Predicting Fecundity of Fathead Minnows (Pimephales promelas) Exposed to Endocrine-Disrupting Chemicals Using a MATLAB®-Based Model of Oocyte Growth Dynamics.
ABSTRACT: Fish spawning is often used as an integrated measure of reproductive toxicity, and an indicator of aquatic ecosystem health in the context of forecasting potential population-level effects considered important for ecological risk assessment. Consequently, there is a need for flexible, widely-applicable, biologically-based models that can predict changes in fecundity in response to chemical exposures, based on readily measured biochemical endpoints, such as plasma vitellogenin (VTG) concentrations, as input parameters. Herein we describe a MATLAB® version of an oocyte growth dynamics model for fathead minnows (Pimephales promelas) with a graphical user interface based upon a previously published model developed with MCSim software and evaluated with data from fathead minnows exposed to an androgenic chemical, 17?-trenbolone. We extended the evaluation of our new model to include six chemicals that inhibit enzymes involved in steroid biosynthesis: fadrozole, ketoconazole, propiconazole, prochloraz, fenarimol, and trilostane. In addition, for unexposed fathead minnows from group spawning design studies, and those exposed to the six chemicals, we evaluated whether the model is capable of predicting the average number of eggs per spawn and the average number of spawns per female, which was not evaluated previously. The new model is significantly improved in terms of ease of use, platform independence, and utility for providing output in a format that can be used as input into a population dynamics model. Model-predicted minimum and maximum cumulative fecundity over time encompassed the observed data for fadrozole and most propiconazole, prochloraz, fenarimol and trilostane treatments, but did not consistently replicate results from ketoconazole treatments. For average fecundity (eggs•female(-1)•day(-1)), eggs per spawn, and the number of spawns per female, the range of model-predicted values generally encompassed the experimentally observed values. Overall, we found that the model predicts reproduction metrics robustly and its predictions capture the variability in the experimentally observed data.
Project description:Cytochrome P450 aromatase catalyzes conversion of C19 androgens to C18 estrogens and is critical for normal reproduction in female vertebrates. Fadrozole is a model aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. However, little is known about the early impacts of aromatase inhibition on steroid production and gene expression in fish. Adult female fathead minnows (Pimephales promelas) were exposed via water to 0, 5, or 50µg fadrozole/L for a time-course of 0.5, 1, 2, 4, and 6h, or 0 or 50µg fadrozole/L for a time-course of 6, 12, and 24h. We examined ex vivo ovarian 17?-estradiol (E2) and testosterone (T) production, and plasma E2 concentrations from each study. Expression profiles of genes known or hypothesized to be impacted by fadrozole including aromatase (cytochrome P450 [cyp] 19a1a), steriodogenic acute regulatory protein (star), cytochrome P450 side-chain cleavage (cyp11a), cytochrome P450 17 alpha hydroxylase/17,20 lyase (cyp17), and follicle stimulating hormone receptor (fshr) were measured in the ovaries by quantitative real-time polymerase chain reaction (QPCR). In addition, broader ovarian gene expression was examined using a 15k fathead minnow microarray. The 5µg/L exposure significantly reduced ex vivo E2 production by 6h. In the 50µg/L treatment, ex vivo E2 production was significantly reduced after just 2h of exposure and remained depressed at all time-points examined through 24h. Plasma E2 concentrations were significantly reduced as early as 4h after initiation of exposure to either 5 or 50µg fadrozole/L and remained depressed throughout 24h in the 50µg/L exposure. Ex vivo T concentrations remained unchanged throughout the time-course. Expression of transcripts involved in steroidogenesis increased within the first 24h suggesting rapid induction of a mechanism to compensate for fadrozole inhibition of aromatase. Microarray results also showed fadrozole exposure caused concentration- and time-dependent changes in gene expression profiles in many HPG-axis pathways as early as 4h. This study provides insights into the very rapid effects of aromatase inhibition on steroidogenic processes in fish.
Project description:Mosquitoes function as important vectors for many diseases globally and can have substantial negative economic, environmental, and health impacts. Specifically, West Nile virus (WNv) is a significant and increasing threat to wildlife populations and human health throughout North America. Mosquito control is an important means of controlling the spread of WNv, as the virus is primarily spread between avian and mosquito vectors. This is of particular concern for avian host species such as the Greater sage-grouse (Centrocercus urophasianus), in which WNv negatively impacts fitness parameters. Most mosquito control methods focus on the larval stages. In North America, control efforts are largely limited to larvicides, which require repeated application and have potentially negative ecological impacts. There are multiple potential advantages to using indigenous fish species as an alternative for larval control including lowered environmental impact, decreased costs in terms of time and financial inputs, and the potential for the establishment of self-sustaining fish populations. We tested the efficacy of using fathead minnows (Pimephales promelas) as biological control for mosquito populations in livestock reservoirs of semiarid rangelands. We introduced minnows into 10 treatment reservoirs and monitored an additional 6 non-treated reservoirs as controls over 3 years. Adult mosquitoes of species known to transmit WNv (e.g., Culex tarsalis) were captured at each site and mosquito larvae were also present at all sites. Stable isotope analysis confirmed that introduced fathead minnows were feeding at the mosquito larvae trophic level in all but one treatment pond. Treatment ponds demonstrated suppressed levels of mosquito larva over each season compared to controls with a model-predicted 114% decrease in larva density within treatment ponds. Minnows established self-sustaining populations throughout the study in all reservoirs that maintained sufficient water levels. Minnow survival was not influenced by water quality. Though minnows did not completely eradicate mosquito larvae, minnows are a promising alternative to controlling mosquito larvae density within reservoirs. We caution that careful site selection is critical to avoid potential negative impacts, but suggest the introduction of fathead minnows in reservoirs can dramatically reduce mosquito larva abundance and potentially help mitigate vector-borne disease transmission.
Project description:Aromatase, a member of the cytochrome P450 superfamily, is a key enzyme in estrogen synthesis and is responsible for the aromatization of androgens into estrogens. Here, we used an integrated approach to better understand the effects of aromatase inhibition as well as the mechanisms involved in the adaptation and recovery process. We exposed female fathead minnows (Pimephales promelas) to 30 μg/L of a model aromatase inhibitor, fadrozole, during 8 days (exposure phase). Fish were then held in clean water for 8 more days (recovery phase). Samples were collected at 1, 2, 4, and 8 days of both the exposure and the recovery phase. We used transcriptomics, metabolomics, and network inference to understand changes and infer connections at the transcript and metabolite level in the ovary. Other apical endpoints such as plasma estradiol, testosterone, and vitellogenin levels were also measured. An integrated analysis of the data revealed changes in gene expression consistent with increased testosterone. Metabolites such as glycogen and taurine were strongly correlated with increased testosterone levels. Comparison of in vivo and ex vivo data suggested the accumulation of steroidogenic enzymes, including aromatase, as a mechanism to compensate for aromatase inhibition. We exposed female fathead minnows (Pimephales promelas) to 30 μg/L of a model aromatase inhibitor, fadrozole, during 8 days (exposure phase). Fish were then held in clean water for 8 more days (recovery phase). Samples were collected at 1, 2, 4, and 8 days of both the exposure and the recovery phase.
Project description:Yeast-like symbiotes (YLS), harbored in the abdomen fat-body cells of the rice brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), are vital to the growth and reproduction of their host. It is feasible to manipulate BPH infestation on rice by inhibiting YLS using fungicide. In this study, the fungicide propiconazole was injected into the hemolymph of BPH thorax via microinjection to investigate its effect on YLS, especially the dominant species, Hypomyces chrysospermus, and their host BPH. Propiconazole markedly reduced the total number of YLS and H. chrysospermus in BPH hemolymph and fat body, thereby leading to an obvious higher mortality and lower fecundity of BPH than the negative control (PBS, phosphate buffer solution). After microinjecting propiconazole, the survival rate of BPH nymphs at the 5th instar was significantly lower than that obtained after PBS treatment. Eight days after propiconazole microinjection, the BPH survival rate dropped to 40%, only half of BPH survival rate treated with PBS microinjection. For female adults (1-day-old), there were significant differences in the survival rates between BPHs treated with propiconazole and those treated with PBS at days 5-8. The fecundity of BPH decreased significantly by microinjecting propiconazole and averaged only 229 eggs per female, which was 20% less than that of the negative control. Furthermore, we reared BPH on the susceptible variety TN1 sprayed with propiconazole to prove the feasibility manipulating field occurrence of BPH by inhibiting YLS using fungicides. The number of YLS and H. chrysospermus in BPH obviously declined. Subsequently, the survival rate and fecundity of BPH significantly decreased after feeding on rice treated with propiconazole. Meanwhile, the propiconazole residue was detected in the hemolymph and gut of BPH by HPLC analysis within 1 day of feeding. Inhibiting YLS using fungicides was a novel and effective way to control BPH infestation.
Project description:One objective in developing adverse outcome pathways (AOPs) is to connect biological changes that are relevant to risk assessors (i.e., fecundity) to molecular and cellular-level alterations that might be detectable at earlier stages of a chemical exposure. Here, we examined biochemical responses of fathead minnows (Pimephales promelas) to inform an AOP relevant to spironolactone's activation of the androgen receptor, as well as explore other biological impacts possibly unrelated to this receptor. Liquid chromatography with high resolution mass spectrometry (LC-MS) was used to measure changes in endogenous polar metabolites in livers of male and female fish that were exposed to five water concentrations of spironolactone (0, 0.05, 0.5, 5, or 50?gL-1) for 21days. Metabolite profiles were affected at the two highest concentrations (5 and 50?gL-1), but not in the lower-level exposures, which agreed with earlier reported results of reduced female fecundity and plasma vitellogenin (VTG) levels. We then applied partial least squares regression to assess whether metabolite alterations covaried with changes in fecundity, VTG gene expression and protein concentrations, and plasma 17?-estradiol and testosterone concentrations. Metabolite profiles significantly covaried with all measured endpoints in females, but only with plasma testosterone in males. Fecundity reductions occurred in parallel with changes in metabolites important in osmoregulation (e.g., betaine), membrane transport (e.g., l-carnitine), and biosynthesis of carnitine (e.g., methionine) and VTG (e.g., glutamate). Based on a network analysis program (i.e., mummichog), spironolactone also affected amino acid, tryptophan, and fatty acid metabolism. Thus, by identifying possible key events related to changes in biochemical pathways, this approach built upon an established AOP describing spironolactone's androgenic properties and highlighted broader implications potentially unrelated to androgen receptor activation, which could form a basis for the development of an AOP network.
Project description:<h4>Background</h4>Endocrine disrupting chemicals (e.g., estrogens, androgens and their mimics) are known to affect reproduction in fish. 17?-ethynylestradiol is a synthetic estrogen used in birth control pills. 17?-trenbolone is a relatively stable metabolite of trenbolone acetate, a synthetic androgen used as a growth promoter in livestock. Both 17?-ethynylestradiol and 17?-trenbolone have been found in the aquatic environment and affect fish reproduction. In this study, we developed a physiologically-based computational model for female fathead minnows (FHM, Pimephales promelas), a small fish species used in ecotoxicology, to simulate how estrogens (i.e., 17?-ethynylestradiol) or androgens (i.e., 17?-trenbolone) affect reproductive endpoints such as plasma concentrations of steroid hormones (e.g., 17?-estradiol and testosterone) and vitellogenin (a precursor to egg yolk proteins).<h4>Results</h4>Using Markov Chain Monte Carlo simulations, the model was calibrated with data from unexposed, 17?-ethynylestradiol-exposed, and 17?-trenbolone-exposed FHMs. Four Markov chains were simulated, and the chains for each calibrated model parameter (26 in total) converged within 20,000 iterations. With the converged parameter values, we evaluated the model's predictive ability by simulating a variety of independent experimental data. The model predictions agreed with the experimental data well.<h4>Conclusions</h4>The physiologically-based computational model represents the hypothalamic-pituitary-gonadal axis in adult female FHM robustly. The model is useful to estimate how estrogens (e.g., 17?-ethynylestradiol) or androgens (e.g., 17?-trenbolone) affect plasma concentrations of 17?-estradiol, testosterone and vitellogenin, which are important determinants of fecundity in fish.
Project description:We examined whether exposure to municipal wastewater effluent (MWWE) compromised the stress performance of laboratory-reared fathead minnows (Pimephales promelas) in a field setting. Adult minnows were caged at two sites upstream and three sites downstream of wastewater treatments plants (WWTPs) discharging MWWE into the Bow River, Calgary, Alberta, Canada. At each site one group of fish was sampled after a 26 day exposure to MWWE, while another group was subjected to 1-min air exposure followed by 60-min confinement and then sampled. Fish morphometrics and proximate composition were measured, and whole-body cortisol, glucose and lactate levels assessed as markers of the stress response. The whole-body protein, glycogen and lipid content were higher at the site closest to a WWTP outfall relative to the other downstream and upstream sites. There were no significant differences in whole-body cortisol levels in minnows sampled at sites either upstream or downstream of WWTPs. Acute stressor exposure significantly elevated whole-body cortisol levels in all groups, and this response was not modified by the location of the sampling sites. The whole-body metabolite profile, including glucose and lactate levels, were significantly higher in fish caged immediately downstream from WWTP inputs relative to upstream sites. There was an acute-stressor-mediated increase in whole-body lactate, but not glucose, levels and this response was independent of sampling site. The results reveal that the capacity to evoke an acute stress response was not compromised in fathead minnows caged for 26 days downstream of WWTPs in the Bow River. However, there were changes in the whole-body proximate composition and metabolite levels immediately downstream from the WWTP outfall suggesting greater accumulation of energy stores in these fish. Taken together, our results suggest that environmental factors in addition to contaminants, including higher water temperature and nutrient availability, influence the impact of MWWEs on fish stress performance.
Project description:Understanding population-level effects of environmental stressors on aquatic biota requires knowledge of the direct adverse effects of pollutants on individuals and species interactions that relate to survival and reproduction. Here, we connect behavioural assays with survival trials and a modelling approach to quantify changes in antipredator escape performance of a larval freshwater fish following exposure to an environmental oestrogen, and predict changes in population abundance. We quantified the effects of short-term (21 days) exposure to 17?-oestradiol (E2) on the antipredator escape performance of larval fathead minnows (Pimephales promelas) and the probability of predation by a natural predator, the bluegill sunfish (Lepomis macrochirus). Compared with unexposed minnows, minnows exposed to environmentally relevant concentrations of E2 that approach total oestrogenic activity of wastewater-dominated environments (38 and 103 ng l-1) had delayed response times and slower escape speeds, and were more susceptible to predation. Incorporating these data into a stage-structured population model demonstrated that enhanced predation mortality at the larval stage can result in population declines. These results indicate that subtle, sub-lethal shifts in the behaviour of individuals due to human-mediated environmental change can impact species interactions with measurable population-level effects. Such changes have the potential to alter higher-order trophic interactions and disrupt aquatic communities.
Project description:During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20) of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas) and brassy minnows (Hybognathus hankinsoni). Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (? 30-32 nm), with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV). The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV) isolated from bluegill (Lepomis macrochirus). Based on complete polyprotein analysis, the FHMPV shared 58% (P1), 33% (P2) and 43% (P3) amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus) within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.
Project description:In this study, female fathead minnows (FHM) were exposed to waterbourne phenanthrene (201.8 Âµg/L) or a solvent control for 7 weeks. Fish were tested for behavioral differences in a modified behavioral test prior to euthansia. Hypothalami were excised and stored for microarray analyses. Fish were exposed to one dose of phenanthrene. Female and male hypothalami were analyzed (n=8 per group, control vs. treatment); liver was also analyzed. N=7 control and 8 phenanthrene. In this study, a subchronic exposure to phenanthrene was investigated