Expanding non-invasive approaches for fish-health monitoring: A survey of the epidermal mucous metabolomes of phylogenetically diverse freshwater fish species.

There is a pressing need for more-holistic approaches to fisheries assessments along with growing demand to reduce the health impacts of sample collections. Metabolomic tools enable the use of sample matrices that can be collected with minimal impact on the organism (e.g., blood, urine, and mucus) and provide high-throughput, untargeted biochemical information without the requirement of a sequenced genome. These qualities make metabolomics ideal for monitoring a wide range of fish species, particularly those under protected status. In the current study, we surveyed the relative abundances of 120 endogenous metabolites in epidermal mucus across eight freshwater fish species belonging to seven phylogenetic orders. Principal component analysis was used to provide an overview of the data set, revealing strong interspecies relationships in the epidermal mucous metabolome. Normalized relative abundances of individual endogenous metabolites were then used to identify commonalities across multiple species, as well as those metabolites that showed notable species specificity. For example, taurine was measured in high relative abundance in the epidermal mucus of common carp (Cyprinus carpio), northern pike (Esox lucius), golden shiner (Notemigonus crysoleucas), rainbow trout (Oncorhynchus mykiss), and rainbow smelt (Osmerus mordax), whereas γ-amino butyric acid (GABA) exhibited a uniquely high relative abundance in flathead catfish (Pylodictis olivaris). Finally, hierarchical cluster analysis was used to evaluate species relatedness as characterized by both the epidermal mucous metabolome (phenotype) and genetic phylogeny (genotype). This comparison revealed species for which relatedness in the epidermal mucous metabolome composition closely aligns with phylogenetic relatedness (e.g., N. crysoleucas and C. carpio), as well as species for which these two measures are not well aligned (e.g., P. olivaris and Polyodon spathula). These, and other findings reported here, highlight novel areas for future research with fish, including development of epidermal mucous-based markers for non-invasive health monitoring, sex determination, and hypoxia tolerance.

Prioritization of Contaminants of Emerging Concern in Wastewater Treatment Plant Discharges Using Chemical:Gene Interactions in Caged Fish.

We examined whether contaminants present in surface waters could be prioritized for further assessment by linking the presence of specific chemicals to gene expression changes in exposed fish. Fathead minnows were deployed in cages for 2, 4, or 8 days at three locations near two different wastewater treatment plant discharge sites in the Saint Louis Bay, Duluth, MN and one upstream reference site. The biological impact of 51 chemicals detected in the surface water of 133 targeted chemicals was determined using biochemical endpoints, exposure activity ratios for biological and estrogenic responses, known chemical:gene interactions from biological pathways and knowledge bases, and analysis of the covariance of ovary gene expression with surface water chemistry. Thirty-two chemicals were significantly linked by covariance with expressed genes. No estrogenic impact on biochemical endpoints was observed in male or female minnows. However, bisphenol A (BPA) was identified by chemical:gene covariation as the most impactful estrogenic chemical across all exposure sites. This was consistent with identification of estrogenic effects on gene expression, high BPA exposure activity ratios across all test sites, and historical analysis of the study area. Gene expression analysis also indicated the presence of nontargeted chemicals including chemotherapeutics consistent with a local hospital waste stream. Overall impacts on gene expression appeared to be related to changes in treatment plant function during rain events. This approach appears useful in examining the impacts of complex mixtures on fish and offers a potential route in linking chemical exposure to adverse outcomes that may reduce population sustainability.

Transcriptional regulatory dynamics of the hypothalamic-pituitary-gonadal axis and its peripheral pathways as impacted by the 3-beta HSD inhibitor trilostane in zebrafish (Danio rerio).

To study mechanisms underlying generalized effects of 3ß hydroxysteroid dehydrogenase (HSD3B) inhibition, reproductively mature zebrafish (Danio rerio) were exposed to trilostane at two dosages for 24, 48, or 96 h and their gonadal RNA samples profiled with Agilent zebrafish microarrays. Trilostane had substantial impact on the transcriptional dynamics of zebrafish, as reflected by a number of differentially expressed genes (DEGs) including transcription factors (TFs), altered TF networks, signaling pathways, and Gene Ontology (GO) biological processes. Changes in gene expression between a treatment and its control were mostly moderate, ranging from 1.3 to 2.0 fold. Expression of genes coding for HSD3B and many of its transcriptional regulators remained unchanged, suggesting transcriptional up-regulation is not a primary compensatory mechanism for HSD3B enzyme inhibition. While some trilostane-responsive TFs appear to share cellular functions linked to endocrine disruption, there are also many other DEGs not directly linked to steroidogenesis. Of the 65 significant TF networks, little similarity, and therefore little cross-talk, existed between them and the hypothalamic-pituitary-gonadal (HPG) axis. The most enriched GO biological processes are regulations of transcription, phosphorylation, and protein kinase activity. Most of the impacted TFs and TF networks are involved in cellular proliferation, differentiation, migration, and apoptosis. While these functions are fairly broad, their underlying TF networks may be useful to development of generalized toxicological screening methods. These findings suggest that trilostane-induced effects on fish endocrine functions are not confined to the HPG-axis alone. Its impact on corticosteroid synthesis could also have contributed to some system wide transcriptional changes in zebrafish observed in this study.

Expression signatures for a model androgen and antiandrogen in the fathead minnow (Pimephales promelas) ovary.

Trenbolone, an anabolic androgen, and flutamide, an antiandrogen, are prototypical model compounds for agonism and antagonism of the androgen receptor. We hypothesized that 48 h exposures of female fathead minnows (Pimephales promelas) to environmentally relevant concentrations of these chemicals would alter genes regulated by the androgen receptor and that a mixture of the two compounds would block the effects. Gene expression in the ovaries was analyzed using a fathead minnow-specific 22,000-gene microarray. Flutamide altered abouttwicethe number of genes astrenbolone, most of which appeared to be through pathways not associated with the androgen receptor. A group of 70 genes, of which we could identify 37, were reciprocally regulated by trenbolone and flutamide. These are candidates for specific biomarkers for androgen receptor mediated gene expression. Four genes stand out as specifically related to reproduction: sperm associated antigen 8 (SPAG8), CASP8 and FADD-like apoptosis regulator (CFLAR), corticotropin releasing hormone (CRH), and 3beta-hydroxysteroid dehydrogenases (3beta-HSD). Three notable transcriptional regulators including myelocytomatosis viral oncogene homologue (MYC), Yin Yang 1 (YY1), and interferon regulator factor 1 (IRF1) may function as early molecular switches to control phenotypic changes in ovary tissue architecture and function in response to androgen or antiandrogen exposure.