Table 1. Sex, size, and toe pad area of geckos used in experimental trials. Toe pad area is an estimate of maximal area of scansors visible on scans. Individual Weight (g) Toe Pad Area (cm2) day 1 day 2 day 3 day 5 day 6 day 7 day 8 day 9 day 12 day 14 tokay 1 tokay 2 tokay 3 tokay 4 tokay 5 tokay 6 tokay 7 tokay 8 tokay 9 tokay 10 tokay 11 tokay 12 tokay 14 tokay 17 tokay 18 tokay 19 Sex Female Female Male Male Female Female Female Female Male Male Male Male Male Male Male Female Female Male Female Male Female Female Female Male Male Female 5.30 5.58 4.96 4.72 5.48 7.26 5.50 6.90 6.2 3.9 68.80 59.34 59.14 73.24 54.30 78.16 69.22 79.90 73.30 47.8 42.8 44.9 46.4 43.8 50.0 39.2 0.591 0.64 0.72 0.66 0.65 0.73 0.84 0.72 0.79 1.0 4.65 4.95 4.87 5.46 3.93 5.31 4.49 5.27 4.91 4.9 4.06 4.32 4.47 4.57 5.55 4.15 Factors Affecting Adhesion observed at the lowest humidity (35%). Although there was a trend suggesting the effect of humidity was stronger for Day geckos, there was no significant difference between species in the rate of change in clinging force with humidity (species*rh F3,27 = 2.28, P = 0.1). In order to determine if the humidity effect observed at 12uC varied with temperature, we did one last set of trials measuring clinging ability at 32uC at 35% and 80% RH. Surprisingly, at 32uC, clinging ability did not vary in response to humidity as it did at 12uC (Fig. 2.). Instead, the clinging ability of Tokay geckos was significantly higher at 35% RH compared to 80% RH (13.7563.0 N vs. 2.4862.0 N; F1,15 = 38.9, P,0.0001), while the clinging ability of Day geckos did not vary significantly between 35% and 80% RH (0.37560.2 N vs. 0.32760.36 N; F1,15 = 0.05, P = 0.8261). Moreover, at 32uC clinging ability was among the lowest measured in any set of trials. Discussion A preponderance of evidence suggests that geckos stick to substrates as a consequence of the formation of a large number of intimate setal-substrate contacts engaging van der Waals attraction. One prediction about performance that we can derive from this mechanism is that adhesion should be temperature insensitive over a biologically meaningful range [1]. However, previous work, though limited in scope, is equivocal on this prediction [9,10]. Admittedly, such a simple prediction ignores important details of adhesion at the whole organism scale. For example, adhesion may involve more than just the sum of the setal-substrate interactions; the contributions of elaborate subcutaneous vascular, muscular and skeletal elements in adhesion and release is currently completely unexplored [2,10]. Presumably, these components would be subject to thermal dependencies typical of ectotherms [13]. Our experiment measured adhesion at different temperatures using a single protocol with two species that have been previously studied (G. gecko and P. dubia). Although we found evidence of a strong effect of temperature, the direction of the effect was counterintuitive given the thermal biology of geckos and it violated the prediction given by van der Waals interactions presumed responsible for adhesion. Consequently, we wanted to examine whether other factors (e.g., humidity) could explain the variation in clinging performance we observed. We found evidence, unexpectedly, that humidity is likely an important determinant of clinging force in the geckos we studied. Below we explore implications of our results for understanding both the factors affecting adhesion in geckos, as well as for inferring mechanisms that underlie such performance. Restricting attention just to temperature effects (Table 2 and Fig. 1), leads to the conclusion that adhesion is highly temperature sensitive, in both species, with greatest adhesion observed at the lowest test temperature. However, RH varied by approximately 15% between the two temperature extremes (12uC and 32uC; Fig. 1). It is important to note that relative humidity was not controlled in previous studies that examined temperature effects on adhesion in these two species, but differences in protocol are intriguing. Bergmann and Irschick [10], who found no evidence of a temperature effect on clinging ability of P. dubia heated or cooled geckos in an environmental chamber and then performed clinging tests at room temperature and ambient humidity conditions of their laboratory. Alternatively, Losos [9], performed clinging tests on G. gecko inside a walk-in environmental chamber set to one of 9 different temperatures. As in our design, relative humidity in the latter study presumably varied with temperature, and a strong effect of temperature on clinging ability was demonstrated. doi:10.1371/journal.pone.0002192.t001 5.5360.51N, respectively, F1,15 29.9, P,0.0001), but maximal force per unit toepad area was similar between species (7.8361.72 N/cm2 and 6.3461.62 N/cm2, Day and Tokay respectively; F1,15 = 1.6, P = 0.23). Adjusted maximal clinging force was significantly different across temperatures (F4,56 = 8.09, P,0.0001), and there was a significant difference between species in the effect of temperature on clinging force (species*temperature F4,56 = 2.91, P = 0.03) leading us to analyze the effect of temperature separately for each species. Although the effect of temperature was significant for both Day and Tokay geckos (F4,28 = 28.49, P,0.0001 and F4,28 = 5.05, P = 0.003, respectively), the trend for an increase in clinging ability with decreasing temperature appeared stronger for Day geckos (Fig. 1). For example, the clinging ability of Tokay geckos at the intermediate temperature (22uC) was close to but not quite significantly different from clinging ability at the lowest temperature (12uC; matched pairs t-Test, t = 2.23, P = 0.056) but clinging force at 12uC was significantly higher than at 22uC for Day geckos (12uC; matched pairs t-Test, t = 10.78, P,0.0001). Humidity effects At 12uC, clinging ability increased significantly with humidity (Fig. 2; F3,27 = 4.61, P = 0.01), such that forces measured at the highest humidity (80%) were nearly twice as high as the forces PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2192