three sets of experiments were done varying the standard arrangement of the competing embryo (SIM1, REV1, REV2; the tester embryo), which was always in homozygosis. Embryos were collected from grape juice-sucrose-agar plates supplemented with yeast paste using FlyStuff Small Embryo Collection Cages. Using dissecting needles, 100 embryos were deposited on small grape juice-sucrose-agar cube according to one of three starting proportions for the two competing embryos (1:3, 1:1, 3:1). The cube was subsequently introduced into a vial of fresh medium; each assay was replicated 10 times. Separate previous pilot experiments for the strains REC, SIM1, and INV1, using 100 embryos, indicated that the rate of survival was approximately 40% and therefore enough to detect differences between the strains to be compared; each assay was replicated 10 times. In total, 42 different competition settings were set up (7 tester by tested combinations62 genotype conditions for the tested chromosome63 starting proportions). Given the number of embryos used across competition experiments (42,000), only those involving the same tester w2 embryo (e.g. SIM1/SIM1) were performed simultaneously, which determined how the contrasts were done (three for each genotype condition of the tested chromosome and particular starting proportion, i.e. 18 in total). No bias in sex ratio was assumed in all cases. Progenies from each cross were scored after 15 days to ensure the emergence of all surviving imagoes and the relative viability between the two competing genotypes estimated as (n196n2)/(n16n29), where n1 and n2, and n19 and n29, are the number of embryos and imagoes, respectively, of the two competing genotypes. Values of relative viability were log2-transformed. Male fertility tests The effects of the induced disruption on male fertility were assayed by examining progeny size, sperm performance, and mating ability. For the first test, we exposed single four-day-old virgin females carrying the ultraconserved region CG15121– CG16894 in its intact form (SIM1, REV1, REV2) to single two- day-old males with (INV1, INV2) or without (REC) the disrupted ultraconserved region. Males were discarded after 24 hours while females were transferred daily to vials with fresh food over a 10-day period. The number of replicates ranged from seven to nine. To evaluate sperm performance in the carriers of the disrupted ultraconserved region, we followed a similar experimental design to that for monitoring progeny size with the exception that the females mated in the first day were exposed to single males of their own strain in subsequent days. The progeny sired by the first and second males was scored based on eye color. For those days during which progeny from both parents were detected, i.e. those oviposited roughly the same day, we estimated the fraction of red-eyed individuals (necessarily sired by INV1, INV2, or REC males) in relation to the total. To account for the effect of the order of the males used, we performed identical experiments but this time the first male possessed the same genotype as the female while the second male was from the strains INV1, INV2, or REC. The number of replicates for each combination of genotypes ranged from four to nine. As for the comparison of mating abilities, we exposed single two-day-old males to 10 four-day-old virgin females for different time periods (1 hr, 3 hr, 6 hr). Afterwards, the females were transferred individually into vials with fresh food. After 15 days, 10 vials were examined for the presence of progeny, which indicates that at least one successful fertilization event occurred, and the number of females successfully fertilized recorded. Ten replicates were done per strain and time period combination. Collinearity Conservation in Eukaryotes Response to volatile compounds The ‘‘dipstick’’ method was used [65]. Briefly, virgin individuals from INV and REC strains were separated by sex and transferred by aspiration in groups of five to marked empty plastic vials (O.D.6H: 25695 mm) 24 hr after emergence. The vials were marked at 3 and 6 cm from the bottom. Fisherbrand Q-tips dipped into the odorant dilutions to be tested were introduced into the vials up to the 6 cm mark, and secured with a cotton plug to avoid contact with the walls of the vial. After a 15-second recovery period, the number of flies in the bottom compartment was recorded 10 times every five seconds and the avoidance scored estimated as the average over those 10 measurements. A score of 2.5 indicates indifference to the odorant tested while values .2.5 and ,2.5 indicate repulsion and attraction, respectively. All tests were performed between 2 and 6 pm after starving the flies for no less than four hours, and the vials were always placed sideways to prevent interfering with the geotactic response. For each strain, 10 groups of five individuals for each of the sexes were analyzed. We tested three odorants, two of them usually considered to elicit an attracting response (ethanol, Gold Shield; acetone, Fisher Chemical) while the other is considered to be a repellant (benzaldehyde; Sigma Aldrich B1334 Benzaldehyde-ReagentPlus). Since odorant response can be concentration dependent, we tested two concentrations deferring by several orders of magnitude. These concentrations (vol/vol) were: ethanol, 1023 and 1020.5; acetone, 1024.5 and 1021.5; and benzaldehyde, 1023 and 1020.5. Only fresh and thoroughly mixed dilutions were used to prevent oxidation, which is particularly relevant in the case of benzalde- hyde. As a control, we used distilled water, which is known to attract starved flies [65]. General homeostasis tests Four proxies, including gravity response and survival to three stressors (heat-shock, desiccation, and starvation), were assayed. In all cases, males and females were separated after emergence and left for 24 hours in vials with fresh food. Subsequently, all individuals were transferred to new vials by aspiration for performing the pertinent tests. Negative gravitaxis was measured essentially as reported [66]. Twenty flies per sex and strain were transferred individually into a 250 ml glass volumetric cylinder. Flies were knocked down by tapping the cylinder ten times on a pad, and the height reached by each fly in 20 s recorded using the volumetric scale, which was divided by the maximum height. In our experience, flies performed more consistently after some training, reason why only measurements from a third trial were recorded. For the heat-shock resistance test [67], five groups of 20 flies per sex and strain were transferred in pairs into 3 ml Pyrex vials and then incubated in water baths at 35uC for 30 m (as a pre- conditioning step) and then at 39uC for another 30 m. The mobility of the flies was restricted to the submerged portion of the vial with cotton plugs. After the heat-shock treatment, flies were collected in vials with food that had been incubated at 25uC overnight. Next morning, the fraction of flies alive within each group was recorded. For the desiccation resistance test [68], groups of five flies, from a total of 20 per sex and strain, were transferred into empty vials. The mobility of the flies was restricted to the lower third of the vial using foam plugs, over which 3 g of Drierite desiccant were added. Next, the vials were sealed with Parafilm to maintain low humidity. Flies were checked every hour and the elapsed time-to-death recorded. As for the starvation resistance test, 20 flies per sex and strain were transferred into glass vials containing 2 ml of 1% agar dissolved in water to ensure normal humidity conditions. Flies were examined every 12 hr and the elapsed time-to death recorded. PLoS Genetics | www.plosgenetics.org 8 February 2012 | Volume 8 | Issue 2 | e1002475