Imaging and Measurements

Nymphs were removed from colony jars by tapping the harborage on the side of a plastic bin near the top, with CO₂ anesthesia flowing at the bottom of the bin. Nymphs that landed in the bin were collected after being anesthetized and killed by freezing. Thawed nymphs of similar sizes were arranged in groups of 10–15 on a Petri dish lined with filter paper at the bottom so their pronota were clearly visible. Nymphs were photographed using a 12-megapixel Olympus TG-5 (IM005, Olympus Corporation, Tokyo, Japan) camera on a tripod. An extra light was pointed at the dish. Photographs were often taken using both the fill-in flash setting and the LED light setting; the better photograph of the two was selected for analysis. All images were analyzed with ImageJ software (Schneider et al. 2012), a popular choice for image processing (Mutanen and Pretorius 2007, Teale et al. 2009, Seiter et al. 2010, Wieferich et al. 2013). A pair of calipers with millimeter marks (Model 62379–531, VWR International Inc., West Chester, PA) were included in each photograph to calibrate pixels/mm. The pronotal length and width of each nymph was measured by drawing a line and using the measure function. After each nymph was measured, it was sexed according to the methods of Ross and Cochran (1960).