Transmission of mitochondrial mutations and action of purifying selection in Drosophila melanogaster

Abstract

It is not known how selection affects mutations in the multiple copies of the mitochondrial genome. We transferred cytoplasm between D. melanogaster embryos carrying mitochondrial mutations to create heteroplasmic lines transmitting two mitochondrial genotypes. Increased temperature imposed selection against a temperature-sensitive mutation affecting cytochrome oxidase, driving decreases in the abundance of the mutant genome over successive generations. Selection did not influence the health or fertility of the flies but acted during midoogenesis to influence competition between the genomes. Mitochondria might incur an advantage through selective localization, survival or proliferation, yet timing and insensitivity to park mutation suggest that preferential proliferation underlies selection. Selection drove complete replacement of the temperature-sensitive mitochondrial genome by a wild-type genome but also stabilized the multigenerational transmission of two genomes carrying complementing detrimental mutations. While they are so balanced, these stably transmitted mutations have no detrimental phenotype, but their segregation could contribute to disease phenotypes and somatic aging.

Figure 1

Stable transmission of genetically marked mt genomes in the background of mt:ND2^del1. A) Mutants at the BglII and XhoI sites (yellow highlight) of mtDNA used in this study (see Supplementary Table 1 for more detailed phenotypes of these mutants). D. melanogaster mtDNA contains one BglII and one XhoI site in the coding regions of ND2 and CoI, respectively; no PCR product was detected with the wt mt:ND2 specific primers when mt:ND2^del1 sequence was used as template. B) Stable transmission of heteroplasmy over multiple generations for the indicated three mtDNA genotypes in the background of mt:ND2^del1. Various heteroplasmic lines were established using mt:ND2^del1 flies as recipient. qPCR defined the proportion of donor mt genotypes to total mtDNA in four independent lineages (colored lines). Growth was at 25°C, except for the lines with mt:CoI^T300I (which were tested at 22°C, the permissive temperature).

Figure 2

Segregation of mitochondrial genotypes in progeny. Individual progeny from mothers with a preponderance of the mt:ND2^del1 genotype (see mother 1 and 2 in Table 1) were assayed by qPCR for the abundance of a second genome (i.e. the donor genome). The histograms show the number of early progeny within different abundance intervals for the donor genome. The black bar indicates the number of progeny lacking PCR detectable donor genomes (homoplasmic). See Supplementary Figure 1 for more progeny collected from mother 1 on successive days.

Figure 3

Purifying selection against the ts genome. A) At the restrictive temperature, abundance of the ts genome declined over multiple generations when co-resident with either wt or mt:ND2^del1 at 29°C. Several independent linages are shown (colors: see Supplementary Figure 5 for additional lines). B) Reduction in the abundance of the ts genome over one generation (adult to adult) at 29°C. Abundance in mothers is shown on the x axis and the decline in abundance in a group (30 individuals) of their progeny is shown on the y axis. C) The abundance distribution of the ts genome in individual flies (n=33) of two mt:CoI^T300I/mt:ND2^del lines after 32 generations at 29°C.

Figure 4

Temperature shift of mothers followed by analysis of mt genome abundance in eggs reveals the timing of selection in the germline. A) Schematic drawing of an ovariole with the germarium at the anterior tip and egg chambers of increasing age (modified from^,). A group of primordial germ cells differentiates directly into cystoblasts to initiate the first wave of oogenesis at the larval/pupal junction. Subsequently, cystoblasts generated by asymmetric division of germline stem cells follow the depicted progression to continue the production of oocytes. The duration of oogenesis (from the cystoblast to a mature egg) is ~6 days at 29°C. B) Measuring selection during oogenesis. Heteroplasmic mothers (mt:CoI^T301I/mt:ND2^del1) were shifted from 22°C to 29°C at the beginning of larval stages (blue diamonds), as 3^rd instar wandering larva (red squares), or as newly eclosed adults (green triangles), and abundance of the ts genome in their eggs was measured by qPCR. C) Young adult mothers (mt:CoI^T301I/mt:ND2^del1) were shifted from 22°C to 29°C and the abundance of ts genomes was measured in their eggs collected on successive days. Data from two of the ten mothers used in the experiment are shown. D) The progeny from individual heteroplasmic mothers (mt:CoI^T301I/mt:ND2^del1) were shifted from 22°C to 29°C at the larval/pupal transition stages, held at 29°C for 1–7 days and then downshifted to 22°C. The abundance of the ts genome in the original mother and the eggs of her double shifted progeny was measured by qPCR. Results from the progeny of five mothers are shown here. The right panel shows the average level of selection after a series of days at 29°C. E) Selection against the ts genome still occurred in a line with reduced Parkin activity (nanos-Gal4 driven germline RNAi knockdown) and a parkin null mutation. For both lines, declines in the abundance of the ts genome were measured in five mothers and their eggs (the mothers were shifted from 22°C to 29°C at the larval/pupal transition stages). The percentage of decline was calculated by dividing the absolute decline by the initial abundance the ts genome in the mothers. The error bars represent standard deviation.