Preparation and Imaging of Insect Labia

The material was dissected to separate the labia from the head and cleaned in detergent using an ultrasonic cleaner. Then, the standard procedure was applied [54 (link)]: dehydration with a series of baths in 80%, 90%, and 96% ethanol solutions, for 20 min each, and two baths of 99.8% ethanol solution for 30 min each. The labia were glued with carbon adhesive discs on the aluminium pin stubs, which then were coated with a film of gold (30 nm) using the Q150T ES sputter coater with the rotary planetary stage (Quorum Technologies Ltd., Laughton, UK). SEM micrographs (Figure 2, Figure 3 and Figure 4) were obtained using a Phenom XL field emission scanning electron microscope (Phenom-World B.V., Eindhoven, The Netherlands) at 15 kV accelerating voltage and with a BackScatter Detector (BSD) and Secondary Electron Detector (SED) and Hitachi UHR FE-SEM SU8010 (High Technologies, Tokyo, Japan) with a secondary-electron detector (ESD) at 5, 7, and 10 kV accelerating voltage. To obtain high-quality figures, fragments of labia were imaged at high magnifications and combined using the Image Composite Editor (panoramic image stitcher) and the graphic editor Adobe Photoshop CS6. In a few cases, a series of images at different focal distances were taken and combined using the software mentioned above to attain the appropriate depth of field.