Fatty acid profile variations after exposure to textile industry effluents in Indian Major Carps

Abstract Indian major carps are the widely consumed fish species of Pakistan, being a cheap source of proteins and unsaturated fatty acids, they are good for cardiovascular health. Water pollution due to discharge of untreated industrial waste water into water bodies contaminates this precious source of nutrients. The present study therefore, was aimed to assess deterioration of fatty acid profile of three Indian major carp species due to different concentrations of industrial wastes. The water samples were collected from the river Chenab at the site where it receives industrial wastewater via Chakbandi drain. After exposure to 1.5%, 3.0%, and 4.5% dilutions of collected water in different aquaria it was observed that proportion of unsaturated fatty acids in selected fish species were decreased significantly as the intensity of the dose increased (P 0.05). Conversely the level of saturated fatty acids increased with the increasing dose of treatment (P 0.05). These findings suggest that untreated wastewater not only deteriorate the fatty acid profile of aquatic animals but also these toxic substances can reach human body through fish meat and pose further health hazards. Therefore, it is highly recommended that industrial effluents should be treated before they are dumped into water bodies.

Resumo As carpas indianas são as espécies de peixes mais consumidas no Paquistão, sendo uma fonte barata de proteínas e de ácidos graxos insaturados e boa para a saúde cardiovascular. A poluição da água por causa do descarte de resíduos industriais não tratados em corpos dágua contamina essa preciosa fonte de nutrientes. Portanto, o presente estudo teve como objetivo avaliar a deterioração do perfil de ácidos graxos de três principais espécies de carpas indianas em diferentes concentrações de resíduos industriais. As amostras de água foram coletadas do rio Chenab no local onde recebe esgoto industrial via dreno de Chakbandi. Após a exposição a diluições de 1,5%, 3% e 4,5% da água coletada em diferentes aquários, foi observado que a proporção de ácidos graxos insaturados em espécies de peixes selecionadas diminuiu significativamente com o aumento da intensidade da dose (P 0,05). Por outro lado, o nível de ácidos graxos saturados aumentou com a elevação da dose de tratamento (P 0,05). Essas descobertas sugerem que águas residuais não tratadas não apenas deterioram o perfil de ácidos graxos dos animais aquáticos, mas também essas substâncias tóxicas podem atingir o corpo humano por meio da carne de peixe e representar mais riscos à saúde. Portanto, é recomendável que os efluentes industriais sejam tratados antes de serem despejados em corpos dágua.

Fatty acid profile variations after exposure to textile industry effluents in Indian Major Carps / Variações do perfil de ácidos graxos após exposição a efluentes da indústria têxtil nas principais carpas indianas

Abstract Indian major carps are the widely consumed fish species of Pakistan, being a cheap source of proteins and unsaturated fatty acids, they are good for cardiovascular health. Water pollution due to discharge of untreated industrial waste water into water bodies contaminates this precious source of nutrients. The present study therefore, was aimed to assess deterioration of fatty acid profile of three Indian major carp species due to different concentrations of industrial wastes. The water samples were collected from the river Chenab at the site where it receives industrial wastewater via Chakbandi drain. After exposure to 1.5%, 3.0%, and 4.5% dilutions of collected water in different aquaria it was observed that proportion of unsaturated fatty acids in selected fish species were decreased significantly as the intensity of the dose increased (P < 0.05). Conversely the level of saturated fatty acids increased with the increasing dose of treatment (P < 0.05). These findings suggest that untreated wastewater not only deteriorate the fatty acid profile of aquatic animals but also these toxic substances can reach human body through fish meat and pose further health hazards. Therefore, it is highly recommended that industrial effluents should be treated before they are dumped into water bodies.

Resumo As carpas indianas são as espécies de peixes mais consumidas no Paquistão, sendo uma fonte barata de proteínas e de ácidos graxos insaturados e boa para a saúde cardiovascular. A poluição da água por causa do descarte de resíduos industriais não tratados em corpos d'água contamina essa preciosa fonte de nutrientes. Portanto, o presente estudo teve como objetivo avaliar a deterioração do perfil de ácidos graxos de três principais espécies de carpas indianas em diferentes concentrações de resíduos industriais. As amostras de água foram coletadas do rio Chenab no local onde recebe esgoto industrial via dreno de Chakbandi. Após a exposição a diluições de 1,5%, 3% e 4,5% da água coletada em diferentes aquários, foi observado que a proporção de ácidos graxos insaturados em espécies de peixes selecionadas diminuiu significativamente com o aumento da intensidade da dose (P < 0,05). Por outro lado, o nível de ácidos graxos saturados aumentou com a elevação da dose de tratamento (P < 0,05). Essas descobertas sugerem que águas residuais não tratadas não apenas deterioram o perfil de ácidos graxos dos animais aquáticos, mas também essas substâncias tóxicas podem atingir o corpo humano por meio da carne de peixe e representar mais riscos à saúde. Portanto, é recomendável que os efluentes industriais sejam tratados antes de serem despejados em corpos d'água.

Isolation of Aeromonas salmonicida subspecies salmonicida from aquaculture environment in India: Polyphasic identification, virulence characterization, and antibiotic susceptibility.

This study reports the polyphasic identification, characterization of virulence potential, and antibiotic susceptibility of Aeromonas salmonicida subspecies salmonicida COFCAU_AS, isolated from an aquaculture system in India. The physiological, biochemical, 16s rRNA gene sequencing and PAAS PCR test identified the strain as Aeromonas salmonicida. The MIY PCR tests established the subspecies as 'salmonicida'. The in vitro tests showed the isolated bacterium as haemolytic with casein, lipid, starch, and gelatin hydrolysis activity, indicating its pathogenic attributes. It also showed the ability to produce slime and biofilm, and additionally, it possessed an A-layer surface protein. In vivo pathogenicity test was performed to determine the LD50 dose of the bacterium in Labeo rohita fingerlings (14.42 ± 1.01 g), which was found to be 106.9 cells fish-1. The bacteria-challenged fingerlings showed skin lesions, erythema at the base of the fins, dropsy, and ulcer. Almost identical clinical signs and mortalities were observed when the same LD50 dose was injected into other Indian major carp species, L. catla and Cirrhinus mrigala. Out of the twelve virulent genes screened, the presence of nine genes viz., aerA, act, ast, alt, hlyA, vapA, exsA, fstA, and lip were detected, whereas ascV, ascC, and ela genes were absent. The A. salmonicida subsp. salmonicida COFCAU_AS was resistant to antibiotics such as penicillin G, rifampicin, ampicillin, and vancomycin while highly sensitive to amoxiclav, nalidixic acid, chloramphenicol, ciprofloxacin, and tetracycline. In summary, we have isolated a virulent A. salmonicida subsp. salmonicida from a tropical aquaculture pond which can cause significant mortality and morbidity in Indian major carp species.

Comparative analysis of unwashed and single washed mince gel from Indian major carps.

The gelling properties and quality characteristics of unwashed and single washed mince of catla, rohu and mrigal have been investigated to find out suitability of Indian major carps for the preparation of mince gel. The higher moisture content and lower protein content was reported in the single washed mince. The single washing of mince did not improve the gel strength. The gel strength showed significant difference (p < 0.05) and decreased in single washed mince than its unwashed counterparts in catla and mrigal except rohu. It has been observed that gel did not set at pre-incubation temperature of 40 °C for 30 min treatment. SDS-PAGE patterns of proteins did not show any loss of myosin heavy chain (MHC) in single washed mince of Indian major carps. Texture profile analysis showed higher hardness in washed mince gel of Indian major carps while, non-significant difference (p > 0.05) was observed in cohesiveness, adhesiveness and elasticity properties. The whiteness index of washed mince showed improvement. The overall study indicated that mince gels can be made from unwashed mince of Indian major carps, alleviating the problems of waste water disposal leading to production of more value added products with better nutritional value.

Labeo rohita Mx1 exhibits the critical structural motifs of the family of large GTPases of mammals and is activated by rhabdovirus vaccination and bacterial RNA stimulations.

Myxovirus resistance (Mx) proteins belonging to the dynamin superfamily of high molecular weight GTPases exist in various isoforms and play crucial role in innate immunity. In addition to the isoforms, Mx1 also plays important role in exerting its anti-viral actions against a broad range of animal RNA viruses. In rohu (Labeo rohita), mx1 full-length cDNA sequence consists of 2440 nucleotides (nt) encoding 628 amino acids (aa) polypeptide of 71.289 kDa. Structurally, it belongs to the family of large GTPases with one DYNc domain (13-257aa) comprising of dynamin family motifs (LPRGSGIVTR) and the tripartite GTP-binding motifs (GDQSSGKS, DLPG and TKPD) at the N-terminal and one GED domain (537-628aa) at C-terminus. Rohu Mx1 is closely related to zebrafish Mx1 and is widely expressed in gill, liver, kidney, spleen and blood. In response to rhabdovirus vaccinations, poly I:C stimulation and bacterial infections, mx1 gene expression in rohu was significantly (p < 0.05) induced in majority of the tested organs/tissues. Stimulation of rohu gill cell line with bacterial RNA also induced mx1 gene expression. Together these data suggest the important role of Mx1 in innate immunity in rohu against wide spectrum of fish pathogens.

The occurrence of known Myxobolus and Thelohanellus species (Myxozoa, Myxosporea) from Indian major carps with the description of Myxobolus bandyopadhyayi n. sp. in West Bengal.

Myxosporean infection of Indian major carps (rohu, Labeo rohita; catla, Gibelion catla; mrigal, Cirrhinus mrigala) was examined from two fish farms and two fish markets in West Bengal, India. One Thelohanellus and four Myxobolus species were detected from the fins and scales of the investigated species. Comprehensive morphological and molecular biological studies revealed four already known species, Thelohanellus caudatus from the fins of rohu, Myxobolus dermiscalis from the scales of rohu, Myxobolus chakravartyi from the fins of catla, and Myxobolus rewensis from the fins of mrigal. This study complemented the species description of M. chakravartyi and M. rewensis with the missing molecular data. Moreover, based on morphometrics and ssrDNA sequence data, a new species was documented from the scales of rohu, and named Myxobolus bandyopadhyayi n. sp.

Toll-interacting protein in the freshwater fish Labeo rohita exhibits conserved structural motifs of higher eukaryotes and is distinctly expressed in pathogen-associated molecular pattern stimulations and bacterial infections.

Toll-interacting protein (Tollip) is a critical regulator of TOLL- like receptor (TLR)-signaling pathway. It is predominantly associated with TLR2 and TLR4 during acute inflammatory conditions and inhibits the TLR-mediated nuclear factor-kappa activation by suppressing the autophosphorylation of interleukin-1 receptor-associated kinase and its kinase activity. This article describes the Tollip of Labeo rohita (LrTollip), a highly valuable freshwater fish from the Indian subcontinent. The full-length LrTollip complementary DNA (1412 nucleotides) encodes a 276-amino acid (aa) protein, depicting a highly conserved target of the Myb1 (Tom1)-binding domain (TBD; 1-53 aa), conserved core domain 2 (C2; 54-151 aa), and coupling of ubiquitin to endoplasmic reticulum degradation (CUE; 231-273 aa) domains of mouse and human counterparts. The key amino acids exerting the critical functions of Tollip, such as phospholipids recognition and ubiquitination, are present in the C2 and CUE domains of LrTollip, respectively. LrTollip is widely expressed in the kidneys, gills, spleen, liver, and blood, and among these tested tissues, the highest expression is observed in blood. In response to TLR ligands and NOD-like receptor (NLR) ligands stimulations and Aeromonas hydrophila, Edwardsiella tarda, and Bacillus subtilis infections, LrTollip gene expression is induced in various organs/tissues with remarkable difference in their kinetics. These data together suggest the important role of LrTollip in TLR- and NLR-signal transduction pathways and immune-related diseases in fish.

Current prospects and challenges in fish vaccine development in India with special reference to Aeromonas hydrophila vaccine.

Infectious diseases are adversely affecting aquaculture practices throughout world and Asian countries are no exception. Indian aquaculture practices are facing serious setback due to a variety of infectious agent's which are responsible for severe mortality and morbidity of all the cultured freshwater fish species leading to severe economic losses. The emergence of antibiotic resistant pattern, residual effect and environmental degradation due to indiscriminate use of antibiotics has necessitates the development of suitable alternate prophylaxis measures for better protection. In this regard, vaccine(s) has proved to be an effective strategy against pathogens to improve the fish production. Over the years numerous studies have been conducted to develop vaccine(s) against different pathogens. While most of the efforts are made to develop vaccine against bacterial pathogens especially against Aeromoniasis and Edwardsiellosis, few attempts have also been made against certain other bacterial, parasitic and fungal pathogens as well. Despite various successful experimental attempts, till date no vaccines against any of the pathogens are commercially available for Indian aquaculture. This review principally focuses on the current state of art in the development of vaccine against different microbial pathogens in general and Aeromonas hydrophila in particular since the bacterium is a major pathogen which is involved in a number of disease conditions in all the cultured fish species in India. Herein in this review, details of various experimental approaches made to find out a potential vaccine candidate which in turn can induce protective immune responses in host alongwith the constraints associated with it in developing a suitable vaccine against this bacterium and its market potential have been illustrated from an Indian perspective.

Influence of a diet containing plant ingredients at different levels on growth performance, carcass biochemical composition, and blood parameters in Indian major carps grown in polyculture earthen ponds.

The effect of feeding levels of plant ingredient-based diet on growth, body composition, and serological constituents of Indian major carps was determined in pond culture condition. Juveniles of Indian major carps (IMCs), Catla catla (catla, 65.87 ± 2.45 g), Labeo rohita (rohu, 64.67 ± 2.15 g), and Cirrhinus mrigala (mrigal, 39.58 ± 3.49 g) were fed 1%, 1.5%, 2%, and 2.5% of the body weight for a period of 150 days. At the end, the total production was significantly higher at 2.0% feeding level and did not change thereafter. The nutrient utilization parameters were significantly (P < 0.001) affected by the feeding level and decreased both linearly and quadratically with the higher level of feed. The SGR in terms of wet weight, dry weight, protein, and lipid increased up to 2% feeding level and plateaued thereafter. The whole body crude protein content of all the three species was the lowest at 1% feeding level and the whole body lipid content increased with increased feeding levels. Tissue protein gain and lipid gain of IMCs were the highest at 2% feeding level. Blood parameter did not indicate any disease or stress condition due to feeding treatments. Considering the growth and nutrition utilization and health of fish, it can be concluded that optimum feeding level of all plant ingredient-based feed of IMC could be 2% of the body weight in pond culture condition.

Next-generation sequencing reveals significant variations in bacterial compositions across the gastrointestinal tracts of the Indian major carps, rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus cirrhosis).

Bacterial communities strongly influence the digestion, health and immune status of fish. This study investigates the microbial distribution of the anterior, middle and distal gut sections of three economically important carp species in Bangladesh, rohu, catla and mrigal (commonly known as Indian major carps), using 16S rRNA-based Illumina sequencing technology. The alpha-diversity measurement with one-way ANOVA indicated high species richness, Shannon and Simpson indices in the middle and distal gut, while the anterior gut of IMCs had the lowest diversity. At the phylum level, there was high abundance of Proteobacteria in the GITs of rohu and mrigal, whereas Fusobacteria was dominant in the anterior and middle guts of catla. At the genus level, diverse microbial communities were identified across the three GIT sections, with six indicator genera found in rohu, catla and mrigal, as revealed by linear discriminant analysis (LDA) at a 0·05 level of significance. Of the 218 genera identified, only 33 were common across the anterior, middle and distal guts of all three species. Bacterial diversity was significantly higher (P < 0·05) in mrigal, followed by catla and rohu, respectively. Alongside the common bacteria Aeromonas, Enterobacter and Serratia, the overwhelming abundance of Cetobacterium, Shewanella and Plesiomonas warrants further investigation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study investigates the microbial communities of the gastrointestinal tracts (GITs) of three Indian major carp (IMC) species-rohu, catla and mrigal, obtained from a polyculture pond under the same feeding regime. Diverse microbial communities were found, with significantly different relative abundances and diversities of phyla and genera. The results provide valuable information on GIT microbial communities that may be useful for nutrition and health management in IMCs.

Optimization of protein in supplementary feeds for pond raised cyprinids.

Labeo rohita, Gibelion catla, Cirrhinus mrigala, Ctenopharyngodon idella and Hypophthalmichthys molitrix were grown together for 365 days in triplicate ponds. Six iso-caloric feeds having varying protein levels i.e. 22, 24, 26, 28, 30 and 32% were prepared and supplemented to fish at 2% fish weight daily. Significantly, higher final average weight (793 ± 197 g) and total length gain (398 ± 40 mm) was recorded for Ctenopharyndodon idella. Net fish yield enhanced as the level of digestible protein (DP) in the supplementary diets increased and plateaued at 28% DP level, thereafter significant reduction (P<0.05) in fish weight increments was experienced at any surplus protein level. Among the supplementary feeds, 28% DP level resulted in maximum net fish yield (4304 ± 33 kg ha-1 year-1) and a significantly higher overall nitrogen conversion ratio (NCR) of 1:5.2 ± 0.14. Higher level of DP in supplementary feeds resulted in reduced fish growth.

Consequences of oxidative damage and mitochondrial dysfunction on the fatty acid profile of muscle of Indian Major Carps considering metal toxicity.

Current study aims to find interrelation between mitochondrial enzyme function and fatty acid profile in fish muscle and role of antioxidant agents to maintain their balance in response to metal accumulation. Fishes (Labeo rohita, Catla catla, Cirrhinus cirrhosus) were collected from two sites (Nalban Bheri and Diamond Harbour, India). Concentrations of metals (lead, cadmium, copper, nickel, zinc), enzymatic and non-enzymatic antioxidant activity (malondialdehyde, superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione S-transferase), muscle enzyme activity (acetylcholinesterase, succinate dehydrogenase, lactate dehydrogenase, Ca2+ATPase, AMP-deaminase, lipoamide reductase, cytochrome C oxidase, aldolase) and fatty acid composition in muscle tissues were analyzed. Metal concentrations were significantly higher (P < 0.05) in fish muscles from Nalban compared to those in Diamond Harbour. Increased activity of antioxidant enzymes was noted with diminished mitochondrial enzymes activity and altered fatty acid composition in response to higher metal accumulation. Higher metal concentration in fish muscle of Nalban seems to significantly (P < 0.05) affect poly and monounsaturated fatty acid content, possibly due to oxidative damage and accumulation of hazardous reactive oxygen species (ROS) molecules. Changes in fatty acid contents following metal accumulation were observed to be species specific. Current study is the first correlative study to illuminate the level of oxidative damage and possible consequences on muscle cellular integrity, mitochondrial functionality and flesh quality against bioaccumulation of different metals in carps. Future studies are needed to quantify the relative contributions of enzymatic and low-molecular-mass antioxidants in protecting mitochondrial function and maintenance of proper fatty acid oxidation during acclimation to long term metal exposure.

Use of statistical analysis to validate ecogenotoxicology findings arising from various comet assay components.

Cirrhinus mrigala, Labeo rohita, and Catla catla are economically important fish for human consumption in Pakistan, but industrial and sewage pollution has drastically reduced their population in the River Chenab. Statistics are an important tool to analyze and interpret comet assay results. The specific aims of the study were to determine the DNA damage in Cirrhinus mrigala, Labeo rohita, and Catla catla due to chemical pollution and to assess the validity of statistical analyses to determine the viability of the comet assay for a possible use with these freshwater fish species as a good indicator of pollution load and habitat degradation. Comet assay results indicated a significant (P < 0.05) degree of DNA fragmentation in Cirrhinus mrigala followed by Labeo rohita and Catla catla in respect to comet head diameter, comet tail length, and % DNA damage. Regression analysis and correlation matrices conducted among the parameters of the comet assay affirmed the precision and the legitimacy of the results. The present study, therefore, strongly recommends that genotoxicological studies conduct appropriate analysis of the various components of comet assays to offer better interpretation of the assay data.

A report on two new myxozoan parasites infecting gills of fingerlings of Indian major carps cultured in nursery ponds in Punjab (India).

This paper presents the morphological characteristics of two new species belonging to the genus Myxobolus viz M. knobii n. sp. and M. majraiensis n. sp., parasites infecting gill lamellae and gill filaments of fingerlings of Cirrhinus mrigala (Ham.) and Catla catla (Ham.) respectively collected from nursery ponds located in the village Fagan Majra, District Fatehgarh Sahib, Punjab (India). A total of 90 fingerlings were examined (40 Catla catla and 50 Cirrhinus mrigala). Out of which, 32 C. mrigala and 19 C. catla fishes were found infected. The total prevalence rate was recorded as 56.66%. The age of the fish was recorded as 2-3 months and length of the fish ranged 4-4.5 cm. The plasmodia of M. knobii n. sp. were round to irregular, 0.6-1.0 mm in diameter. Myxospores oval to spherical, pot-shaped in frontal view with a prominent knob at anterior end, measured 5.83 × 4.29 µm in size. Polar capsules were equal, broadly pyriform, 1.95 × 1.70 µm in size having 3-4 polar filament coils. The plasmodia of M. majraiensis n. sp. were round to oval, 0.8-1.5 µm in diameter. Myxospores egg-shaped in frontal view also having prominent knob at the anterior end, measured 8.58 × 5.2 µm in size. Polar capsules were equal, pyriform, 3.47 × 1.80 µm in size having 5-6 polar filament coils. Gill plasmodium index showed heavy infection in both of the species. This is the first report of myxobolid infection in the fingerlings of Indian major carps in nursery ponds in Punjab (India).

Species diversity of the genus Thelohanellus Kudo, 1933 (Myxozoa: Bivalvulida) parasitizing fishes in Indian subcontinent.

A synopsis of 52 nominal species of the genus Thelohanellus Kudo, 1933 reported to infect freshwater fishes in India is presented. These species infect various organs such as gills, fins, scales, skin, muscles, gall bladder, kidney etc. The diagnostic features of the genus include tear-shaped myxospores with smooth shell valves and single polar capsule. The members are histozoic in their nature of parasitism infecting fresh water fishes. The present manuscript is aimed to compile the list of reported species in India along with essential information on morphometrics of vegetative stage, host, organ preference and pathogenic effects if any. Maximum number of species are recorded from state of West Bengal (25) followed by 23 in Punjab (out of which 8 were already reported from other states of India), Andhra Pradesh (7), Orissa (3), Manipur (1) and Haryana (1). Majority of the species have been recorded to infect major and minor carps (43 species) and 9 species in cat fishes. Out of 43 species infecting Indian major carps, T. batae infected the epithelial lining of gill filament of Labeo bata and also the wall of the duodenum and pectoral fin of Wallago attu in Harike Wetland (Punjab). Organ distribution of the species belonging to the genus Thelohanellus indicated that the plasmodia of 27 species were located in the gills, 14 species in the fins, 7 species in the gall bladder, 4 species in muscles and 3 species in kidney, 2 each in scale and skin on the head region and 1 species each in brain, eye, spleen, liver and wall of the duodenum. Recent studies conducted by the authors and also by few earlier studies in West Bengal have documented many species of Thelohanellus to cause serious gill haemorrhagic disease and dermal lesions. Histological studies on the gills of cultured carps in Punjab infected with members of Thelohanellus have indicated that these parasites cause 15-20 % damage to respiratory surface leading to localized loss of function.

Molecular characterization of Myxoboluscatmrigalae (Myxosporea: Myxobolidae) infecting the gill lamellae of carp Cirrhinusmrigala (Hamilton).

The present study attempted sequencing the 18S rRNA gene of Myxoboluscatmrigalae infecting the gill lamellae of carp, Cirrhinusmrigala and compared its genetic homology and phylogenetic characteristics with 18S rRNA genes of other Myxobolus spp. The infected fish had up to 3 small, creamy white plasmodia per gill filament with 30-50 spores each. The spore size was 17.90 ± 0.70 × 7.40 ± 0.40 µm. The sporoplasm contained two large nuclei of size 0.57 ± 0.09 µm and no iodinophilous vacuole. The DNA sequence of M.catmrigalae was clustered phylogenetically with other Myxobolus spp. infecting the gills of cyprinids available in GenBank, which showed 77-87 % homogeneity. On the phylogenetic tree, M.catmrigalae (KC933944) was clustered with M.pavlovskii (HM991164) infecting the gill lamellae of silver carp, Hypophthalmichthysmolitrix. The species most closely related to M.catmrigalae in GenBank was M.pavlovskii (AF507973) infecting the gill lamellae of big head carp, Aristichthysnobilis with 87 % homogeneity. This is the first report on molecular characterization of gill lamellae infecting M. catmrigalae.

Microelectrophoretic study of environmentally induced DNA damage in fish and its use for early toxicity screening of freshwater bodies.

This study investigates the potential of the comet and micronucleus assays of fish DNA as a means of screening the toxicity of aquatic environments. Catla catla and Cirrhinus mrigala collected from the River Chenab in Pakistan were used as a case study for the application of comet and micronucleus techniques. Comet and micronucleus assays were used to compare DNA damage in C. catla and C. mrigala collected from polluted areas of the River Chenab and farmed fish. Atomic absorption spectrophotometry showed an acute level of toxicity from Cd, Cu, Mn, Zn, Pb, Cr, Sn, and Hg in river water. Comet assay showed significant (p < 0.05) DNA damage in C. catla representing 17.33 ± 2.42, 11.53 ± 2.14, and 14.17% DNA in the comet tail, averaged from three sites of the polluted area of the river. Tail moment was observed as 10.06 ± 2.71, 3.11 ± 0.74, and 14.70 ± 1.89, while olive moment was 8.85 ± 1.84, 3.83 ± 0.76, and 7.11 ± 0.73, respectively. Highly significant (p < 0.01) damage was reported in C. mrigala as 37.29 ± 2.51, 34.96 ± 2.53, and 38.80 ± 2.42% DNA in comet tail, tail moment was 23.48 ± 3.90, 19.78 ± 4.26, and 14.30 ± 1.82, and olive moment was 16.22 ± 2.04, 13.83 ± 1.96, and10.99 ± 0.90. Significant (p < 0.05) differences were observed in genotoxicity between farmed and polluted area fish. Micronucleus assay showed a similar picture of significant difference in respect to single and double micronucleus induction: i.e., 23.20 ± 4.19 and 2.80 ± 1.07‰ in C. catla and 44.80 ± 3.73 and 06.20 ± 0.97‰, respectively, in C. mrigala. Nuclear abnormalities were found as 6.00 ± 0.84 and 09.60 ± 1.72/thousand cells, respectively, in both species. The results of this study suggest that these novel fish DNA damage assays can be used as an expedient toxicity screening for aquatic environments.

First report on fish cysteine as a biomarker of contamination in the River Chenab, Pakistan.

The eastern and southern parts of the Faisalabad city produce considerable quantities of industrial and municipal pollutants, much of which is drained into the River Chenab, reducing the productivity of fauna and flora in the river. This study was aimed to determine whether cysteine is useful as a biomarker of exposure to polluted fresh water. The amino acid profile of fish muscle was analyzed by paper chromatography in Cirrhinus mrigala and Labeo rohita from the River Chenab to determine habitat related variations due to the pollution from industrial and domestic sources. C. mrigala showed higher level of metal contamination in muscle tissues for Sn, Cr, Pb, Zn, Mn, Cu, and Cd when compared to L. rohita. Both fish species collected from polluted areas of the river Chenab showed significantly (P < 0.01) higher levels of metals in comparison to upstream and farmed fish. Farmed C. mrigala showed cysteine concentrations in the muscle tissue as 22 ± 1 mg/g dry weight, but concentrations increased to 45 ± 2 mg/g dry weight for fish from a mildly polluted section of the river, and further increased to 83 ± 2 mg/g dry weight in more heavily polluted sections. Cysteine concentration in farmed L. rohita was detected as 28 ± 2 and 25 ± 4 mg/g dry weight, respectively for farmed fish and fish from a mildly polluted section of the river, and then increased to 94 ± 3 mg/g dry weight for fish from highly polluted water. C. mrigala from a mildly polluted area of the river also had higher levels of cysteine in the muscle, along with increases in aspartic acid, glutamic acid, and alanine. Elevated concentrations of cysteine seem to be associated with a threat to these fish species in polluted sections of the river, and thus may be used as a biomarker.

Population genetic characterization and family reconstruction in brood bank collections of the Indian major carp Labeo rohita (Cyprinidae:Cypriniformes).

The founder stock of a captive breeding program is prone to changes in genetic structure due to inbreeding and genetic drift. Genetic characterization of the founder population using suitable molecular markers may help monitor periodic changes in the genetic structure in future. To develop benchmark information about the genetic structure we analyzed six microsatellite loci in the Brodbank collections of rohu (Labeo rohita) originated from three major rivers-the Jamuna, the Padma and the Halda. A total of 28 alleles were detected in 90 individuals with an average of 4.6 alleles per locus. The average observed heterozygosity ranged from 0.655 to 0.705 and the expected heterozygosity ranged from 0.702 to 0.725. The mean F IS values were 0.103, 0.106 and 0.018 for the Jamuna, Padma and Halda fishes respectively. The population pair-wise F ST values ranged from 0.0057 to 0.0278. Structure analysis grouped the fishes of the three rivers into two clusters. The numbers of half-sib families were 5, 5 and 4 and the numbers of full-sib families were 12, 10 and 18 for the Halda, Jamuna and the Padma samples respectively. Bottleneck was detected in all the river samples. We recommend to collect more fish from different locations of the major rivers to broaden the genetic variability of the founder stocks of the Brood bank.

Effect of ice storage on the functional properties of proteins from a few species of fresh water fish (Indian major carps) with special emphasis on gel forming ability.

In the present study the effect of ice storage on physico-chemical and functional properties of proteins from Indian major carps with special emphasis on gel forming ability have been assessed for a period of 22 days. The solubility profile of proteins in high ionic strength buffer and calcium adenosine triphosphatase (ATPase) enzyme activity reduced significantly (p < 0.05), while that of total volatile base nitrogen (TVB-N) increased significantly (p < 0.05) at the end of 22 days of ice storage. The major protein fraction showed association-dissociation-denaturation phenomenon during ice storage as revealed by gel filtration profile and viscosity measurements. The gel forming ability of three fish species both in fresh and during different periods of ice storage was assessed by measuring the gel strength of heat induced gel. Among the three species the gel strength of the gel obtained from Catla catla and Cirrhinus mrigala was higher (586 and 561 g.cm) than the gel obtained from Labeo rohita (395 g.cm) in fresh condition. The gel forming ability of three species was significantly affected (p < 0.05) during ice storage. The TVB-N values of fish meat as a function of ice storage was within the prescribed limit up to 17 days of the ice storage.