A comparative metabolomics study on anadromous clupeid Tenualosa ilisha for better understanding the influence of habitat on nutritional composition.

INTRODUCTION: Fish inhabiting different aquatic habitats adapts to the environment by metabolomic readjustments. Understanding the combined activities of all the metabolic pathways (metabolome) helps in better understanding the complex interactions between gene and environment. OBJECTIVES: The anadromous migratory Tenualosa ilisha is a high value food fish comprising the dominant fishery of the rivers Padma and Hooghly. The present study aimed at understanding the influence of the two habitats on the nutritional composition of hilsa. METHODS: Metabolite profiling was carried out by GC/MS. De novo assembly of hilsa liver transcriptome was generated under Illumina HiSeq platform and multivariate analysis was employed for correlation and comparison. RESULTS: GC/MS fingerprinting showed C16:0, C18:1, C20:5 and C22:6 to be the predominant fatty acids present in hilsa liver, which were also found to be significantly higher in Hooghly hilsa. Comparative transcriptome analysis revealed that the differentially expressed genes were mainly associated with 'lipid metabolism' and 'amino acid metabolism' pathways. Multivariate analysis between the metabolites amino acid, fatty acid and corresponding gene expression showed that few genes of amino acid metabolism (EZH1, ALAS2 and ALDH4A1) positively correlated with individual amino acids (lysine, glycine and glutamate) in Hooghly hilsa. Similarly, the key genes for LC-PUFA biosynthesis (ELOVL5, FADS2, CPT1) showed positive correlation with individual LC-PUFAs (C18:3, C20:4, C20:5, C22:6), indicating higher LC-PUFA biosynthesis potential in Hooghly hilsa. CONCLUSION: Comparative metabolomic study in hilsa from the two different habitats showed that the habitats influence the nutritional composition as evidenced by high abundance of amino acids lysine, leucine and arginine and LC-PUFAs C18:3, C20:4, C20:5, C22:6 in Hooghly hilsa.

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 profiling of white muscle from freshwater catfish Rita rita.

Muscle tissues contribute 34-48 % of the total body mass in fish. Proteomic analysis enables better understanding of the skeletal muscle physiology and metabolism. A proteome map reflects the general fingerprinting of the fish species and has the potential to identify novel proteins which could serve as biomarkers for many aspects of aquaculture including fish physiology and growth, flesh quality, food safety and aquatic environmental monitoring. The freshwater catfish Rita rita of the family Bagridae inhabiting the tropical rivers and estuaries is an important food fish with high nutritive value and is also considered a species of choice in riverine pollution monitoring. Omics information that could enhance utility of this species in molecular research is meager. Therefore, in the present study, proteomic analysis of Rita rita muscle has been carried out and functional genomics data have been generated. A reference muscle proteome has been developed, and 23 protein spots, representing 18 proteins, have been identified by MALDI-TOF/TOF-MS and LC-MS/MS. Besides, transcript information on a battery of heat shock proteins (Hsps) has been generated. The functional genomics information generated could act as the baseline data for further molecular research on this species.

Amino Acid Composition of Thirty Food Fishes of the Ganga Riverine Environment for Addressing Amino Acid Requirement through Fish Supplementation.

Amino acids are significant biomolecules that govern the major metabolic processes and act as precursors for macromolecules such as proteins that are crucial to life. Fish is an integral component of human nutrition and a dietary source of high-quality animal proteins and amino acids. In this context, the crude protein and amino acid compositions of food fish from different landing stations of the Ganga river have been determined. The Kjeldahl method was utilized to determine the crude protein content and the amino acids were analyzed using high-performance liquid chromatography (HPLC); data on 30 food fish were assessed. The study showed that among the fish studied, Eleotris fusca, Macrobrachium malcomsonii, and Mystus cavasius were rich in most of the amino acids important for human nutrition, such as glycine, glutamic acid, cysteine, threonine, phenylalanine, methionine, lysine, leucine, isoleucine, histidine, and valine. Further, it was observed that the daily consumption of these fish (approximately 50 g) can fulfil the daily requirement of these individual amino acids for an adult human with a body weight of 60 kg. Therefore, the amino acid composition analyzed in the present study could be utilized for recommendation by clinicians according to the requirement for specific amino acids, and fish can be prescribed as a natural supplement against the amino acid requirement.

Toxic Metal Element Concentration in 31 Food Fishes from River Ganga: Risk Assessment on Human Consumption.

Consumption of toxic metal contaminated fish is a significant risk to human health. The Ganga river is one of the vital river systems in India, and it nurtures a rich biodiversity of flora and fauna. In the present study, screening of potential toxic metal elements (Cd, Cr, Mn, Cu, Pb, Ni, Zn, and As) was undertaken in 31 food fishes, especially the small indigenous fishes (SIFs) from the lower stretch of river Ganga by inductively coupled plasma mass spectrometry (ICP-MS). The concentration of toxic metal elements varied among different fish species. Among the toxic metal elements studied, Cr, Zn, and As were found to be dominant in Eleotris fusca; Cd, Ni, and Pb were highest in Securicula gora; Cu was highest in Cabdio morar; and Mn was highest in Coilia dussumieri. The average pollution load index values (APLI) for all the fishes analyzed were less than one except for Eleotris fusca, which indicated no serious toxic metal element pollution load. The estimated daily intake values (EDI) for the toxic metal elements were found to be within the permissible maximum tolerable daily intake (MTDI). Among the fishes studied, only a few species showed hazard index (HI) greater than one, indicating non-carcinogenic health risks. Similarly, the target carcinogenic values (TCR) for most of the toxic metal elements were below the permissible limit (10-4) in the fishes that assures minimal cancer risk. This study provides a comprehensive data on the composition of potential toxic metal elements of 31 food fishes from the lower stretch of the river Ganga, the first of its kind, and suggests the necessity of periodic monitoring of these in the aquatic ecosystem.

Emerging contaminant triclosan incites endocrine disruption, reproductive impairments and oxidative stress in the commercially important carp, Catla (Labeo catla): An insight through molecular, histopathological and bioinformatic approach.

Triclosan (TCS), a broad-spectrum antimicrobial agent is ubiquitous in aquatic ecosystems; however, the mechanisms regarding TCS-induced reproductive toxicity in the teleost still remains uncertain. In this context, Labeo catla were subjected to sub-lethal doses of TCS for 30 days and variations in expression of genes and hormones comprising the hypothalamic-pituitary-gonadal (HPG) axis along with alterations in sex steroids were evaluated. Moreover, manifestation of oxidative stress, histopathological alterations, in silico docking and the potential to bioaccumulate were also investigated. Exposure to TCS may lead to an inevitable onset of the steroidogenic pathway through its interaction at several loci along the reproductive axis: TCS stimulated synthesis of kisspeptin 2 (Kiss 2) mRNAs which in turn prompts the hypothalamus to secrete gonadotropin-releasing hormone (GnRH), resulting in elevated serum 17ß-estradiol (E2) as a consequence; TCS exposure increased aromatase synthesis by brain, which by converting androgens to oestrogens may raise E2 levels; Moreover, TCS treatment resulted in elevated production of GnRH and gonadotropins by the hypothalamus and pituitary, respectively resulting in the induction of E2. The elevation in serum E2 may be linked to abnormally elevated levels of vitellogenin (Vtg) with harmful consequences evident as hypertrophy of hepatocytes and increment in hepatosomatic indices. Additionally, molecular docking studies revealed potential interactions with multiple targets viz. Vtg and luteinizing hormone (LH). Furthermore, TCS exposure induced oxidative stress and caused extensive damage to tissue architecture. This study elucidated molecular mechanisms underlying TCS-induced reproductive toxicity and the need for regulated use and efficient alternatives which could suffice for TCS.

Endocrine disruptive toxicity of cypermethrin in Labeo catla: Involvement of genes and proteins related to the HPG axis.

Cypermethrin (CYP) is a synthetic pyrethroid abundantly used in agriculture and aquaculture. It is an established potent endocrine disruptor to fish, yet the molecular mechanism behind its reproductive toxicity remains unclear. In this study, fish Labeo catla (Catla) was exposed to environmentally relevant concentration of CYP (0.7 µg/L) and 0.14 µg/L for 30 days. The changes in circulating sex steroids, genes, and hormones linked to the hypothalamic-pituitary-gonadal (HPG) axis, stress response and associated histological alterations were studied. Significant decline (P < 0.05) in serum 17 beta (ß) estradiol (E2), 11 ketotestosterone (11-KT), and brain (FSH and GnRH) were observed in 0.7 µg/L dose of CYP. These effects may be due to the down-regulated expression of the upstream genes of the HPG axis i.e. Kiss 1 and Kiss 2, which further downregulates the expression of the GnRH gene. The decreased level of E2 and 11-KT also affects the vitellogenin (Vtg) gene expression, reducing the production of Vtg, a crucial protein for ovarian development. Principal component analysis (PCA) revealed the relationship between CYP and the biosynthesis of sex steroids. The toxic effect of CYP was also visible in antioxidant enzyme assay and related histological alterations. Overall, the study elucidated that long-term exposure to CYP, even at an environmentally relevant dose, may affect reproductive potential and fish recruitment. The study provides important insights into molecular mechanisms underlying CYP-induced endocrine disruption in fish, and it also raises questions about CYP's potential toxicity at environmentally relevant concentration in terms of understanding ecological risk.

Sustainable biodiesel production from microalgae Graesiella emersonii through valorization of garden wastes-based vermicompost.

Biodiesel production from microalgae has gained significant interest recently due to the growing energy demand and non-renewable nature of petroleum. However, high cost of production and environmental health related issues like excess use of inorganic fertilizers, eutrophication are the major constraints in commercial-scale biodiesel production. Besides this, solid wastes (garden-based) management is also a global concern. In the present study, to overcome these limitations vermicompost extract was tested as nutrient source to enhance growth performance and lipid production from a freshwater microalga (Graesiella emersonii MN877773). Garden wastes were first converted into vermicompost manure and its extract (aerobic and anaerobically digested) was prepared. The efficacy of the extract was then tested in combination with BG11 medium. The mixotrophic cultivation of microalgae in anaerobically digested vermicompost extract at 50:50 combination with BG11 medium enhanced the cell biomass (0.64 g d. wt. L-1) and lipid productivity (3.18 mg L-1 day-1) of microalgae by two times. Moreover, the combination also improved the saturated (methyl palmitate) and monounsaturated fatty acids (oleic acid) content in the test algae. The quality of biodiesel also complies with all the properties of biodiesel standard provided by India, the USA, and Europe except the cold filter plugging property. The combination was also found to improve the cell biomass (0.041 g L-1) as compared to BG11 medium in mass-scale cultivation. Hence, the study proved that G. emersonii grown in media supplemented with garden waste-based vermicompost extract had significant potential for mass-scale biodiesel and bioproduct production.

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.

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.

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.

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.

Micronutrient Composition of 35 Food Fishes from India and Their Significance in Human Nutrition.

The micronutrients (vitamins and minerals) are required in small amounts but are essential for health, development, and growth. Micronutrient deficiencies, which affect over two billion people around the globe, are the leading cause of many ailments including mental retardation, preventable blindness, and death during childbirth. Fish is an important dietary source of micronutrients and plays important role in human nutrition. In the present investigation, micronutrient composition of 35 food fishes (includes both finfishes and shellfishes) was investigated from varying aquatic habitats. Macrominerals (Na, K, Ca, Mg) and trace elements (Fe, Cu, Zn, Mn, Se) were determined by either atomic absorption spectroscopy (AAS) or inductively coupled plasma mass spectrometry (ICP-MS)/atomic emission spectrometry (ICP-AES). Phosphorus content was determined either spectrophotometrically or by ICP-AES. Fat-soluble vitamins (A, D, E, K) were analyzed by high-performance liquid chromatography (HPLC). The analysis showed that, in general, the marine fishes were rich in sodium and potassium; small indigenous fishes (SIFs) in calcium, iron, and manganese; coldwater fishes in selenium; and the brackishwater fishes in phosphorous. The marine fishes Sardinella longiceps and Epinephelus spp. and the SIFs were rich in all fat-soluble vitamins. All these recommendations were made according to the potential contribution (daily value %) of the species to the recommended daily allowance (RDA). Information on the micronutrients generated would enhance the utility of fish in both community and clinical nutrition.

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.

Amino Acid compositions of 27 food fishes and their importance in clinical nutrition.

Proteins and amino acids are important biomolecules which regulate key metabolic pathways and serve as precursors for synthesis of biologically important substances; moreover, amino acids are building blocks of proteins. Fish is an important dietary source of quality animal proteins and amino acids and play important role in human nutrition. In the present investigation, crude protein content and amino acid compositions of important food fishes from different habitats have been studied. Crude protein content was determined by Kjeldahl method and amino acid composition was analyzed by high performance liquid chromatography and information on 27 food fishes was generated. The analysis showed that the cold water species are rich in lysine and aspartic acid, marine fishes in leucine, small indigenous fishes in histidine, and the carps and catfishes in glutamic acid and glycine. The enriched nutrition knowledge base would enhance the utility of fish as a source of quality animal proteins and amino acids and aid in their inclusion in dietary counseling and patient guidance for specific nutritional needs.