Extended Data Fig. 3: Point mutations have some effect on adult emergence, but do not lead to major changes in baseline Atpα expression or Na⁺/K⁺-ATPase activity.

a, Percentages of emerging adults of the knock-in and control lines on standard Drosophila medium (n = 7–8 vials, 100 eggs per vial, mean ± s.e.m.). Survival of the knock-in lines and control lines QAN (engineered control) and QAN* (w¹¹¹⁸ wild type) was compared using one-way ANOVA (P < 0.001). Survival differed between QAN* and some of the knock-in lines, but not between the engineered control line QAN and any of the knock-in lines except VSN (post hoc Tukey’s tests (letters)). b, Atpα expression was not different among the engineered Drosophila knock-in lines or w¹¹¹⁸ wild-type flies (QAN*). Atpα transcript level differences were assayed by qPCR. Expression was assayed in three biological replicates (symbols represent the mean ± s.e.m.), with two technical replicates per biological replicate (averaged for each biological replicate), of five- to six-day-old females as fold change standardized against rpl32 expression in QAN* flies. The expression fold change between genotypes was compared using one-way ANOVA (P = 0.3197). c, None of the sequential Atpα genotypes found along the monarch lineage affected base-line levels of pump activity in a sodium pump enzymatic assay using extracts of fly heads (one-way ANOVA, P = 0.1377; symbols represent the mean ± s.e.m. of 3–7 biological replicates). Further information on experimental design and statistical test results is in the Source Data. ns, not significant.