(A) We measured whether the COIIG177S mtDNA mutation affected the mating success of males. We measured eggs laid by virgin wildtype females that were either unmated, or mated with 2–5 day old wildtype, or COIIG177S mutant males at 29°C. We determined the fraction of eggs hatched by counting unhatched eggs and larva 24 hrs after eggs were laid. All experiments were done in replicates of 6 per group. Error bars represent standard error of the mean. Our results show that the number of eggs laid after mating to wildtype mtDNA males is not significantly different from those mated to COIIG177S mutant mtDNA males; in the latter case, most of the eggs are unfertilized and do not hatch. (B,C) We present maximum projection representative images of DAPI stained testis from 2–5 day old wildtype mtDNA male flies grown at 29°C at early (B), middle (B’), and late (B’’) needle stage of sperm development. Note that the sperm are organized during early needle stage (arrow) and then break up into individual sperm by late needle stage (arrowheads). We also present maximum projection images of DAPI stained testis from COIIG177S mutant mtDNA male flies grown at 29°C at early (C), middle (C’), and late (C’’) needle stage of sperm development. Note also that the sperm in COIIG177S mutant are ‘clumped’ and disorganized early in the needle stage and remain so through remainder of spermiogenesis (arrow). Scale bar, 20 μm. (D,E) Representative DAPI stained images of whole testis (outlined in dotted line) from 2–5 day old virgin wildtype mtDNA (D) and COIIG177S mutant mtDNA males (E) raised at 29°C. For orientation, in both images, we identify the tip of the testis (where germ stem cells reside) as well as the seminal vesicle (the storage organ for mature sperm). Note the much smaller seminal vesicle size in the mutant males. Scale bar, 50 μm. (F) Quantification of the seminal vesicle size, as measured by cross-sectional area, normalized to wildtype. Average calculated from 5–7 testes. Error bars represent standard error of the mean.