The mito-nuclear incompatibility alters flight and climbing ability. (A) Flight distributions of mito-nuclear introgression strains. The distribution of (simw⁵⁰¹); OreR is downshifted and displays the lowest average flight score. (simw⁵⁰¹); Aut and (ore); Aut distributions do not differ significantly (P_Wilcox=0.33). All other comparisons between genotypes are significantly different (pairwise contrasts in supplementary material Table S4). (B) Flight distributions of transgenic rescue strains. (simw⁵⁰¹); Aatm^Ore displays the lowest distribution of height scores. The distribution of (simw⁵⁰¹); Aatm^Ore differs significantly from (simw⁵⁰¹); Aatm^Aut and (simw⁵⁰¹); Aatm^Ore_V275A (pairwise contrasts in supplementary material Table S4). The distributions of (simw⁵⁰¹); Aatm^Aut and (simw⁵⁰¹); Aatm^Ore_V275A do not differ (P_Wilcox>0.7). (C) Climbing profile of mito-nuclear introgression strains. (simw⁵⁰¹); OreR and (ore); OreR distributions do not differ significantly (P_Wilcox>0.1). All other comparisons between genotypes are significantly different (pairwise contrasts in supplementary material Table S4). (D) Climbing profile of transgenic rescue strains. The distribution of (simw⁵⁰¹); Aatm^Ore differs significantly from (simw⁵⁰¹); Aatm^Aut and (simw⁵⁰¹); Aatm^Ore_V275A (pairwise contrasts in supplementary material Table S4). The distributions of (simw⁵⁰¹); Aatm^Aut and (simw⁵⁰¹); Aatm^Ore_V275A are marginally different (P_Wilcox=0.04). All P-values associated with Wilcoxon tests are Bonferroni-corrected to account for multiple testing. Different letters signify significant differences in pairwise contrasts.

Characterization of mitochondrial morphology in the mito-nuclear introgression strains. TEMs of indirect flight muscle reveal structural abnormalities in (simw⁵⁰¹); OreR. (A-D) (simw⁵⁰¹); OreR display a loose cristae structure and matrix gaps. (simw⁵⁰¹); OreR contained the most mitochondria per unit area, and several examples of swirled concentric cristae structures were observed (A,D). (E,F) (ore); OreR displayed some loose cristae structure and matrix gaps, and on average contained the largest mitochondria. (G-J) (simw⁵⁰¹); Aut and (ore); Aut displayed tight cristae packing and healthy mitochondrial morphology. (K) Quantification of mitochondrial morphology parameters in mito-nuclear introgression strains. Approximately 100 mitochondria per genotype were analyzed to generate size data. Different superscript letters signify significant differences in pairwise comparisons (values for P_Tukey pairwise contrasts are displayed in supplementary material Table S5). Mitochondria were counted in 50 μm×50 μm sections to generate density data. The average number of mitochondria per unit area of >3 sections are displayed. Scale bars: 2 μm (A,B,E-G,I); 500 nm (C,D,H,J).

Characterization of mitochondrial morphology in the transgenic rescue strains. The incompatible mito-nuclear pair results in mitochondrial morphological defects. (A-D) Large matrix holes and unidentified inclusions were observed in (simw⁵⁰¹); Aatm^Ore. On average (simw⁵⁰¹); Aatm^Ore contains the smallest mitochondria, but the most per unit area. (E,F) (simw⁵⁰¹); Aatm^Aut displays some matrix holes, but maintains a relatively low ratio of empty matrix space to mitochondrial area. (G,H) The transgenic rescue genotype (simw⁵⁰¹); Aatm^Ore_V275A displays a healthy cristae structure and normal mitochondrial morphology. (I) Quantification of mitochondrial morphology parameters in transgenic rescue strains. Approximately 100 mitochondria per genotype were analyzed to generate size data. Different letters signify significant differences in pairwise comparisons (values for P_Tukey pairwise contrasts are displayed in supplementary material Table S5). Mitochondria were counted in 50 μm×50 μm sections to generate density data. The average number of mitochondria per unit area of >3 sections are displayed. Scale bars: 2 μm (A,C,E,G); 500 nm (B,D,F,H).

OXPHOS enzyme abundance and activity is reduced in mitochondrially translated complexes containing the most tyrosines. BN-PAGE analysis of mitochondrial protein isolates. (A) Example of BN-PAGE gel for transgenic rescue strains (note: full lanes moved from original gel locations for genotype consistency, no manipulations within lanes). Quantification of individual band intensity corresponding to Complex I, III and V relative to BSA. Across all complexes (simw⁵⁰¹); Aatm^Ore displayed reduced protein levels to varied degrees (individual contrasts in supplementary material Table S7). (B) Enzymatic activity of OXPHOS complexes. (simw⁵⁰¹); Aatm^Ore has significantly reduced activity of Complex I compared to (simw⁵⁰¹); Aatm^Aut and (simw⁵⁰¹); Aatm^Ore_V275A (P_Tukey=3.18e-5 and P_Tukey<0.05, respectively). The (simw⁵⁰¹); Aatm^Ore_V275A rescue strain has marginally lower Complex I activity compared with (simw⁵⁰¹); Aatm^Aut (P_Tukey<0.05). Complex II and Complex III activity does not differ between the rescue strains (P_ANOVA>0.8, each complex). Complex IV activity is reduced by the incompatibility in (simw⁵⁰¹); Aatm^Ore (P_ANOVA<0.01). There is no difference in citrate synthase among the rescue genotypes (P_ANOVA=0.7). The complexes with more mitochondrially encoded tyrosines display the most severe defect (Complexes I and IV). Enzymes that are completely nuclearly encoded (Complex II and citrate synthase) or those with few mitochondrially encoded tyrosines (Complex III) show little disruption of enzyme capacity. *P<0.05; **P<0.01; ***P<0.001.