MITOCHONDRIAL VARIATION FOR THE EVOLUTIONARY ECOLOGIST

My current research is shaped by my ARC DECRA program, which tests how mitochondrial genetic variation has cascading effects on mitochondrial performance, physiological function, and finally behavioural display quality. The genetic lines of fruit flies I have developed allow me to explore how population-level variation in mitochondrial DNA sequence alters mitochondrial performance, in turn affecting the myriad processes dependent on mitochondrial respiration and signalling. I also use these lines to explore more fundamental evolutionary theory, such as the Mother's Curse Hypothesis.

Through this empirical work and an upcoming collaborative review paper (working title of "Mitochondrial bioenergetics for ecologists"), I hope to provide non-cellular-biologists a solid foundation in the proximate mechanisms underlying mitochondrial variation and the ultimate evolutionary forces that shape it.

A very basic overview of the scope of my research on Drosophila. I explore how mitochondrial genotype and phenotype affect performance of critical physiological pathways, which in turn influence the quality of a behavioural display.

FUNCTIONAL MITOCHONDRIAL DNA

Variation in mitochondrial DNA sequence has long been thought to be "neutral," meaning that differences in mitochondrial DNA found among populations have no net effect on the functionality of those mitochondria (or individuals that possess them). However, research within the Dowling Lab and others has revealed that even a few base pair differences in the mitochondrial genome (which, in animals, is already tiny compared to the nuclear genome) can have startlingly large effects on important traits like lifespan and fertility.

Even more surprisingly, a mitochondrial DNA variant can have different effects on males and females. Because mitochondria are maternally inherited, genetic mutations that have negative effects on males but neutral (or positive) effects on females can become prevalent in a population. Natural selection cannot act directly on harmful mutations in male mitochondria because fathers cannot pass their mitochondria to their offspring. The saying "survival of the fittest" still applies to males--but the mitochondrial genes that may make them more "fit" will not get passed on to the next generation, so there is no opportunity for selection. This has been called the "Mother's Curse" hypothesis. It's a bit of an overly dramatic name (and not particularly accurate), but it's catchy!

I am using new mito-nuclear fly lines that I have developed to test how mitochondrial DNA variation affects performance at the levels of the cell and the organism. I am also testing the Mother's Curse hypothesis by searching for evidence of male-harming mutations.

You can read the science portion of my relevant ARC DECRA proposal here.

DROPPING LIKE FLIES

(Negative geotaxis)

To date, most research on the functional effects of mitochondrial genetic variation have explored either  life-history traits (fecundity, lifespan) or molecular traits (gene expression). I aim to look more closely at physiological traits in between the broad and the narrow.

The first of such traits I have measured comprises both behavioural and physiological components: a test of negative geotaxis, or the propensity for flies to re-orient themselves and climb upward after being knocked to the bottom of their vials. This is a well-established measurement used in studies of motor and neural capacity--flies show diminished negative geotaxis behaviour with increasing age and oxidative stress.

I put together a new apparatus to test negative geotaxis by dropping flies through PVC pipes, knocking them down against a sticky surface of blue tack. The usual method of inducing negative geotaxis is tapping vials against a surface by hand. My apparatus offers a more controlled means to knock the flies down.

I have used this system to test for differences in performance among lines of flies with different mito-nuclear genotypes. Early results indicated a strong effect of parental age on performance, so I am currently processing data of a follow-up experiment targeted at isolating this effect.

The video (top) demonstrates the new negative geotaxis apparatus, lovingly termed the Droso Tower of Terror. I can then use fly positions at a set time (4s after impact; middle image) and ImageJ particle detectors (bottom image) to measure fly performance in terms of height climbed and numbers of flies that fail to climb at all.

Because mitochondria are only passed on from the mother, I crossed FEMALES of the target mitochondrial type (red) with MALES of the target nuclear type (blue). Then, I repeatedly "backcross" female offspring with males of the target nuclear type until all vestiges of the original red nuclear DNA has been bred out (~16 generations).

MITO-NUCLEAR FLY LINES

I have developed a new panel (group) of genetic fruit fly lines that  allow me to vary either mitochondrial or nuclear genomes while controlling for all other genetic variation. The Dowling Lab has historically maintained a panel of 13 fly lines that share the same nuclear DNA (they are nuclear clones), but that each possess a unique mitochondrial DNA sequence (called a "haplotype"). These 13 types were originally collected from wild fruit flies around the world. This "mitoline panel" has been used to test what happens when mitochondrial DNA varies, but nuclear DNA remains the same.

A key weakness of this historical panel is that because only one nuclear DNA sequence is shared across all the lines of flies, we cannot tell whether the patterns we observe only exist with THAT nuclear genome--or whether they may be more universal. The nuclear and mitochondrial genomes are known to interact, and it is possible that a mitochondrial sequence that performs well with one nuclear genome will perform poorly with another.

I have therefore used a line-breeding scheme (not unlike that which created red canaries from yellow) to create THREE additional replicated populations of the 13 mitolines, each with one of three new nuclear genomes.

STAY TUNED FOR UPCOMING PROJECTS...

I am currently setting up the experimental infrastructure for tests of lifespan in these mito-nuclear lines of flies using a new, high-throughput apparatus.