Factors Affecting Adhesion Figure 3. Adhesion during variable temperature, uncontrolled humidity trials (black solid lines), and variable relative humidity constant temperature trials (12uC; red-dotted lines) showing convergence of maximal clinging force with increasing temperature and decreasing humidity. Error bars are 62 SEM. doi:10.1371/journal.pone.0002192.g003 maintained at 2561uC and set to a 12 hour photoperiod with UVA/UVB full spectrum lights. Heating tape along the underside of each tank allowed geckos to thermoregulate within the range of body temperatures typical for free-ranging geckos (,25–35uC). Temperature trials We used a walk in environmental chamber maintained to within61uC of five randomly presented experimental tempera- tures, 12, 17, 22, 27, and 32uC. Each set of trials was initiated approximately two hours into the species-specific diel-cycle (9am for P. dubia and 9pm for G. gecko). Geckos were placed inside the environmental chamber from 1-2 hours prior to all experimental procedures to allow body temperature equilibration. No more than a single temperature was tested on any given day. We used a specially designed apparatus (Fig. 4) to hold a clinging substrate (glass in our experiment) and a motor driven force gauge (Shimpo FGV-10X) which moved relative to the substrate. Digital output from the force gauge was collected by a LabVIEWH program allowing us to save traces of each adhesion trial. For all trials, we report values with the substrate in the vertical position. Each gecko was fitted with specially designed harnesses positioned around the pelvis and allowing unimpeded movement of legs and minimizing abrasion to the gecko’s skin. At the start of each test, the harness was attached to the force gauge which was subsequently driven downward by the motor. Geckos were introduced to the substrate and induced to take step with each foot, providing a standardized posture with all four feet and footpads fully in contact with the substrate. Each individual was then pulled parallel to the surface in a smooth motion until all four feet began to slide indicating the end of the test (referred to hereafter as a ‘pull’). Geckos were pulled three times in succession to estimate maximum clinging force. Between each gecko, the glass was cleaned with ethanol and dried before the next set of pulls. Humidity trials Subsequent to completing all the temperature trials, we became concerned that variation in the relative humidity of the environmental chamber at different temperatures might be a contributing factor to variation in estimates of maximum clinging ability. Consequently, we initiated a second set of trials to estimate the effect of humidity at the temperature where recorded clinging forces were maximal for both species (12uC). Humidity trials followed the protocol described above for temperature trials, except that temperature was maintained constant but relative PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2192