Negative geotaxis assay

Adult negative geotaxis (hereafter simply “geotaxis”) was measured using an assay modified from Patel and Tamanoi (2006) and Sofola et al. (2010). Briefly, five replicates of 15 flies of each genotype were collected 48 hr post eclosion, and aged for 3 d in single-sex, single-genotype, vials. Following aging, the groups of flies were transferred into empty 25 × 95 mm shell vials marked with a height of 5 cm and allowed 30 sec to recover. Following recovery, flies were tapped down to the bottom of the vial and allowed 10 sec to climb. After 10 sec, the number of flies above and below the 5 cm mark was counted by eye. For each vial, three tap down trials were performed, each at 1 min intervals. A performance index (PI), an estimate of the probability that a fly will climb, was calculated for each genotype following Sofola et al. (2010). PI values range from zero to one; values close to one indicate that flies have a high geotaxic response, while values close to zero indicate that flies have a poor geotaxic response (Sofola et al. 2010). PI was calculated as PI = 1/2 (n_TOTAL+ n_TOP − n_BOTTOM / n_TOTAL), where n_TOTAL is the total number of flies, n_TOP is the number of flies above the 5 cm line, and n_BOTTOM is the number of flies below the 5 cm line (Sofola et al. 2010). Due to the variation in success across lines, the total number of animals assayed varied between 2168 flies and 1923 flies, for the 0 and 50% comparison and the 30 and 80% comparison, respectively.

Because cSOD-null flies are strikingly poor climbers, we modified the geotaxis assay by using a shorter climbing distance than typically employed (Patel and Tamanoi 2006; Sofola et al. 2010). However, this modification could reduce the ability of the assay to detect small geotaxic differences in high-performance flies (i.e., animals possessing 30, 50, and 80% cSOD activity). Such a reduction in resolving power could lead us to detect no difference across backgrounds, even if small differences exist. Therefore, we conducted a second locomotor assay, the countercurrent climbing assay (Petersen et al. 2013), to more closely assess potential performance differences across genotypes.