Sanger sequencing (Figure 2B–2C and Figure S8). Further expression profiling confirmed the absence of local position effects at the inversion breakpoints (see below). Lastly, engineered chromosomes were maintained in homozygosis thus preventing the otherwise unavoidable accumulation of detrimental mutations if kept in heterozygosis over a balancer chromosome. Most of the strains that were not discarded were included in one or more downstream analyses. Phenotypic consequences of disrupting the ultraconserved region CG15121–CG16894 The disruption of the ultraconserved region CG15121–CG16894 did not lead to viability impairment in the progeny of the carriers (INV1, INV2) as compared to that of non-carriers based on two proxies examined (Figure S7). Cursory examination of embryos, larva, and pupa did not detect any obvious morphological defect either. We then explored the possibility of a detrimental effect in heterozygous condition before reaching adult eclosion, either because of the disruption of the collinear block, meiotic distortion due to the presence of a chromosomal inverted rearrangement, or both. The comparison of different chromosome combinations revealed no departures from the expected Mendelian ratios (1:2:1) and absence of sex-specific effects (Figure S9). Further, we assessed differential viability during early stages (i.e. prior to imago emergence), when competition among individuals is specially intense [45,46], and when most of the genes putatively targeted by HCNEs exhibit high levels of expression. Differences among carriers of different 2R chromosomes were detected in frequency- dependent competition experiments involving different pairwise combinations of embryos, but those differences were not consistently shown by the carriers of the disrupted ultraconserved region (INV1, INV2) compared to those that carry it in its intact form (REC) across different genetic backgrounds (Figure S10). Together, these results do not point out to any obvious detrimental effect on viability, and therefore on stages of the life cycle that encompass key developmental transitions, as a result of disrupting the ultraconserved region CG15121–CG16894. Next, we examined the effect of disrupting the ultraconserved region CG15121–CG16894 after imago emergence since many of the protein-coding genes included in the region under study are expressed during adulthood, often in a sex-dependent fashion (Figure S1). Specifically, the region under study is populated with male-biased genes in expression, 11 of them preferentially expressed in testes [22] and four, all of them Obp genes, present in the seminal fluid (Figure 1; Table S2). We tested for differences in several parameters of male fertility: progeny size, sperm performance, and mating ability. The comparison of males from the strains INV1, INV2, and REC, which are all red-eyed so that differences due to differential pigmentation can be factored out, revealed that although there are differences in progeny size (Figure S11A), strains INV1 and INV2 are more different from each other than either of them is to strain REC. Double-mating experiments did not reveal any substantial difference in sperm performance (Figure S11B) and mating ability (Figure S12). Further, given the presence of nine odorant-binding and one odorant-receptor protein-coding genes in the ultraconserved region CG15121– CG16894, we analyzed the odorant abilities of the different strains. We examined the response to three volatile compounds (ethanol, acetone, and benzaldehyde) and found statistically significant differences in five out of 12 sex-by-strain combinations. Impor- tantly, for females exposed to ethanol at a concentration of 1023 and males exposed to acetone at a concentration of 1024.5, strains INV1 and INV2 exhibited a coherent pattern of differentiation from strain REC (Figure 3; Table S14), involving in all cases an Collinearity Conservation in Eukaryotes Figure 3. Olfactory response of strains with (INV1, INV2) and without (REC) the disrupted ultraconserved region CG15121– CG16894 to three different volatile compounds. Blue, females; red, males. The ‘‘dipstick’’ method was used in all cases. An avoidance score equal to 2.5 indicates indifference while values ,2.5 and .2.5 are interpreted as attractant and repellent responses, respectively. Ethanol, acetone, and benzaldehyde were assayed at two different concentra- tions (vol/vol) deferring by several orders of magnitude in order to test the response of the strains in very different conditions. Distilled water was used as a reference for the default response when no compound is added. For females, we found statistically significant differences (Kruskal-Wallis, d.f. = 2 in all cases) across strains in response to distilled water (P,0.0107), ethanol (concentration: 1023, P,0.0056), ethanol (concentration: 1020.5, P,0.0230), and acetone (concentration: 1024.5, P,0.0002), while in males, the differences were confined to ethanol (concentration: 1023, P,0.0183) and acetone (concentration: 1024.5, P,0.0182). INV1 and INV2 showed significant differences in the same direction in relation to REC after accounting for multiple tests in the case of distilled water (females), ethanol (concentration: 1023, females), and acetone (concentration: 1024.5, males) (Table S14). Error bars indicate 95% CI. doi:10.1371/journal.pgen.1002475.g003 attenuated attraction to the chemical in question. This attenuated response does not result from an overall impairment of the odorant abilities of the flies as shown by the response to the repellent compound benzaldehyde. Analyses of different proxies for the general adult homeostasis (negative gravitaxis, heat-shock resistance, desiccation resistance, and starvation resistance) did not uncover any other difference between the strains with and without the disrupted ultraconserved region CG15121–CG16894 (Figure S13). Lastly, we tested whether the disruption of the ultraconserved region CG15121–CG16894 resulted in a perturbation of gene expression. We performed a microarray-based characterization of the transcriptome of six lines (REC, INV1, INV2, SIM1, REV1, and REV2) during adulthood, the stage in which we found evidence of phenotypic differences associated with the disruption generated. At FDR 0.01, we found a very limited number of differentially expressed transcripts both in males and females (0.07% -11/16,637- and 6.2% -1,033/16,637-, respectively; Dataset S1 and Table S16). Further analyses confirmed the similarity of the expression profiles between equivalent strains generated by our procedure (first three planned contrasts in Table S17; Datasets S1 and S2). Likewise, these analyses indicated that the most statistically significant differences in mRNA abundance found are associated with differences in pigmentation (last three planned contrasts in Table S17; Dataset S2), in good agreement with the clustering of expression profiles among strains (Figure 4). The inspection of the chromosomal distribution of the differences PLoS Genetics | www.plosgenetics.org 5 February 2012 | Volume 8 | Issue 2 | e1002475