At the start of the experiment, 120 fish (standard length:
mean þ SD ¼ 26.79 þ 3.49 mm) were distributed equally over the
12 tanks (35 24 cm and 24 cm high), half of which contained
methyl-testosterone (Sigma), 0.5 mg dissolved in 0.0125 ml ethanol
per litre water (a concentration shown to induce nuptial coloration
and adult-like aggressive and sexual behaviour in A. rivulatus:
Groothuis 1993), and half of which contained the solvent only,
0.0125 ml ethanol per litre water. Twice a week, we removed most
of the water, leaving only a small layer, and replaced it with clean
water, followed by renewal of the hormone treatment. Treatment
lasted until the behavioural test started. For logistical reasons, only
a limited number of fish could be tested each day, resulting in
a variable treatment period of 8e12 weeks. This period of exposure
to treatment was chosen based on a previous study (A. F. H. Ros &
T. G. G. Groothuis, unpublished data) showing that juvenile fish
show adult coloration and adult-like agonistic and courtship
behaviour after this period of testosterone treatment and that the
effects on both coloration and behaviour disappear after cessation
of treatment.