Sex in troubled waters: Widespread agricultural contaminant disrupts reproductive behaviour in fish
Introduction
Chemical pollutants have accumulated in ecosystems globally, endangering wildlife, ecosystem function and human health (Schwarzenbach et al., 2006). One class of chemical pollutant, known as endocrine disrupting chemicals (EDCs), comprises environmental contaminants with the capacity to disrupt the natural hormonal functioning of organisms (Colborn et al., 1993). Endocrine disruptors are of particular concern given their extreme potency, with exposure to concentrations as low as nanograms per litre having deleterious effects, as well as the propensity of some EDCs to bioaccumulate, persist temporally and act transgenerationally (Diamanti-Kandarakis et al., 2009). Conventionally, studies in ecotoxicology have focussed on direct mortality and chronic sub-lethal effects of EDCs on development and reproduction (Melvin and Wilson, 2013). However, EDCs can also induce alarming changes in behaviour. Indeed, the particular sensitivity of behaviour to EDCs has driven recent interest in behavioural ecotoxicology as a tool for investigating endocrine disruption at environmentally relevant pollutant concentrations (reviewed in Melvin and Wilson, 2013). Existing studies in behavioural ecotoxicology typically focus on EDCs that disrupt gonadal steroid signalling by interacting with vertebrate estrogen or androgen receptors, as chemical interference with this pathway has the potential to disrupt sexual selection (e.g., Saaristo et al., 2009). However, the vast majority of these efforts have concentrated on EDCs with estrogenic activity. This is surprising because the handful of studies that have considered androgenic EDCs suggest that they are also capable of markedly altering animal behaviour (e.g., Hoffmann and Kloas, 2012).
An androgenic EDC of particular concern is 17β-trenbolone, the most bioactive metabolite of trenbolone acetate, a hormonal growth promotant used extensively in livestock production around the world (Kolodziej et al., 2013). Trenbolone acetate is a powerful steroid, with androgenic and anabolic potency 15–50 times greater than testosterone (Kolodziej et al., 2013, Neumann, 1976). Its metabolite 17β-trenbolone acts as a powerful androgen receptor agonist in the environment, is highly temporally persistent (with a half-life of approximately 260 days; Schiffer et al., 2001) and has been repeatedly detected in aquatic environments associated with feedlot operations. Detected concentrations of 17β-trenbolone range from ≤ 20 ng/L in diffuse run-off (Durhan et al., 2006), to as high as 162 ng/L in fields directly receiving effluent (Gall et al., 2011). Recent studies report that exposure to 17β-trenbolone adversely impacts physiological and morphological endpoints in fish species (e.g., Morthorst et al., 2010). However, despite the potency and widespread global use of 17β-trenbolone, very little is known about its effects on behaviour. This is concerning as the ability of animals to produce and maintain behaviour appropriate to their environment is fundamental for survival and reproduction, so that disruption of these behaviours can have dire ecological and evolutionary consequences (reviewed in Candolin and Wong, 2012).
The mating system of the guppy, Poecilia reticulata, is ideal for investigating the effects of 17β-trenbolone on reproductive behaviour. The guppy is a small, live-bearing freshwater fish native to north-eastern South America that has a widespread global distribution, precipitated by numerous deliberate and accidental introductions (Lindholm et al., 2005). Importantly, throughout their range, guppies have the potential to be exposed to 17β-trenbolone, as they are known to inhabit water bodies receiving agricultural waste (e.g., Araújo et al., 2009, López-Rojas and Bonilla-Rivero, 2000, Widianarko et al., 2000). Male guppies employ two alternate mating strategies, either soliciting copulations from females through courtship (‘sigmoid displays’) or coercing copulations through unsolicited ‘sneaking’ behaviour (Luyten and Liley, 1991). The latter involves males surreptitiously approaching females from behind to insert their gonopodium (a modified anal fin serving as an intromittent organ) into the female's genital pore (Luyten and Liley, 1991). Female guppies are choosy and are known, for example, to prefer males possessing greater orange pigmentation (Houde, 1987). By preferentially associating with certain males over others, females are able to directly influence mating outcomes (Shohet and Watt, 2004).
Here we test the hypothesis that short-term (21-day) exposure to an environmentally relevant concentration of 17β-trenbolone (22 ng/L) alters male and female reproductive behaviour in guppies. A short-term exposure duration was employed as agricultural pollutants often enter aquatic habitats in pulses and these temporally discrete contamination events can have persistent consequences (García et al., 2011, Morthorst et al., 2010).
Section snippets
Ethical statement
The research detailed in this paper was approved by the Biological Sciences Animal Ethics Committee of Monash University (permit number: BSCI/2013/09) and complies with all relevant State and Federal laws of Australia.
Animal housing
This study used laboratory-reared descendants of wild caught guppies from Alligator Creek (19° 26ʹ 18ʺ S, 146° 57ʹ 01ʺ E), Queensland, Australia. Sexually mature guppies reared in large mixed-sex holding tanks (90 cm × 45 cm × 45 cm) were assumed to be non-virginal given the
Male behaviour
The number of courting events performed by males was associated with treatment, male orange pigmentation, male Condition Index and female length. Relative to males in the control treatment (i.e., unexposed males paired with unexposed females), both unexposed and exposed males performed fewer courting events when paired with exposed females (partial Wald test: z = − 2.219, p = 0.027 and z = − 2.409, p = 0.016, respectively; Fig. 1A). Increased male orange pigmentation was associated with an increase in
Discussion
This research is the first to document altered male reproductive behaviour following exposure to 17β-trenbolone at an environmentally relevant concentration. Males paired with exposed females performed fewer courtship bouts than did males in the control treatment. Male exposure to 17β-trenbolone led to an increase in sneaking behaviour when paired with unexposed females. However, this finding was reversed when males were paired with exposed females, with unexposed males sneaking more than
Conclusion
This study reports that short-term (21-day) exposure to an environmentally relevant concentration (22 ng/L) of the androgenic endocrine disruptor 17β-trenbolone can alter reproductive behaviour and morphology in the guppy. This is the first study to show altered reproductive behaviours in male animals resulting from an environmentally realistic exposure to 17β-trenbolone. Given the prevalence and potent biological activity of 17β-trenbolone, the ongoing multidisciplinary scrutiny of this EDC is
Acknowledgments
We thank John Endler and his research group for supplying fish for this study.
Funding statement
This study was supported by a Discovery Grant from the Australian Research Council (DP130100385) (awarded to BBMW) and an Academy of Finland Postdoctoral Researcher Fellowship (265629) (to MS). At the initiation of this study, the Centre for Aquatic Pollution Identification and Management (CAPIM) received foundation funding from the Victorian Science Agenda Investment Fund managed by the Department of
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