Eanes Lab - Experimental Population Genetics

Selection on Metabolic Pathways

We are interested in how natural selection on life history variation  molds molecular variation in specific pathways, with special interest in the major cross roads of metabolism, the glycolytic pathway. Our primary model is Drosophila melanogaster.  Many of the enzymes directing this flux are polymorphic for amino acid polymorphisms in natural populations of D. melanogaster, and these variants often show allele frequency clines with latitude (Sezgin et al. 2004). Our projects are aimed at assessing the patterns of molecular variation at various points and assessing their impact on metabolic pool levels and flight metabolism by mutagenesis of individual genes using P-element excision (see Merritt et al. 2006). We are also interested in the extent to which individual enzymes differ in their levels of polymorphism and rates of molecular evolution (see Flowers et al. 2007).

Insect Flight

The glycolytic pathway is the best understood in biology. For this pathway, it is proposed that individual enzyme levels are matched to the maximum metabolic flux demands of a particular cell type .    An extreme example of this essential matching is insect flight, where the highest mass-specific oxygen consumption rates among animals are observed. In the flight muscle, high enzyme levels are needed for the rapid turnover of ATP associated with the 100-fold or greater increase in oxygen consumption over resting states .  Wing beat frequency (WBF) is closely correlated with oxygen consumption and  directly reflects the rate of ATP hydrolysis and glycolytic flux. This tight connection between pathway flux and ATP turnover makes the measurement of WBF an ideal system for studying metabolic flux control.

We use P-element excision-derived knockouts in D. melanogaster to experimentally lower the in vivo activity levels of seven enzymes in this pathway and examine individual enzyme influence on tethered flight performance measured as wing beat frequency (WBF) and ability to sustain free flight (Eanes et al. 2006). Our results find the classic regulatory enzyme, glycogen phosphorylase, is nearest to capacity or saturation, while other enzymes, especially those termed near-equilibrium, possess excess capacity. These latter enzymes show extreme genetic dominance and low flux control that extends down to markedly reduced enzyme levels. The genetic results are consistent with physiological studies using in vitro estimates that predict near-equilibrium enzymes carry excess capacity. The possibility is that this excess capacity is exploited under demand states different from the experimental test conditions.

Reproductive Diapause

There is a described phenotype in Drosophila melanogaster females that is called a reproductive diapause. Females if placed upon emergence on a short day length and low temperature show a response where egg production stops and developing eggs are absorbed. This trait is genetically variable in natural populations, and we have data showing that this trait is geographically variable with northern populations showing a much higher frequency of induction (Schmidt et al. 2005). I have a joint NSF-sponsored grant with Paul Schmidt at the University of Pennsylvania to study the ecology and genetics of reproductive diapause and the responsibility of this lab is to map the genes for the trait using QTL mapping techniques. 

Ethanol Tolerance`

Drosophila melanogaster has been used as a model in studies of alcohol metabolism and tolerance. Nevertheless, while the pathways involved in ethanol metabolism are somewhat known, their relative importance and the relative importance of individual enzymatic steps in the control of those pathways are unknown. We use the experimental power of transposon-derived knockouts to modulate the enzyme activity of relevant pathways and evaluate the metabolic control associated with individual steps. This level of control will likely vary between steps, and will implicate those enzymes where activity variation will be responsive to natural selection for ethanol tolerance, a trait shown to increase with temperate latitudes in D. melanogaster. We study the relationship of allelic variation in these enzymes to latitudinal change and test for patterns of long-term molecular adaptation in multispecies contrasts of molecular variation. 

Our studies have the  potential to add to an understanding of the metabolic elimination of alcohols in Drosophila and the adaptations associated with those pathways.  Furthermore, the cosmopolitan spread and adaptation of D. melanogaster to alcohol environments is a longstanding textbook paradigm in evolutionary genetics, and this paradigm will be expanded by this system-wide exploration. Fruit flies feed on yeast, and this ecological niche exposes them to numerous toxic fermentation products including alcohols. In particular, it is believed that the tolerance of D. melanogaster to alcohols is an evolved trait since other members of the melanogaster subgroup show very low tolerance and avoid alcohols. In contrast, D. melanogaster readily utilizes ethanol as a carbon source and individual tolerance is highest in temperate climates.