Identification of resilin in the leg of cockroach, Periplaneta americana: confirmation by a simple method using pH dependence of UV fluorescence

We have examined the tarsus (foot) and tibial segments of the cockroach leg to identify structures that contain the elastic protein resilin. The presence of resilin was tested using the conventional criteria of fluorescent emission at 420 nm under UV illumination and histological staining of wholemount tissues by toluidine blue. We have also developed a simple method of confirming identification of resilin through changes in its fluorescence that occur with alteration of pH of the surrounding medium. Using a commonly available excitation filter that only passes light at >330 nm, we found that the emission was present at neutral pH but was eliminated at low pH. It then reversibly reappeared when medium of higher pH was restored. This effect is attributable to a known shift in the absorption maximum of amino acids of resilin that occurs in acidic media (from 330 to 285 nm). The accuracy of this method of identification was confirmed by examination of ligaments of the wing hinge, which has previously been shown to contain resilin in a number of insects. Using these techniques, we have identified resilin in association with ligaments at the tibio-tarsal joint and in the articulation between the fourth and fifth tarsal segments of the leg. The anatomical arrangement of these ligaments suggests that they could aid in the generation of leg movements during walking by functioning as elastic antagonists to the actions of leg muscles. The method of identification we have devised could readily be applied to aid in the localization of resilin in other animals.