Assessment of DNA damage induced by endosulfan in grass carp (Ctenopharyngodon idella Valenciennes, 1844).

Endosulfan is an organochlorine pesticide, which is commonly used throughout the world. It accumulates in the environment and may cause significant damage to the ecosystems, particularly to the aquatic environments. The present study was conducted to evaluate the genotoxic effect of endosulfan on the grass carp (Ctenopharyngodon idella) blood. The fish were exposed to three different concentrations, 0.75 ppb/day, 1.0 ppb/day, and 1.5ppb/day of endosulfan for 7, 14, 21, and 28 days. The study was a randomized control trial and the control group was not exposed to endosulfan. The results showed that after 7 days, the level of DNA damage in all the concentrations was significant (P < 0.05), while after 14, 21, and 28 days' trials, highly significant (P < 0.000) level of DNA damage was observed. Hence, time- and dose-dependent DNA damage was observed in fish DNA by comet assay. It is concluded from our results that with the increase in endosulfan concentration and exposure duration, the level of DNA damage also increased. As the current study showed the severe genotoxic effect of endosulfan in Ctenopharyngodon idella, therefore, the imprudent and indiscriminate use of endosulfan should be controlled and monitored by the concerned government authorities.

Physiological status of silver carp (Hypophthalmichthys molitrix) in the Illinois River: An assessment of fish at the leading edge of the invasion front.

Silver carp (Hypophthalmichthys molitrix) are invasive to North America, and their range has expanded within the Mississippi River Basin, seemingly unchecked, since their introduction in the late 1970s, with the exception of the upper reaches of the Illinois River. With the imminent threat of their movement into the Great Lakes, the goal of the present study was to assess whether differences in the physiological status between silver carp at the leading edge of their invasion front and core population sites could explain their lack of expansion upstream toward Lake Michigan over the past decade. A transcriptomic approach using RNA sequencing and analysis of plasma variables were used to quantify differences among fish at the leading edge and two downstream core population sites. Leading-edge fish exhibited upregulation of genes associated with xenobiotic defense (e.g., ATP-binding cassette C1 [abcc1], abcc2, abcc6), decreased cell integrity (i.e., macroautophagy and apoptosis; autophagy-related protein 9A [atg9a], caspase 3b [casp3b]), and cholesterol metabolism (e.g., abca1, apolipoprotein A1 [apoa1], sterol O-acyltransferase [soat1]) and downregulation of genes associated with DNA repair (e.g., tumor suppressor p53-binding protein 1 [tp53bp1]) compared to core population sites. Transcriptomic profiles of leading-edge fish were consistent with fish inhabiting a polluted environment and suggest that poorer water quality conditions upstream of the leading edge may represent a non-permanent barrier to silver carp range expansion. The present study provides potential molecular targets for monitoring the physiological status of silver carp over time and in response to future improvements in water quality upstream of their leading edge.

Immunosuppression, oxidative stress, and glycometabolism disorder caused by cadmium in common carp (Cyprinus carpio L.): Application of transcriptome analysis in risk assessment of environmental contaminant cadmium.

Cadmium (Cd), a hazardous environmental contaminant with irreversible toxicity to fish, has been detected in aquatic environment of many countries. The common carp is one of the most widely distributed fish in the world, so we used common carp to assess environmental contaminant risk. In present study, we investigated effects of Cd on immune function, oxidative defense, and glycometabolism in the spleens of common carp by transcriptome analysis. Obtained 3794 differentially expressed genes (including 1848 up-regulated and 1946 down-regulated genes) were enriched using databases of Kyoto Encyclopedia of Genes and Genomes, and Gene Ontology in David bioinformatics software (version 6.8). The pathways and gene functions of immune, oxidative defense, and glycometabolism were obtained and identified. Some relative genes were validated using qRT-PCR and gene expression of IL-1ß, INF-γ, IL-6, Cxcl18b, HO-1a, CAT, GPx1, GCK, and FBA decreased; and gene expression of B4GALT1, GPAT3, and CYP26B1 increased. Our results indicated that Cd exposure led to immunosuppression, oxidative stress, and glycometabolism disorder in the common carp spleens. The present study gives a novel insight and method on environmental risk assessment.

Historical demography of common carp estimated from individuals collected from various parts of the world using the pairwise sequentially markovian coalescent approach.

The inference of historical demography of a species is helpful for understanding species' differentiation and its population dynamics. However, such inference has been previously difficult due to the lack of proper analytical methods and availability of genetic data. A recently developed method called Pairwise Sequentially Markovian Coalescent (PSMC) offers the capability for estimation of the trajectories of historical populations over considerable time periods using genomic sequences. In this study, we applied this approach to infer the historical demography of the common carp using samples collected from Europe, Asia and the Americas. Comparison between Asian and European common carp populations showed that the last glacial period starting 100 ka BP likely caused a significant decline in population size of the wild common carp in Europe, while it did not have much of an impact on its counterparts in Asia. This was probably caused by differences in glacial activities in East Asia and Europe, and suggesting a separation of the European and Asian clades before the last glacial maximum. The North American clade which is an invasive population shared a similar demographic history as those from Europe, consistent with the idea that the North American common carp probably had European ancestral origins. Our analysis represents the first reconstruction of the historical population demography of the common carp, which is important to elucidate the separation of European and Asian common carp clades during the Quaternary glaciation, as well as the dispersal of common carp across the world.

A full-body transcriptome and proteome resource for the European common carp.

BACKGROUND: The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies. RESULTS: Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data. CONCLUSIONS: The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

In silico assessment of primers for eDNA studies using PrimerTree and application to characterize the biodiversity surrounding the Cuyahoga River.

Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semi-aquatic animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity.

QTL mapping for economically important traits of common carp (Cyprinus carpio L.).

Quantitative trait loci (QTL) were analyzed for three economically important traits, i.e., body weight (BW), body length (BL), and body thickness (BT), in an F1 family of common carp holding the 190 progeny. A genetic linkage map spanning 3,301 cM in 50 linkage groups with 627 markers and an average distance of 5.6 cM was utilized for QTL mapping. Sixteen QTLs associated with all three growth-related traits were scattered across ten linkage groups, LG6, LG10, LG17, LG19, LG25, LG27, LG28, LG29, LG30, and LG39. Six QTLs for BW and five each for BL and BT explained phenotypic variance in the range 17.0-32.1%. All the nearest markers of QTLs were found to be significantly (p ≤ 0.05) related with the trait. Among these QTLs, a total of four, two (qBW30 and qBW39) related with BW, one (qBL39) associated with BL, and one (qBT29) related to BT, were found to be the major QTLs with a phenotypic variance of >20%. qBW30 and qBW39 with the nearest markers HLJ1691 and HLJ1843, respectively, show significant values of 0.0038 and 0.0031, correspondingly. QTLs qBL39 and qBT29 were found to have significant values of 0.0047 and 0.0015, respectively. Three QTLs (qBW27, qBW30, qBW39) of BW, two for BL (qBL19, qBL39), and two for BT (qBT6, qBT25) found in this study were similar to populations with different genetic backgrounds. In this study, the genomic region controlling economically important traits were located. These genomic regions will be the major sources for the discovery of important genes and pathways associated with growth-related traits in common carp.

Optimization of liquid media and biosafety assessment for algae-lysing bacterium NP23.

To control algal bloom caused by nutrient pollution, a wild-type algae-lysing bacterium was isolated from the Baiguishan reservoir in Henan province of China and identified as Enterobacter sp. strain NP23. Algal culture medium was optimized by applying a Placket-Burman design to obtain a high cell concentration of NP23. Three minerals (i.e., 0.6% KNO3, 0.001% MnSO4·H2O, and 0.3% K2HPO4) were found to be independent factors critical for obtaining the highest cell concentration of 10(13) CFU/mL, which was 10(4) times that of the control. In the algae-lysing experiment, the strain exhibited a high lysis rate for the 4 algae test species, namely, Chlorella vulgari, Scenedesmus, Microcystis wesenbergii, and Chlorella pyrenoidosa. Acute toxicity and mutagenicity tests showed that the bacterium NP23 had no toxic and mutagenic effects on fish, even in large doses such as 10(7) or 10(9) CFU/mL. Thus, Enterobacter sp. strain NP23 has strong potential application in the microbial algae-lysing project.

MH II-DAB gene expression in grass carp Ctenopharyngodon idella (Valenciennes) after infection with the ciliate parasite, Ichthyophthirius multifiliis.

The grass carp, Ctenopharyngodon idella (Valenciennes), is one of the most extensively aquacultured freshwater fish in China. However, because of the lack of effective control measures and the high-density culture environment, considerable economic losses are caused by infection of C. idella with the parasitic ciliate, Ichthyophthirius multifiliis. The major histocompatibility (MH) DAB gene belongs to antigen-presented genes in the class II genomic region, which is associated with parasite resistance. To understand the relationship of the DAB gene with I. multifiliis infection in grass carp, the expression profiles of MH II-DAB were studied in tissues using real-time quantitative polymerase chain reaction. The results showed that expression of the MH II-DAB gene was up-regulated in head kidney after I. multifiliis infection, and the expression peak appeared earlier in the study (case) group than in the control group. The obvious up-regulation peak of MH II-DAB gene was found at days 2 and 4 in skin; at 12 h to day 4 in spleen; at 12 h and days 1 and 6 in gill; and at day 10 in blood, whereas the MH II-DAB gene was down-regulated in liver and intestines after I. multifiliis infection. These results have implications for better understanding C. idella resistance to I. multifiliis infection.

Assessment of global proteome dynamics in carp: a model for investigating environmental stress.

Fish have to respond to a range of natural and man-made environmental stressors, which can lead to molecular changes within their tissues. Many studies focused on environmental stress in fish have examined the change in protein abundance or mRNA level. However, it is well-known that there is a disconnect between mRNA and protein expression. In order to bridge this gap, protein turnover must also be considered. We have developed an experimental strategy to determine the synthesis rates of individual proteins in the tissues of fish on a proteome-wide scale. This approach has been applied to the common carp ( Cyprinus carpio ), a key model species for investigating environmentally induced physiological plasticity. We have calculated the rates of protein synthesis for over a thousand individual proteins from the skeletal muscle and liver of carp. The median synthesis rate of proteins from liver was higher than that of skeletal muscle. The analysis further revealed that the same protein can have a different rate of synthesis depending on the tissue type. Our strategy permits a full investigation of proteome dynamics in fish and will have relevance to the fields of integrative biology and ecotoxicology.

Sublethal toxicity of esbiothrin relationship with total antioxidant status and in vivo genotoxicity assessment in fish (Cyprinus carpio L., 1758) using the micronucleus test and comet assay.

Esbiothrin, synthetic pyrethroid with quick activity against insects, is widely used against household pests and in public health. Despite widespread use, data on ecotoxicity and genotoxic effects are extremely scarce. The aim of the present study is to evaluate the genotoxic potential of esbiothrin on a model fish species Cyprinus carpio L., 1758 (Pisces: Cyprinidae, koi) using the micronucleus test and comet assay in peripheral blood erythrocytes. Effects of two sublethal exposure concentrations on plasma total antioxidant status (TAS mmol/L), and Hct values were examined. On the basis of the 96 h LC50 data from U.S. EPA ecotox database (32 µg/L) two sublethal exposure concentrations (5 and 10 µg/L) were used together with ethyl methanesulfonate (EMS) (5 mg/L) as positive control. Five fish were used for each dose/duration group (24, 48, and 72 h) under controlled laboratory conditions. The fish showed behavioral changes at the higher dose. Plasma TAS (mmol/L) levels decreased in 24 h; an increase was observed slightly for 48 and obviously for 72 h in both exposure doses. Similarly, hematocrit (Hct) values differed between exposure duration but no significant differences in mean values were found between groups of the same exposure time. The general trend was a rise after 48 h, which decreased afterwards. Our results revealed significant increases in the frequencies of micronuclei and levels of DNA strand breaks and thus demonstrated the genotoxic potential of this pesticide on fish, a nontarget organism of the aquatic ecosystem. To our knowledge this is the first study to report observable genotoxic effects of esbiothrin on fish.

An assessment of androgenic/anti-androgenic effects of GH transgenic carp by Hershberger assay.

OBJECTIVE: To evaluate the androgenic and anti-androgenic effects of GH (growth hormone) transgenic carp in male rats. METHODS: Hershberger assay was carried out in castrated male SD rats aged 4-5 weeks. Testosterone propionate (TP) (0.4 mg/kg BW) was administrated for a positive control, GH transgenic carp (3.0 g/kg BW)+TP (0.4 mg/kg BW), parental carp (3.0 g/kg BW) + TP (0.4 mg/kg BW), and flutamide (Flu) (3.0 g/kg BW) were used for negative controls, and vehicle was administered orally for a blank control. All groups were administrated for 10 consecutive days. At the end of the test, animals were anesthetized, then weights of accessory sex organ were measured. Serum testosterone (T), luteinizing hormone (LH), and Follicle-Stimulating Hormone (FSH) levels were detected. RESULTS: The weights ratios of the accessory sex organs and body weights showed no significant differences between the solvent control and the GH transgenic carp-treated groups. Serum concentrations of FSH, LH, and T of the rats treated with GH transgenic carp + TP showed no significant changes, compared with those treated with TP only. CONCLUSION: GH transgenic carp does not have any androgenic agonist or antagonist properties in vivo screening tests.

Genotoxicity assessment of carp (Cyprinus carpio L.) fingerlings by tissue DNA damage and micronucleus test, after environmental exposure to fenitrothion.

The purpose of this study was to evaluate the adverse effects of sublethal doses of fenitrothion, an organophosphothionate insecticide on brain, gill, liver, and muscle tissues as a ratio of 8-OHdG to dG to indicate the DNA damage and erythrocyte micronucleus frequency for genotoxicity of carp (Cyprinus carpio L.) fingerlings. In our study, the mean weights and lengths of the fish (n = 4-12) were 31.13 ± 14.24 g and 12.53 ± 1.41, respectively. Before the experiment, fish were maintained in aerated dechlorinated tap water at 21.8 ± 1 °C and fed daily with commercial feed at a rate of 2% of their body weights. Experiments were conducted under static conditions in the aquaria. Technical grade (95%) fenitrothion was diluted in acetone to give a dosing solution of 10 mg/L. The increased lesions/106 DNA bases (p < 0.05) of liver tissue of exposure group (0.49 ± 0.18) was observed when compared to control group (0.28 ± 0.30). There was not any significant differences between brain tissues, no damage were detectable in gill and muscle tissues of control groups, and in exposure groups altered levels of damage were detected for gill (0.06 ± 0.05) and muscle (0.16 ± 0.21) tissues. The increased micronucleus frequencies (%) in erythrocytes of carp following the exposure to 48 h fenitrothion (6.43 ± 3.89; p<0.05) was observed when compared to control group (1.29 ± 1.03). The available data indicate that there is still lack of well-established dose-response relationships between occupational or environmental exposures and the induction of 8-OHdG. Such biomarkers may be used in assessing adverse/toxic effects of pesticides as environmental stressors.

Epizootic haematopoietic necrosis virus--an assessment of the likelihood of introduction and establishment in England and Wales.

Epizootic haematopoietic necrosis virus (EHNV) is an iridovirus that affects perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss). It emerged in Australia in the 1980s and has not been discovered elsewhere. It causes a high level of mortality in perch resulting in steep population declines. The main possible routes of introduction of the virus to England and Wales are the importation of infected live fish or carcasses. However, no trade in live susceptible species is permitted under current legislation, and no importation of carcasses currently takes place. The virus is hardy and low levels of challenge can infect perch. Therefore, mechanical transmission through the importation of non-susceptible fish species should be considered as a potential route of introduction and establishment. Carp (Cyprinus carpio) have been imported to the UK from Australia for release into still-water fisheries. A qualitative risk assessment concluded that the likelihood of EHNV introduction and establishment in England and Wales with the importation of a consignment of carp was very low. The level of uncertainty at a number of steps in the risk assessment scenario tree was high, notably the likelihood that carp become contaminated with the virus and whether effective contact (resulting in pathogen transmission) is made between the introduced carp and susceptible species in England and Wales. The virus would only establish when the water temperature is greater than 12 degrees C. Analysis of 10 years of data from two rivers in south-west England indicated that establishment could occur over a period of at least 14 weeks a year in southern England (when average water temperature exceed 12 degrees C). Imports of live fish from Australia need to be evaluated on a case-by-case basis to determine which, if any, sanitary measures are required to reduce the assessed risk to an acceptable level.

Classical crosses of common carp (Cyprinus carpio L.) show co-segregation of antibody response with major histocompatibility class II B genes.

In cyprinids, two paralogous groups of major histocompatibility (MH) class II B genes, DAB1 and DAB3, have been reported but have not been studied in detail. In our study on MH association with immune responsiveness in common carp (Cyprinus carpio L.) we have taken a long-term approach using divergent selection for antibody production. We report the co-segregation of Cyca-DAB1-like and Cyca-DAB3-like genes with antibody response, in backcrosses to high- and low-responsive parental carp lines. We show that the presence of Cyca-DAB1-like, but not Cyca-DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp. Background genes other than Cyca-DAB genes also influenced the level of antibody response. Our data support the hypothesis of a genetic control by Cyca-DAB genes on the antibody response measured. We could not detect an association of the Cyca-DAB genes with disease resistance to the parasite Trypanoplasma borreli. Sequence information, constitutive transcription levels and our co-segregation data indicate that both paralogous Cyca-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. Previously defined differences in allelic diversity between Cyca-DAB1-like genes, especially, identify Cyca-DAB1 as the most interesting DAB gene for further study in common carp.

[Analysis of genetic structure of mirror carp population and correlation of microsatellite markers and economic traits].

The genetic structure of one breeding population of mirror carp(Cyprinus carpio L.) was analysed using thirty-five polymorphic microsatellite markers. The effective number of alleles (Ae), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphism information content(PIC) were all determined. The Hardy-Weinberg equilibrium was checked by chi-square test. For each locus, 2-7 alleles were detected, with a total of 118 alleles for 35 loci . The value of Ae, Ho, He and PIC showed that the genetic variation of the population was not high .The average effective number of alleles and the mean polymorphism information content were 2.16 and 0.42, respectively, and the observed and expected heterozygosity were 0.431 and 0.4736 , respectively.The probability value of chi-square test showed that more than half of the thirty-five loci have significantly (Plt;0.01) deviated from Hardy-Weinberg equilibrium. The correlation of genotype of each locus and individual phenotype data was analysed , and 4 loci seemed associated with body weight and body size . When compared with the result of QTL mapping of common carp, HLJ319 locus was consistent with the mapping result approximately, which was significantly correlated with body size . Several significantly deviated loci were also analysed, and the possible cause of the kind of deviation was discussed .

[Genetic potential analysis of Germany mirror carp (Cyprinus carpio L.) using microsatellite markers].

Using 30 microsatellite markers and combining quantity characters such as body weight, body size and body width, we evaluated the genetic potential of 3 Germany mirror carp populations. Number of effective alleles (Ae), observed (Ho) and expected (He) heterozygosity values and polymorphic information contents (PIC) were all calculated. 287 alleles and 559 genotypes were detected. The DNA fragment length was 109-400 bp. The Hardy-Weinberg Equilibrium was checked and phenomenon of some disequilibrium were studied according to the test of c2. The result showed that the level of genetic variability was moderate, but genetic potential of Shuanglai population was much lower than that Huanxin and Songpu breeding populations. Polymorphic Information Contents (PIC) of the 3 populations of Germany mirror carp were between 0.08787 and 0.5377, both of highly and moderately polymorphic markers were 13. The number of effective alleles (Ae) were between 1.1014 and 6.4665. The observed (Ho) and expected (He) heterozygosity values were 0.0968-0.9892 and 0.0926-0.8554 respectively. The linkage correlation was analyzed using data of body weight, body size and body width and 30 loci. The result shown that there existed 2 loci, HLJ319 and HLJ693 associated with body size. The HLJ693 locus significantly correlated with body weight character. The HLJ677 locus linked with body width. And then the result were verified in Recombinant Inbred Lines (RIL) of common carp. It shown that the HLJ319 locus was significantly linked with body size, the same as the result of common carp QTL location.

Effluent impact assessment using microarray-based analysis in common carp: a systems toxicology approach.

Effluents are a main source of direct and continuous input of pollutants to the aquatic environment, and can cause ecotoxicological effects at different levels of biological organization. Since gene expression responses represent the primary interaction site between environmental contaminants and biota, they provide essential clues to understand how chemical exposure can affect organismal health. The aim of the present study was to investigate the applicability of a microarray approach for unraveling modes of action of whole effluent toxicity and impact assessment. A chronic toxicity test with common carp (Cyprinus carpio) was conducted where fish were exposed to a control and 100% effluent for 21 days under flow-through conditions. Microarray analysis revealed that effluent treatment mainly affected molecular pathways associated with the energy balance of the fish, including changes in carbohydrate and lipid metabolism, as well as digestive enzyme activity. These gene expression responses were in clear agreement with, and provided additional mechanistic information on various cellular and higher level effects observed for the same effluent. Our results demonstrate the benefit of toxicogenomic tools in a "systems toxicology" approach, involving the integration of adverse effects of chemicals and stressors across multiple levels of biological complexity.