Scanning Electron Microscopy and Brochosome Measurements

Leafhopper forewings were prepared for scanning electron microscopy (SEM) by placing 3 forewings from 3 separate individuals from each species dorsal side up on double-sided carbon conductive tape on aluminum SEM stubs. The forewings were sputter coated with a 7 nm layer of gold–palladium (Au–Pd) with a Denton Desk II TSC turbo-pump sputter coater (Denton Vacuum, Inc., Moorestown, NJ) in the Microscopy Suite at the Beckman Institute for Advanced Science and Technology at the University of Illinois Urbana-Champaign. The proximal, medial, and distal portions of the forewings were imaged at 10,000×, 20,000×, or 30,000× magnification using the Microscopy Suite’s FEI Quanta FEG 450 environmental scanning electron microscope (ESEM) (Thermo Fisher Scientific Corp., Waltham, MA) at a high vacuum setting, low voltage 5.0–10.00 kV, and a spot size of 3.0.

For each species, 2 images per wing position (proximal, medial, and distal thirds in relation to the body) and 3 forewings per species were imaged for a total of at least 18 images per species (Fig. 2). The diameter (nm) of the brochosomes were measured from those SEM images for each species using ImageJ Fiji version 2.3.0/1.53f software (Schindelin et al. 2012). To account for the irregular shape of the brochosomes, the diameter measurements were taken at 3 different angles (0°, 45°, and 90°) and the mean was calculated to give a final diameter measurement (Fig. 2). At least 40 brochosomes were measured per image and at least 420 brochosomes were measured per species. The total number of brochosomes measured exceeded 3,900. The mean of these measurements was calculated per wing position and per species. A total of 3 individuals from each of the 8 species were examined.

Measurements of brochosome diameter (nm) using ImageJ. Three diameter (D) measurements were taken at 90° (D1), 45° (D2), 0° (D3), and averaged to provide the brochosome diameter mean per wing position. Circles on leafhopper indicate proximal, medial, and distal portions of the wing relative to the body (top to bottom in the diagram, respectively). Scale bar = 500 nm.