Generation, Analysis and Functional Annotation of Expressed Sequence Tags From the Sheepshead Minnow (\u3ci\u3eCyprinodon variegatus\u3c/i\u3e)

Background: Sheepshead minnow (Cyprinodon variegatus) are small fish capable of withstanding exposure to very low levels of dissolved oxygen, as well as extreme temperatures and salinities. It is an important model in understanding the impacts and biological response to hypoxia and co-occurring compounding stressors such as polycyclic aromatic hydrocarbons, endocrine disrupting chemicals, metals and herbicides. Here, we initiated a project to sequence and analyze over 10,000 ESTs generated from the Sheepshead minnow (Cyprinodon variegatus) as a resource for investigating stressor responses. Results: We sequenced 10,858 EST clones using a normalized cDNA library made from larval, embryonic and adult suppression subtractive hybridization-PCR (SSH) libraries. Post- sequencing processing led to 8,099 high quality sequences. Clustering analysis of these ESTs indentified 4,223 unique sequences containing 1,053 contigs and 3,170 singletons. BLASTX searches produced 1,394 significant (E-value \u3c 10(-5)) hits and further Gene Ontology (GO) analysis annotated 388 of these genes. All the EST sequences were deposited by Expressed Sequence Tags database (dbEST) in GenBank (GenBank: GE329585 to GE337683). Gene discovery and annotations are presented and discussed. This set of ESTs represents a significant proportion of the Sheepshead minnow (Cyprinodon variegatus) transcriptome, and provides a material basis for the development of microarrays useful for further gene expression studies in association with stressors such as hypoxia, cadmium, chromium and pyrene

Widespread alterations upon exposure to the estrogenic endocrine disruptor ethinyl estradiol in the liver proteome of the marine male fish Cyprinodon variegatus

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGQuantitative proteomic changes in the liver of adult males of Sheepshead minnow (Cyprinodon variegatus) upon exposure to ethinyl estradiol (EE2) were assessed to provide an advanced understanding of the metabolic pathways affected by estrogenic endocrine disruption in marine fish, and to identify potential novel molecular biomarkers for the environmental exposure to estrogens. From a total of 3188 identified protein groups (hereafter proteins), 463 showed a statistically significant difference in their abundance between EE2 treatment and solvent control samples. The most affected biological processes upon EE2 exposure were related to ribosomal biogenesis, protein synthesis and transport of nascent proteins to endoplasmic reticulum, and nuclear mRNA catabolism. Within the group of upregulated proteins, a subset of 14 proteins, involved in egg production (Vitellogenin, Zona Pellucida), peptidase activity (Cathepsine E, peptidase S1, Serine/threonine-protein kinase PRP4 homolog, Isoaspartyl peptidase and Whey acidic protein), and nucleic acid binding (Poly [ADP-ribose] polymerase 14) were significantly upregulated with fold-change values higher than 3. In contrast, Collagen alpha-2, involved in the process of response to steroid hormones, among others, was significantly downregulated (fold change = 0.2). This pattern of alterations in the liver proteome of adult males of C. variegatus can be used to identify promising novel biomarkers for the characterization of exposure of marine fish to estrogens. The Whey acidic protein-like showed the highest upregulation in EE2-exposed individuals (21-fold over controls), suggesting the utility of abundance levels of this protein in male liver as a novel biomarker of xenoestrogen exposure.Agencia Estatal de Investigación | Ref. PID2019–107611RB-I00Xunta de Galicia | Ref. ED431C 2020/05Xunta de Galicia | Ref. ED481A-2018/289Xunta de Galicia | Ref. ED431C 2017/4

Impacts of Oil Exposure During Early Life Development Stages In Sheepshead Minnows (\u3ci\u3eCyprinodon variegatus\u3c/i\u3e) Under Different Environmental Factors

The release of approximately 5 million barrels of crude oil into the northern Gulf of Mexico during the 2010 Deepwater Horizon oil spill jeopardized estuarine ecosystem health from Texas to Florida. These estuarine habitats, which serve as nurseries for many important fisheries are also prone to rapid fluctuations in environmental stressors such as oxygen concentration, and salinity. The consequence of combined exposure to crude oil and suboptimal environmental factors during early life stage development of fish is still largely unknown. The objective of this project was to investigate the impacts of exposure to crude oil in combination with varying environmental stressors on Cyprinodon variegatus survival, gene expression, and genotoxicity. The post-larval developmental stage was the most sensitive early life stage to oil and abiotic stress. Median lethal concentrations during the post-larval exposures followed a treatment dependent pattern with the greatest lethal effect seen under hypoxic-high salinity conditions (64.55 µg/L ± 12.81). Real-time PCR analysis identified down-regulation of cyp1a1, epo, and arnt1, target genes involved in the two common defense pathways, the aryl hydrocarbon receptor signaling pathway which modulates metabolism of polycyclic aromatic hydrocarbons (PAHs), and the hypoxia inducible 1-α signaling pathway which is responsible for resilience to hypoxic stress, this was only observed under hypoxic-high salinity environmental conditions in treatments with PAH concentrations greater than 226 µg/L. Top toxicological functions impacted during post-larval development in all treatment comparisons included cholesterol biosynthesis, cardiotoxicity, and hepatoxicity. These findings indicate that the post-larval stage is the most sensitive to oil and environmental stress

Molecular Responses to Deepwater Horizon Oil Spill Contaminants in Estuarine Fish

The 2010 Deepwater Horizon (DWH) oil spill resulted in the oiling of approximately 2100 km of shoreline. During that time, resident organisms had to contend with the effects of multiple contaminants and suboptimal environmental conditions. This research examines the molecular effects of DWH oil spill contaminants in estuarine fish (CyprinodonvariegatusandFundulusgrandis) across multiple life stages, contaminant concentrations, and in conjunction with environmental stressors. Our results indicate that: 1. Oil elicits substantial transcriptional effects across life stages in C. variegatus. In adults, exposure to low concentrations of oil and dispersant results in transcriptional effects related to immunity, circulation processes, and DNA replication and repair. In early life-stage C. variegatus, oil exposure elicits different effects across developmental windows. Embryos mount a muted transcriptional response to oil, while larval stages mount a larger response dominated by dysregulation of transcription related to cholesterol biosynthesis, cardiac development, and immunity. In particular, the magnitude of transcriptional response in larval stages suggests examination of larvae may provide the most sensitive assessment of oil spill impacts. 2. Oil elicits differing transcriptional effects in ecologically similar species. We compared oil-induced transcription in larval C. variegatusand F. grandis and found distinct gene expression patterns in the two species, including opposing expression of genes involved in cholesterol biosynthesis. These results suggest that nuanced differences in molecular effects exist among fish, and should be considered when predicting the ecosystem-level effects of oil contamination. 3. Oil elicits altered DNA methylation patterns in larval C. variegatus. We found that oil exposure resulted in altered methylation at several genomic loci, and that simultaneous exposure to oil and hypoxia results in much greater effects to methylation than oil alone. We also determined that differentially methylated loci were correlated with differentially expressed genes, suggesting that altered methylation influences transcriptional responses following oil and hypoxia exposure. Overall, these results suggest that the sub-lethal impacts of oil spill contaminants constitute a substantial insult to cellular and molecular functions in estuarine fish, and imply that effects to fish health following oil spills could be more widespread and persistent than previously thought

Constructing a fish metabolic network model

We report the construction of a genome-wide fish metabolic network model, MetaFishNet, and its application to analyzing high throughput gene expression data. This model is a stepping stone to broader applications of fish systems biology, for example by guiding study design through comparison with human metabolism and the integration of multiple data types. MetaFishNet resources, including a pathway enrichment analysis tool, are accessible at http://metafishnet.appspot.com

Molecular markers of ecotoxicological interest in the rainbowfish Melanotaenia fluviatilis

The Crimson-spotted rainbowfish (Melanotaenia fluviatilis) from the Murray-Darling basin of Australia is a common indicator species in Australian ecotoxicology. Biochemical changes have been investigated in this species, but not molecular markers of ecotoxicological interest. In this study genes of M. fluviatilis were isolated using a cDNA library and sequences analysed. Of 345 randomly selected clones, 94 shared similarity with 26 different genes in other organisms in public databases. Amongst these, reproductive genes coding for vitellogenin, retinol binding protein, sialyltransferase and zona pellucida protein were considered of interest in ecotoxicology. The vitellogenin gene was selected for study as it has been widely used as a molecular marker of exposure to 17â-estradiol (E2) in teleosts. Gene expression was examined via northern blot, RT-PCR and Real-Time PCR relative t o the housekeeping gene (18S rRNA). The expression of vitellogenin mRNA was observed at 12 hours post-exposure, peaked at 48 hours according to northern blot analysis; and cleared within 4 days, partly consistent with RT-PCR. However, Real-time PCR yielded an inconclusive result, probably due to differences between pooled and individual samples. Vitellogenin in blood plasma was confirmed by western blot, found to be significantly increased and retained in the plasma in fish treated with E2 compared to controls. It was concluded that vitellogenin mRNA is a molecular marker of exposure to 17â-estradiol in the rainbowfish, and could potentially be used as a marker of exposure to environmental estrogenic chemicals. Further investigations of the expression of genes in the cDNA library, could establish other molecular markers of ecotoxicological interest in M. fluviatilis