be negatively affected by high temperature stress.

Expression patterns of heat shock protein genes in Rita rita from natural riverine habitat as biomarker response against environmental pollution.

River pollution is one of the principal environmental concerns and biomonitoring tools can play an important role in pollution assessment in the riverine environment. Heat shock proteins (Hsps) have been found to be suitable tools for monitoring stress response. In the present study, expression analyses of hsp genes (hsp27, hsp47, hsp60, hsp70, hsc70, and hsp90) and selected hsp-regulatory genes (hsf1, hyou1, ask1, jnk) were carried out by RT-qPCR in catfish Rita rita collected from selected stretches of river Ganga to investigate changes in their expression patterns as biomarker response. Water quality characteristics were measured in terms of physico-chemical characteristics (DO, BOD, COD, pH, conductivity), element profile (arsenic, mercury, cadmium, lead, chromium, zinc, copper) and persistent organic pollutants (POPs; HCH, DDT, aldrin, endosulphan, heptachlor). Water quality index was calculated and sampling sites were categorized as good/medium/bad. Multivariate analysis was carried out taking the water quality parameters and the fold changes in hsp gene expression as variables, which showed that hsp47 and hsp70b correlated well with BOD, an indicator of organic pollution. To identify the organic pollutant(s) which could be influencing the expression of hsps, again multivariate analysis was employed taking concentration of POPs and fold changes of hsps, which showed up-regulation of hsp47 and hsp70b (HSP72i) correlated well with concentrations of aldrin and HCH. Synergistic effects of these POPs could be responsible for the up-regulation of said hsps, although individually present in low concentration; thus, indicating synergistic effect of the POPs on hsp47 and hsp70b up-regulation as biomarker response.

Expression patterns and mutation analysis of p53 in fish Rita rita from polluted riverine environment.

The present study was undertaken to investigate the alterations in gene expression patterns and for mutation analysis of p53 in the riverine catfish Rita rita collected from polluted riverine habitat. The partial p53 gene sequence of Rita rita generated showed a high degree of similarities with the DNA binding domains of fishes, mice and human. Transcriptomic analysis, carried out by quantitative real-time Polymerase Chain Reaction (RT-qPCR), showed significant down-regulation of p53 in fishes collected from most of the polluted stretches. Similar trend in protein abundance was observed by western blot analysis. Down-regulation of p53 was more pronounced in gill than liver. Expression patterns of p53 suggest that exposure to a multitude of contaminants in the natural riverine ecosystem could suppress the expression of p53. Genomic DNA showed a low stained smear pattern upon electrophoresis, with no evidence of DNA fragmentation. For mutation analysis PCR-SSCP followed by sequence analysis was carried out, which identified eight mutations; two at codon level and six missense mutations in the DNA binding domain IV and V. Secondary structure prediction showed that these mutations could lead to impairment of protein structure. Thus, the present study indicated that aquatic pollution has impacted these lower vertebrates which are reflected by the down-regulation of tumor suppressor protein (p53) in majority of the stretches studied.

Nutrigenomic studies on hilsa to evaluate flesh quality attributes and genes associated with fatty acid metabolism from the rivers Hooghly and Padma.

The Indian shad hilsa (Tenualosa ilisha), a commercially important food fish rich in oils, enjoys high consumer preference in the South Asian countries owing to its unique flavour and culinary properties. The present study was undertaken with the primary objective of determining the flesh quality attributes of hilsa in terms of nutritive value (gross chemical composition, amino acid, fatty acid and mineral composition), pH, water holding capacity (WHC) and expression of genes associated with fatty acid metabolism and flesh quality. Additionally, comparative studies on the flesh quality attributes in hilsa from two distributaries of river Ganga i.e. Hooghly and Padma were also carried out. A high WHC (>80%) suggested juicy and tender nature of hilsa meat. The protein content was 18-21% in hilsa from both the rivers and essential amino acid lysine, valine and functional amino acids leucine and arginine were significantly higher in Hooghly hilsa (P<0.05). The predominance of umami taste amino acids, glutamic acid and aspartic acid and sweet taste amino acids, serine, glycine and alanine in hilsa from both the rivers could be the contributing factors to its unique flavour. The fat content in hilsa from river Hooghly and Padma were found to be 9.94 and 7.84%, respectively. The concentration of flavouring fatty acids like saturated fatty acids (SFA) (myristic acid) and omega (ω)-3 polyunsaturated fatty acids (PUFAs) (linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) were significantly higher in Hooghly hilsa (P<0.05). Among the genes associated with fatty acid metabolism studied, expression of cluster of differentiation (CD36), acetyl CoA oxidase (ACO), fatty acid synthase (FAS), peroxisome proliferator activated receptor beta (PPARß), peroxisome proliferator activated receptor gamma (PPARγ) and desaturase were significantly higher in Padma hilsa (P<0.05), and the change was <2 fold. Comparative gene expression profiling of flesh quality genes (actin, GAPDH, LDH, TPI) showed similar levels of expression in hilsa from both the rivers (P<0.05). The nutrigenomic information generated on various flesh quality attributes of hilsa has enriched the knowledgebase. Further, from comparative nutrient analysis on hilsa from river Hooghly and Padma, it was observed that Hooghly hilsa is superior in terms of oil content, ω-3 PUFAs EPA and DHA and essential amino acids; however, the expression profile of genes associated with flesh quality were found to be similar. Thus, within the scope of the present study, Hooghly hilsa (medium size category, 500-700g size) was found to be nutritionally superior.

Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress.

BACKGROUND: High ambient temperature is known to affect fish gonadal development and physiology in a variety of ways depending on the severity and duration of exposure; however, the underlying molecular mechanisms are poorly understood. Gonadal gene expression influence the gonadal development, physiology and the quality of egg/sperm produced in teleosts and the mechanistic understanding of spatio-temporal changes in the gonadal gene expression could be instrumental in controlling the fate of egg/sperm and the quality of seed produced. Real time-quantititative polymerase chain reaction (RT-qCR), is a high throughput, sensitive and reproducible methodology used for understanding gene expression patterns by measuring the relative abundance of mRNA transcripts. However, its accuracy relies upon a suitable reference gene whose expression levels remain stable across various experimental conditions. In the present study, we evaluated the suitability of ten potential reference genes to be used as internal controls in RT-qPCR analysis in gonadal tissues (ovary and testis) of minnow Puntius sophore exposed to high temperature stress for different time periods (7 days, 60 days). Expression analysis of ten different constitutively expressed genes viz. 18S ribosomal RNA (18S rRNA), beta actin (ßactin), ß-2 microglobulin (b2mg), eukaryotic elongation factor-1 (eef1), glyceraldehyde-3phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), ribosomal binding protein L13 (rpl13), tubulin (tub), tata box binding protein (tbp), ubiquitin (ubi) was carried out by using RT-qPCR and the stability in their expressions were evaluated by using four different algorithms; namely, delta Ct, BestKeeper, geNorm and NormFinder. RESULTS: In ovary, eef1 was found to be the most suitable reference gene in all the algorithms used. In testis, b2mg was found to be the most suitable reference gene in delta Ct, BestKeeper, NormFinder analysis while tbp and eef1 were found to be the most suitable reference genes in geNorm analysis. CONCLUSIONS: In conclusion, eef1 and b2mg were found to be the most suitable reference genes in ovary and testis, respectively, of Puntius sophore exposed to high temperature stress, and could be used as internal controls for gene expression analysis in gonadal tissues of Puntius sophore.

Identification of potential biomarkers of hepatotoxicity by plasma proteome analysis of arsenic-exposed carp Labeo rohita.

Arsenic (As) is a toxic environmental contaminant and potential human carcinogen. Chronic intake of arsenic-contaminated water and food leads to arsenicosis, a major public health problem in many parts of the world. Early detection of arsenic toxicity would greatly benefit patients; however, the detection of arsenicosis needs to be done early before onset of severe symptoms in which case the tools used for detection have to be both sensitive and reliable. In this context, the present study investigated plasma proteome changes in arsenic-exposed Labeo rohita, with the aim of identifying biomarkers for arsenicosis. Changes in the plasma proteome were investigated using gel-based proteomics technology. Using quantitative image analysis of the 2D proteome profiles, 14 unique spots were identified by MALDI-TOF/TOF MS and/or LC-MS/MS which included Apolipoprotein-A1 (Apo-A1) (6 spots), α-2 macroglobulin-like protein (A2ML) (2 spots), transferrin (TF) (3 spots) and warm-temperature acclimation related 65kDa protein (Wap65). The proteome data are available via ProteomeXchange with identifier PXD003404. Highly abundant protein spots identified in plasma from arsenic-exposed fish i.e. Apo-A1 (>10-fold), A2ML (7-fold) and Wap65 (>2-fold) indicate liver damage. It is proposed that a combination of these proteins could serve as useful biomarkers of hepatotoxicity and chronic liver disease due to arsenic exposure.

hsp90 and hsp47 appear to play an important role in minnow Puntius sophore for surviving in the hot spring run-off aquatic ecosystem.

Changes in the expression of a number of hsp genes in minnow Puntius sophore collected from a hot spring run-off (Atri hot spring in Odisha, India; 20o09'N 85°18'E, 36-38 °C) were investigated to study the upper thermal acclimation response under heat stress, using same species from aquaculture ponds (water temperature 27 °C) as control. Expression of hsp genes was analyzed in both groups using RT-qPCR, which showed up-regulation of hsp90 (2.1-fold) and hsp47 (2.5-fold) in hot spring run-off fishes, whereas there was no alteration in expression of other hsps. As the fish inhabit the hot spring run-off area for very long duration, they could have adapted to the environment. To test this hypothesis, fishes collected from hot spring run-off were divided into two groups; one was heat-shocked at 41 °C/24 h, and the other was acclimatized at 27 °C/24 h. Up-regulation of all the hsps (except hsp78) was observed in the heat-shocked fishes, whereas expression of all hsps was found to be down-regulated to the basal level in fishes maintained at 27 °C/24 h. Pathway analysis showed that the expressions of all the hsps except hsp90 are regulated by the transcription factor heat shock factor 1 (Hsf1). This study showed that hsp90 and hsp47 play an important role in Puntius sophore for surviving in the high-temperature environment of the hot spring run-off. Additionally, we show that plasticity in hsp gene expression is not lost in the hot spring run-off population.

DHA and EPA Content and Fatty Acid Profile of 39 Food Fishes from India.

Docosahexaenoic acid (DHA) is the principal constituent of a variety of cells especially the brain neurons and retinal cells and plays important role in fetal brain development, development of motor skills, and visual acuity in infants, lipid metabolism, and cognitive support and along with eicosapentaenoic acid (EPA) it plays important role in preventing atherosclerosis, dementia, rheumatoid arthritis, Alzheimer's disease, and so forth. Being an essential nutrient, it is to be obtained through diet and therefore searching for affordable sources of these ω-3 polyunsaturated fatty acids (PUFA) is important for consumer guidance and dietary counseling. Fish is an important source of PUFA and has unique advantage that there are many food fish species available and consumers have a wide choice owing to availability and affordability. The Indian subcontinent harbors a rich fish biodiversity which markedly varies in their nutrient composition. Here we report the DHA and EPA content and fatty acid profile of 39 important food fishes (including finfishes, shellfishes, and edible molluscs from both marine water and freshwater) from India. The study showed that fishes Tenualosa ilisha, Sardinella longiceps, Nemipterus japonicus, and Anabas testudineus are rich sources of DHA and EPA. Promotion of these species as DHA rich species would enhance their utility in public health nutrition.

Proteomic changes in the liver of Channa striatus in response to high temperature stress.

The present study was undertaken to investigate the proteomic changes in the liver of murrel Channa striatus exposed to high temperature stress. Fishes were exposed to 36°C for 4 days and liver proteome changes were analyzed using gel- based proteomics, i.e. 2DE, MALDI-TOF/TOF-MS, and validation by transcript analysis. The study showed, besides others, increased abundance of two sets of proteins, the antioxidative enzymes superoxide dismutase (SOD), ferritin, cellular retinol binding protein (CRBP), glutathione-S-transferase (GST), and the chaperones HSP60 and protein disulfide isomerase; this was validated by transcript analysis. The proteome data are available via ProteomeXchange with identifier PXD002608. Further, gene expression analysis was also carried out in the fishes exposed to thermal stress for longer durations (30 days experimental exposure in laboratory and for 30 days beyond, taking Channa collected from a hot spring runoff at 36-38°C); sod, gst, crbp, and hsp60 were found to continue to remain upregulated at eight-, 2.5-, 2.4-, and 2.45-fold, respectively, in the hot spring runoff fish. Pathway analysis showed that the upregulations of the antioxidant enzymes as well as molecular chaperones are induced by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Thus, while short-term heat stress tolerance involves the antioxidative enzymes SOD, ferritin, CRBP, GST, and chaperones HSP60 and protein disulfide isomerase, adaptation under chronic heat stress is associated with SOD, CRBP, GST, and HSP60.

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress.

Quantitative real-time polymerase chain reaction is the most advanced method of quantifying gene expression studies; however, the significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. In the present study, expression analysis of six different constitutively expressed genes viz. 18S ribosomal RNA, glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta actin (ßactin), ribosomal binding protein L13, tubulin and TATA-box-binding protein (tbp) were carried out to test their efficacy as reference genes in three different tissues, namely liver, gill and muscle of murrel Channa striatus exposed to high temperature for variable time periods. The stability and suitability of the genes were determined by using bioinformatic tools: GeNorm, NormFinder and BestKeeper. Based on the results, tub/ßactin could be used as the reference genes for liver and gill tissues and ßactin/gapdh could be the reference genes for muscle tissues in Channa striatus under both short- and long-term thermal stress.