Brain circuit imprints of developmental 17α-Ethinylestradiol exposure in guppies (Poecilia reticulata): persistent effects on anxiety but not on reproductive behaviour.

The effects of endocrine disruptors may vary with the timing of exposure. The physiological implications of adult exposure are present during and shortly after exposure while embryonic exposure can imprint changes manifested in adulthood. In this study, guppy (Poecilia reticulata) embryos were exposed to 2 and 20 ng/L of 17α-ethinylestradiol during development via the mother and reared in clean water from gestation until 6 months of age. As adults, fish exposed to 20 ng/L during development showed significantly altered behaviour in the Novel Tank test, where anxiety is determined as the tendency to remain at the bottom upon introduction into an unfamiliar tank. 17α-ethinylestradiol treatment increased the latency time before swimming to the upper half of the tank and decreased the number of transitions to the upper half. In control females the basal stress behaviour responses were significantly higher than in males, as indicated by longer latency period and fewer and shorter visits to the upper half, supporting the importance of gonadal hormones for the behaviour. The anxiety increased, however, with treatment in both sexes, suggesting that the observed response is not entirely due to feminisation of the males. Shoaling behaviour, analysed as tendency to leave a shoal of littermates, was neither sex-differentiated nor changed by treatment. Also male reproductive behaviour, brain aromatase activity and testes histology, previously shown to respond to oestrogen exposure in adult guppy, were unaffected by the developmental treatment. This suggests that the stress system in the guppy is very sensitive to 17α-ethinylestradiol, which possibly causes an early organisational imprint on the brain circuit that regulates stress reactions.

Anxiogenic behaviour induced by 17α-ethynylestradiol in male guppies (Poecilia reticulata).

Behaviour studies are used in toxicology research as they are excellent tools to measure physiological end-points caused by exogenous chemicals. In mammals both reproductive and non-reproductive behaviours have been used for a long period of time, whereas in teleost fishes non-reproductive behaviours have received little attention compared to reproductive behaviours. Recent advances in measuring stress related behaviours in zebrafish have provided additional tools to understand behaviour toxicology in fish. One species with well documented reproductive behaviour disturbed by different toxicants is the guppy, which is better suited than zebrafish for reproductive behaviour studies and therefore might be a better model organism for comparative behaviour studies in fish toxicology. Here we report new applications for non-reproductive behaviours in guppy and test these behaviours on males treated with the endocrine disruptor 17α-ethynylestradiol at environmentally relevant concentrations. 17α-ethynylestradiol increased freezing and bottom-dwelling when fish were placed in a non-familiar aquarium, but did not significantly affect shoaling behaviour. These results are similar to the anxiogenic behaviours seen in rats treated perinatally with 17α-ethynylestradiol and add more concern to the impacts of endocrine disruptors on aquatic wildlife.

17α-Ethinyl estradiol affects anxiety and shoaling behavior in adult male zebra fish (Danio rerio).

Ethinyl estradiol is a potent endocrine disrupting compound in fish and ubiquitously present in the aquatic environment. In this study, we exposed adult zebra fish (Danio rerio) males to 0, 5 or 25 ng Ethinyl estradiol/L for 14 days and analyzed the effects on non-reproductive behavior. Effects of treatment of the exposed males was shown by vitellogenin induction, while brain aromatase (CYP 19B) activity was not significantly altered. Both concentrations of Ethinyl estradiol significantly altered the behavior in the Novel tank test, where anxiety is determined as the tendency to stay at the bottom when introduced into an unfamiliar environment. The effects were, however, opposite for the two concentrations. Fish that were exposed to 5 ng/L had longer latency before upswim, fewer transitions to the upper half and shorter total time spent in the upper half compared with control fish, while 25 ng Ethinyl estradiol treatment resulted in shorter latency and more and longer visits to the upper half. The swimming activity of 25, but not 5 ng-exposed fish were slightly but significantly reduced, and these fish tended to spend a lot of time at the surface. We also studied the shoaling behavior as the tendency to leave a shoal of littermates trapped behind a Plexiglas barrier at one end of the test tank. The fish treated with Ethinyl estradiol had significantly longer latency before leaving shoal mates and left the shoal fewer times. Further, the fish exposed to 5 ng/L also spent significantly less time away from shoal than control fish. Fertilization frequency was higher in males exposed to 5 ng/L Ethinyl estradiol when compared with control males, while no spawning was observed after treatment with 25 ng/L. The testes from both treatment groups contained a normal distribution of spermatogenesis stages, and no abnormality in testis morphology could be observed. In conclusion, we have observed effects on two behaviors not related to reproduction in zebra fish males after treatment with Ethinyl estradiol, adding to the ecological consequences of contamination of aquatic environments with estrogenic substances.