Factors Affecting Adhesion Figure 1. Experimental trials at variable temperatures without controlling for humidity. Relationship between temperature and body-size corrected adhesion (clinging force [N]; left axis, bars) or relative humidity (%; right axis, solid line). doi:10.1371/journal.pone.0002192.g001 Notably, adhesion in the Losos study [9] was maximal at intermediate temperatures (which corresponded to the highest relative humidity). Consideration of both our temperature and 12uC humidity trials (Fig. 3), in light of the two studies described above, suggests that clinging ability is sensitive to variation in humidity, not temperature. Unfortunately, the humidity response does not appear to be simple. Indeed, the response to variation in humidity at 12uC is not constant but apparently itself is affected by temperature (Fig. 2). In other words, clinging ability by geckos on a smooth hydrophilic surface like glass appears to be sensitive to both temperature and humidity. Interestingly, the effect of humidity on gecko adhesion has never been formally investigated at the whole animal scale that we are aware. However, two studies focusing on elucidating the mechanism underlying the setal-substrate interaction have dem- onstrated that adhesive forces between a single spatula and substrate is humidity dependent (at room temperature) [12,14]. Scaling of the effect of humidity at the nano-scale shows an approximately 1.3 fold increase in adhesion between 35% and 80% RH [12], or a 3-fold difference between 0 and 70% RH [14]. Our study shows that at the whole animal scale the effect of humidity is quite complex. We have observed an approximate doubling of adhesion over 35 to 80% RH at 12uC. On the other hand, the adhesion forces are relatively insensitive to humidity at 35uC. We note, however, there is now an important gap in experiments at the setal scale: humidity manipulations have only been done at a single temperature. The strong influence of humidity on the adhesive forces at a single setal and whole animal level also suggests that capillarity forces may play an important role in adhesion. A strong effect of humidity on adhesion forces has also been observed using atomic force microscopy for hydrophilic tips in contact with hydrophilic surfaces. Alternatively, when one of the materials is hydrophobic, adhesion is humidity independent [15]. Our study raises several paradoxes that cannot be resolved with existing data or theory. First, the humidity dependent adhesion forces at 12uC suggest that setae must be hydrophilic, despite evidence that water droplets do not wet gecko feet and roll off easily upon tilting. Second, it is not clear why humidity has a strong influence on the adhesion forces only at low temperatures. A capillary model predicts that the adhesion forces at the same humidity should be proportional to temperature in units of Kelvin [16]. Therefore, the adhesion forces at 35uC should be only 8% higher than the adhesion forces at 12uC. These predictions are much smaller than experimental observations. In addition, the capillary model predicts the adhesion forces at 35uC should show similar dependence on humidity. Both the van der Waals and capillary forces fail to explain the shear adhesion data at the whole animal level. Resolution of this paradox will likely require examination of the particular way in which water interacts with substrate and seta at the nanoscale level. In addition, the adhesion force measurements at a single setal scale are needed at variable temperatures before we can eliminate the possibility of thermal biophysical constraints influencing the adhesion. PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2192