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Annexe III
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MATERIALS AND METHODS
Egg collection
Gravid females of slider turtle (Trachemys scripta elegans) were collected on June 1-4 2003,
on zoological facility in the South of France located at the “Association Tortues Passion”
(Vergèze, France). Each female was isolated in a plastic box with some water to facilitate
clutch identification. Oviposition was induced by injection of 1ml intra muscular oxytocin
(Ewert & Legler, 1978). Each egg was referenced by a code number on the eggshell for
individual identification and weighed with a Sartorius electronic balance. Finally, eggs were
placed on wet cotton in a cool box to prevent desiccation and moving during transport to the
laboratory.
Incubation of eggs at laboratory
On June 7, 188 fertilized eggs, identified by a white spot on the eggshell, were selected from
26 clutches and equally shared between 19 covered plastic boxes, to prevent water
evaporation. Eggs from the same clutch were dispersed as completely as possible between
boxes to prevent confounding box and clutch effects. Eggs were totally buried in moistened
vermiculite and incubated in two programmable incubators (Memmert, IPP 200-400) at a
constant temperature of 28.9 ± 0.1°C estimated to yield both sexes (Godfrey, Delmas &
Girondot, 2003). Every other day, boxes were rotated to prevent local variations of
temperature in incubators and weighed to adjust water potential.
Experimental design
At time 0 of the incubation time, all 19 boxes contained vermiculite with an intermediate
water potential, maintained until day 5 of incubation. To reproduce the fluctuations of
substrate water potential during the course of incubation, we defined six transfer occasions
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