Environ. Sci. Technol. 2009, 43, 8400–8405
Parentage Outcomes in Response to Estrogen Exposure are Modified by Social Grouping in Zebrafish TOBIAS S. COE,† PATRICK B. HAMILTON,† DAVID HODGSON,‡ GREGORY C. PAULL,† A N D C H A R L E S R . T Y L E R * ,† Ecotoxicology and Aquatic Biology Research Group, University of Exeter, Exeter, United Kingdon, and Centre for Ecology and Conservation, University of Exeter, Cornwall Campus, Penryn, United Kingdom
Received May 12, 2009. Revised manuscript received September 24, 2009. Accepted September 24, 2009.
Evidence has recently emerged that endocrine-disrupting chemicals (EDCs) can affect various behaviors, including dominance and aggression in social groups, including fish. This study investigated the effect of short-term exposure of male adult zebrafish to 17R-ethinylestradiol (EE2) on subsequent reproductive output and parentage in colonies with differing numbers of competing males. It was predicted that impacts of EDCs might differ in social groups of fish of differing size because of the greater costs of maintaining dominance hierarchies in large groups. Adult male zebrafish were exposed for 14 days to clean water, 2 ng/L EE2 or 10 ng/L via the water, prior to placement into colonies in clean water with unexposed females. Exposure to EE2 at the concentrations adopted prior to the breeding trials did not significantly affect subsequent colony reproductive output. The reproductive success of the most reproductively successful (MRS) male within colonies containing two males (relative to controls) was also unaffected. There was, however, a significant impact of previous EE2 exposure in tanks containing four males, resulting in a reduction in paternity for the most successful male. Hence, nonlethal behavioral impacts of even short-term exposure to EDCs can have significant impacts on social dominance hierarchies and population genetic diversity.
Introduction The detrimental impact of exposure to endocrine-disrupting chemicals (EDCs) on wildlife and humans is an issue of global concern (1, 2). Impacts of EDCs are most commonly reported for organisms living in aquatic environments, which can be subject to high levels of EDC input, for example, rivers receiving inputs from wastewater treatment works (WWTW) effluent (3-5). Direct exposure of fish to estrogenic effluents has been shown to adversely affect sexual development (6-8), and incidences of intersex and disruptions in reproductive function in populations of wild fish in U.K. rivers are strongly correlated with exposure to these effluents (9-11). Some of * Corresponding author phone: +44 (0)1392 264450; fax: +44 (0)1392 263434; e-mail:
[email protected]; address: Hatherly Laboratory, Prince of Wales Road, Exeter, Devon EX4 4PS, United Kingdom. † University of Exeter. ‡ University of Exeter, Cornwall Campus. 8400
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the main estrogenic EDCs identified that are discharged through WWTW include natural steroid estrogens and pharmaceutical estrogens used in contraceptive and menopause treatments, e.g., 17R-ethinylestradiol (EE2) and equine estrogens (3, 12). Laboratory exposures to the specific estrogen components contained in WWTW effluents have been shown to have similar impacts on fish. The most potent of these estrogens is EE2, a component of the contraceptive pill that is typically found in WWTW effluents in the low ng/L range (13). Exposure to EE2 has been shown to disrupt sexual development and reduce reproductive capacity in a range of fish species, including zebrafish Danio rerio (14, 15), fathead minnow Pimephales promelas (16, 17), Japanese medaka Oryzias latipes (18, 19), and roach Rutilus rutilus (20). Longterm exposure to EE2 (4-6 ng/L) has even been shown to result in a complete cessation of reproduction, leading to eventual population collapse (21, 22). Attempts to investigate the impacts of EDCs at the population level in the ambient environment have used modeling approaches (especially matrix population models) to estimate how changes in individual life history parameters such as fecundity and egg viability affect demographic rates, particularly population growth rate (23). Such approaches typically predict that exposure to EDCs, including at environmentally relevant concentrations, may result in a reduction in population growth rate, which may lead to subsequent population decline (16, 24, 25). Impacts of this magnitude in wild fish populations, however, have not been reported. A further limitation of these modeling approaches is that they extrapolate from the level of the individual to the population and ignore interactions occurring at the group and subpopulation level. Previous studies have shown that changes in behavior and patterns of parentage induced by exposure to EDCs (specifically for estrogen via reducing the reproductive fitness of males) may have implications for fish populations, resulting in changes in natural patterns of gene flow and reproduction (26-28). Similar reductions in reproductive fitness have been found as a result of increasing group density in the absence of chemical exposure (29). Combined effects of social grouping and EDC exposure on breeding outcome have not be studied previously. In this study, we investigated how the number of males paired with individual females and previous exposure of male zebrafish to EE2, including at an environmentally relevant concentration, affected subsequent breeding dynamics and paternity. Zebrafish Danio rerio was chosen for this study because it has a group-spawning reproductive strategy, similar to that found in many fish species, and is easily manipulated for studies of this nature. Furthermore, its behavior and natural patterns of parentage and reproduction are well-described (29-31) and an extensive suite of DNA microsatellite markers is available for use in parentage assessments (27). EE2 was used as the test chemical as it is the most potent environmental estrogen, disrupting sexual development and causing feminization of male fish, including at concentrations found in WWTW effluents (5, 13, 32, 33), and it is believed to be a key contributor to the feminized responses seen in wild fish in U.K. rivers (34).
Methods Test Fish. Adult zebrafish were cross-bred in the laboratory at the University of Exeter from two existing stocks of fish: the first, a wild Indian Karyotype (WIK), originally obtained from the Max Planck Institute, Tu ¨ bingen, Germany, and the second, an F2 generation of truly wild fish obtained from 10.1021/es902302u CCC: $40.75
2009 American Chemical Society
Published on Web 10/07/2009
FIGURE 1. Experimental system (including temporal block design) for exposure of male zebrafish to EE2, prior to their placement into breeding colonies with unexposed females. Bangladesh via Carl Smith at the University of Leicester. Breeding was carried out by multiple pairings of two adult male F2 wild fish with one adult female WIK fish. The resulting offspring that were used in the subsequent breeding experiments retained the high degree of genetic variability found in the F2 wild Bangladesh fish (35), facilitating parentage assignment and the domestic temperament of the WIK strain. Fish were maintained according to ref 36. Chemicals and Chemical Dosing. The EE2 exposure was run with solvent-free dosing. EE2 with a purity of 98% was obtained from Sigma, U.K. The appropriate volume of stock solution of 20 mg EE2/L acetone (100%) was added into 2.5 L dosing bottles and was left for ∼30 min to allow the acetone to evaporate. Reverse osmosis water was then added to the dosing bottles, and they were placed on magnetic stirrers, where they remained for the duration of the dosing until replenishment of the dosing solution. Concentrations in the dosing bottles were 1 µg/L (for experimental exposure at 10 ng/L) and 0.2 µg/L (for experimental exposure at 2 ng/L). From the dosing bottles, EE2 was delivered independently to the mixing chambers at a rate of 10 mL/h, and clean water was supplied to each mixing chamber at a rate of 1 L/h, with flow rates checked daily. Water was then pumped by peristaltic pump from the mixing chambers into the exposure tanks at a rate of 400 mL/h/exposure tank, with each mixing chamber supplying two exposure tanks. Tap water was filtered by reverse osmosis (RO; Osmonics E625 with cellulose membranes; GE Water and Process Technologies, Trevose, PA) and was then reconstituted with Analar grade mineral salts to concur with U.S. Environmental Protection Agency (EPA) guidelines on standardized synthetic freshwater. Conductivity was 304 ( 7 µS cm-1, and pH was 7.0 ( 0.05. Experimental Procedure. Because of the large number of tanks involved in the overall experiment, the experimental design adopted was a fully crossed, blocked approach (two replicates of each experimental scenario per week), repeated weekly over six weeks to give 12 replicates of each treatment (Figure 1). The weekly intervals are hereafter referred to as the timing of the sequential exposures and were included in subsequent statistical analyses as a variable with six levels. Each week, adult zebrafish from a stock tank were sexed,
and 14 male fish were randomly assigned to each of the three exposure regimes (control-dilution water, 2 ng EE2/L, or 10 ng EE2/L), where they were then exposed for 14 days. In the experimental breeding tanks, there were three male: female ratios, giving a total of nine experimental scenarios (three exposure × three male:female ratios). At the end of the 14 day EE2 exposure, each of the experimental tanks was randomly assigned one of the nine experimental scenarios, and the corresponding number of male fish, from the appropriate exposure, were placed in the tank, with a randomly assigned unexposed female. The fish were allowed to acclimate for 60 h, and then eggs were collected daily, 1-2 h after the artificial dawn for 5 days, using an egg collection system that avoided disturbance of fish in the tanks (37). The eggs were washed and counted, and unfertilized or fungal infected eggs were discarded. Fertilized embryos were then incubated in plastic containers for 24 h to increase DNA quantity and then stored in 100% ethanol for subsequent parentage analysis. The number of fertilized and viable embryos was counted at 1-2 hpf and also at 26 hpf. After 5 days of egg collection, all fish were killed by a lethal dose of benzocaine and destruction of the brain, according to U.K. Home Office Animal License guidelines. The wet weight and fork length of each fish were recorded and a fin-clip taken and stored in 100% ethanol for subsequent parentage analysis. Blood was collected from each fish using heparinised capillary tubes and centrifuged to separate the plasma from the blood cells, and then the plasma was stored at -20 °C. Water Chemistry. Water samples (total volume of 1 L) were collected from each exposure tank twice weekly and stabilized by adding methanol and acetic acid to a final concentrations of 5% and 1%, respectively. Water samples were then passed through Sep-Pack C18 cartridges (Waters Corporation, U.S.A.). EE2 was later eluted, and concentrations of the extracted samples were measured by gas chromatography-mass spectrometry by the Environment Agency, National Laboratory Service, Nottingham (38). The detection limit was 0.140 ng EE2/L. Measured mean concentrations of EE2 were 2.55 ( 0.27 ng/L and 8.36 ( 0.85 ng/L for the 2 ng/L VOL. 43, NO. 21, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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and 10 ng/L nominals, respectively, and non-detectable (less than 0.140 ng/L) in the control tanks. Plasma 11-Ketotestosterone Quantification. 11-Ketotestosetrone (11-KT) is one of the major androgens in male fish and is associated with dominance behavior (39). Where sufficient blood was obtained, 11-KT was quantified in the blood plasma using radioimmunoassay (RIA), according to the method described in Scott et al. (40). The detection limit was 2.35 ng/mL 11-KT. For the statistical analysis, any samples below the detection limit of the assay were assigned a value of half of that of the detection limit (41). Parentage. Parentage analyses were conducted for 10 of the tanks with two males and five of the tanks with four males (cost limiting). Twenty-four embryos from each tank containing two males and 48 from each tank containing four males were randomly selected for parentage analysis, in direct proportion to the number of eggs laid on each day. Thus, if more eggs were laid on a particular day, a greater number were sampled from this day. DNA was extracted from the parental fins and from embryos using ammonium acetate precipitation (42). In total, 1080 embryo and fish samples were analyzed in the parentage studies. Each sample was genotyped using six DNA microsatellite markers, five of which have been used previously for parentage analysis (27). An additional locus, Z4830 (www.zfin.org), was used in order to increase the discriminatory power for the parentage analysis in this study. The PCR protocol for this locus was as for Z249 and Z20450 in Coe et al. (27). Parentage was assigned with the program Probmax, version 1.3 (43). A total of 94.4% of all embryos tested for the tanks with two males were assigned to a single parental pair, and 95.0% of all embryos tested for the tanks with four males were assigned to a single parental pair. Embryos unable to be assigned to a single parental pair were not included in the subsequent statistical analysis. Statistical Analysis. All statistical analyses were conducted with appropriate model simplification, that is the removal of successive nonsignificant terms to produce minimal adequate models (44) using the software R 2.8.1 (45). Standard model checks were used to verify normality and homogeneity of standardized residuals (46). Data for egg counts and egg viability were analyzed using generalized linear models (GLM) with appropriate error structures (quasipoisson for count data, binomial or quasibinomial for proportional data, Gaussian for all other data), accounting for overdispersion where necessary (44). Data for 11-KT concentrations were analyzed using a generalized linear mixed model (GLMM), including the variables of individual fish and timing of exposure as random factors within the model to account for statistical nonindependence. Data for proportional paternity was analyzed using a GLMM, with binomial errors. Tests and error structures used are given with the corresponding results. Interactions between and main effects of explanatory variables were removed from models, sequentially using the criterion of nonsignificance with R ) 0.05. Where given, values are expressed as the mean ( one standard error.
Results Impacts of Number of Males and EE2 Exposure on Egg Numbers and Viability. The mean total number of eggs produced per colony over 5 days was 277 ( 20.3 for colonies with one male, 241 ( 22.4 for colonies with two males, and 147 ( 14.7 for colonies with four males. The number of males within a colony had a significant effect on the number of viable embryos (GLM with quasipoisson errors; F ) 15.570; df ) 2, 89; p < 0.001), with embryo production decreasing with an increasing number of males (Figure 2). Exposure of male fish to EE2 prior to the breeding studies had no significant effect on the number of viable embryos produced 8402
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FIGURE 2. Total egg output in colonies of zebrafish, with different numbers of competing males with different histories to estrogen exposure (no previous exposure or exposure to either 2 or 10 ng EE2/L). The central line within the box is the median. The box represents the upper and lower quartiles, and the whiskers the 95% confidence intervals. Open circles (O) are outliers beyond the 95% confidence intervals. Only the number of males had a significant impact on the average egg count per colony (p < 0.001). per colony (GLM with quasipoisson errors; F ) 0.4283; df ) 2, 89; p ) 0.653), and there was no effect of timing of exposure over the study period (F ) 2.1245; df ) 5, 92; p ) 0.06996). There was no significant statistical interaction between the number of males present in each colony and exposure. A GLM with quasibinomial errors showed that there was no significant effect of either the number of male fish (F ) 0.1737; df ) 2,73; p ) 0.8409) or timing of exposure (F ) 2.127; df ) 4,71; p ) 0.08634) on the proportion of embryos inviable at 26 hpf. However, there was a significant effect of male EE2 exposure on the subsequent proportion of viable embryos at 26 hpf (F ) 5.5062, df ) 2,73; p ) 0.005984). The proportion of viable embryos at 26 hpf was highest in colonies in which males had previously been exposed to 2 ng/L EE2 (Figure 3). There was no significant statistical interaction between the number of males and previous exposure and EE2. Impacts of Exposure and Number of Males on Reproductive Success of Male Fish. In controls, the MRS male in colonies containing two males sired on average 71.9% of the offspring and in colonies containing four males 58.1% of the offspring. The results for the controls demonstrate that the number of males within a colony had a significant effect on the proportional reproductive success of the MRS male (Figure 4). A GLMM with binomial errors found a significant interaction between previous male exposure to EE2 and number of males within a colony (χ2 ) 7.4798, df ) 1, p ) 0.00624) and, while exposure to EE2 did not change the proportional reproductive success of the MRS male in colonies containing two males, it did in colonies containing four males, eroding their success rate (Figure 4). For high EE2 concentration exposure, the MRS male in colonies containing two males sired on average 72.3% of the offspring (versus 71.9% in controls) and 42.4% in colonies containing four males (versus 58.1% in controls). The results demonstrate that the proportional reproductive success of the MRS male was affected by previous exposure to EE2 in the different sized colonies.
FIGURE 5. Proportion of offspring sired by males in colonies with four males, for which experimental males were previously exposed to 10 ng/L EE2 or were unexposed throughout (controls). The central line within the box is the median. The box represents the upper and lower quartiles, and the whiskers the 95% confidence intervals. Open circles (O) are outliers. Parentage success was significantly more even in colonies in which males had been previously exposed to EE2 (p ) 0.01587). FIGURE 3. Proportion of nonviable embryos in colonies of zebrafish, with different numbers of competing males with different histories of estrogen exposure (no previous exposure or exposure to either 2 or 10 ng EE2/L). The central line within the box is the median. The box represents the upper and lower quartiles, and the whiskers the 95% confidence intervals. Open circles (O) are outliers beyond the 95% confidence intervals. Only previous exposure had a significant impact on the proportion of inviable eggs, with the highest proportion of viable eggs in those colonies containing males previously exposed to 2 ng/L EE2 (p ) 0.005984).
control colonies with four males (Mann-Whitney U test, W ) 24, p ) 0.01587; Figure 5). Impacts of EE2 Exposure and Number of Males on 11KT Concentrations in Male Fish. 11-KT concentrations were 4.83 ( 0.80 ng/mL in the controls, 5.57 ( 0.67 ng/mL in fish exposed to 2 ng EE2/L, and 4.11 ( 0.66 in fish exposed to 10 ng EE2/L. There was no effect of previous male exposure (GLMM, χ2 ) 2.407, df ) 2, p ) 0.3001) or the number of males present within a colony (χ2 ) 1.8157, df ) 1, p ) 0.1778) on the concentration of 11-KT in the blood of male fish at termination of the breeding experiment.
Discussion
FIGURE 4. Proportion of offspring sired by the reproductively dominant male within colonies with different numbers of competing males for experimental animals previously exposed to 10 ng EE2 /L or that were unexposed throughout (controls). The central line within the box is the median. The box represents the upper and lower quartiles, and the whiskers the 95% confidence intervals. Open circles (O) are outliers. Significant differences between groups are indicated by letters. As a result of this effect, the distribution of parental success was more even in tanks with four males that had been previously exposed to EE2, with thus an overall change in male parentage outcome in the colony progeny. The coefficient of variation of paternity success (expressed as the standard deviation and mean parental success per colony) was lower in colonies previously exposed to EE2 than in the
Effects of Number of Males and Previous Exposure to EE2 on Egg Output and Embryo Viability. The finding that increasing the number of males in a breeding colony resulted in a decrease in total reproductive output (Figure 2) may have come about from a number (and combination) of factors. These factors include increased total egg predation and/or a suppression of female reproductive output due to increased aggression toward her from competing males. This has been reported previously for zebrafish colonies (31), and highlights that reproductive output in group-spawning fish is significantly affected by the density and sex ratio of the breeding colony. This has implications when comparing responses to chemical exposure on reproduction for different sized and constituted groups of spawning fish. Previous studies investigating the impacts of exposure to EE2 on reproduction in breeding colonies of zebrafish (and for similar exposure concentrations) have exposed male and female fish and have typically found reductions in fecundity (15, 21). Some of these effects have been shown to come about via direct effects on the male physiology but some may potentially also be mediated by effects on the females or indeed as a consequence of effects on interactions between the sexes (47). Estrogen exposure has been shown to affect typical male reproductive behaviors (19, 26, 28, 48), but there has been little focus on the impacts of estrogen exposure on females or the responses of females to exposed males even though these effects (if they occur) may be more important for population level outcomes (49). The results from this study showed that short-term exposure of males prior to being placed in breeding colonies did not affect egg output in the subsequent breeding colonies maintained in clean water (Figure 2). This is in contrast with some of the previous studies that exposed male and female zebrafish to EE2. This suggests that some of the reported effects of EE2 on reproductive output in this species may VOL. 43, NO. 21, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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have been mediated by the females or via effects on interactions between the sexes. However, the lack of any significant effects of EE2 on egg production in this study may rather have been because any effects of the short-term exposure on adult males were lost rapidly after termination of the chemical exposure. Indeed, 7 days after the EE2 exposure, there were no differences in the circulating concentrations of 11-KT in exposed males compared with controls. Male exposure to EE2 had a significant subsequent effect on the proportion of viable embryos (Figure 3). Interestingly, proportional viability was highest in colonies in which males had been previously exposed to 2 ng EE2/L compared with control colonies and for colonies with males previously exposed to 10 ng EE2/L. We do not know the mechanism for this effect, but it is possible that there was a subtle alteration in interactive behaviors facilitating more effective fertilization of the eggs spawned by the female. Alternately, exposure to a low dose of EE2 has been previously shown to increase sperm quality (15), and this may have increased fertilization success or egg viability. Effects of EE2 Exposure on Reproductive Success in Colonies with Different Numbers of Competing Males. In the absence of any chemical treatment, the number of males had a significant impact on the reproductive success of the MRS male within each colony. With more males in a colony competing for a single female, there was a decrease in the proportion of offspring sired by the most dominant male. Similar results have been found in a previous study in which the reproductive success of male zebrafish in colonies of differing densities was examined (29). The effect of exposure to EE2 on the proportion of offspring sired by the MRS male was dependent on the number of males present in a colony. The reproductive success of the MRS male was unaffected in colonies containing two males but was significantly reduced in colonies containingfour males (Figure 4). The erosion of the reproductive success of the MRS male within colonies containing four males meant that there was an effect on the overall parentage distribution between the competing males (Figure 5; the coefficient of variation of paternity success was significantly lower in colonies in which males had been previously exposed to EE2). Comparing these findings directly with previous studies is difficult as the breeding dynamics differ across these studies. In the work of Coe et al. (27) and Colman et al. (48), reduction in reproductive success of the most reproductively successful (MRS) male occurred in colonies where males and females were exposed to EE2 (at a concentration of 10 ng/L (27) or a very high concentration of 50 ng/L (48)) in colonies containing two males and two females, and two males and three females, respectively. It is clear from these results, however, that colony structure (particularly the number of females present and as a result, the sex ratio) has an influence on parentage outcome when colonies are exposed to environmental estrogens. These differences may be manifest by alterations in male-male interactions, male-female interactions (e.g., mate choice), and/or female-female interactions (50, 51). These findings have implications for patterns of parentage in wild populations of fish. While the number of males was experimentally manipulated in this study, previous studies have shown that female zebrafish display mate choice, and males compete for females, with some males establishing territories (30, 31). Thus, it is possible that exposure to estrogens in wild fish may result in disruptions in natural patterns of parentage, particularly as competition between males increases, represented here by the increasing number of males paired with each female. The erosion of the breeding hierarchy between the subdominant males in this breeding scenario is a novel 8404
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finding, and it not only further highlights the intricacies and complexities of reproductive success within group-spawning fish with differing levels of competition but also illustrates how differential effects of chemical treatments can result from different colony structures.
Acknowledgments We thank Jan Shears, Tessa Scown, and Amy Filby at the University of Exeter and Alexander Scott (EE2 radioimmunoassay) at the Centre for Environment, Fisheries and Aquaculture Science, for their assistance with the practical work. C.R.T. and D.H. were funded by the U.K. Environment Agency, Department of the Environment, Food and Rural Affairs, and University of Exeter.
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