This project aims to understand how evolution shapes animal physiology in response to prolonged exposure to juvenile undernutrition, whereby juvenile animals are forced to grow and develop in spite of chronic nutrient shortage. It will address four general questions:(1) In what way can evolution modify metabolism of a growing juvenile animal to alleviate consequences of undernutrition for Darwinian fitness?(2) What changes in allocation of metabolic resources does this adaptation entail?(3) To what degree are these changes mediated by genetic variants in 'master' genes with large effects on multiple aspects of the adaptation?(4) To what degree is evolution of tolerance to poor diet mediated by genetic variants in genes that regulate growth (i.e., the 'demand' for biomass building blocks) versus genes whose products catalyze and regulate their acquisition and allocation (i.e., the 'supply' side of growth)? The project will use lines of Drosophila melanogaster characterized by extraordinary genetically-based tolerance to larval undernutrition, a unique resource generated through >17 years of laboratory experimental evolution. To address the above questions, we will elucidate causal changes in growth regulation and metabolism underlying this highly polygenic and phenotypically complex evolutionary adaptation. First, we will quantify the rate of amino acid turnover and the allocation of resources to different components of biomass (proteins, triglycerides, glycogen, etc.) in the 'Selected' (malnutrition-tolerant) and 'Control' (unselected) larvae. Together with gene expression data, these data will be used to model the core metabolic network of Drosophila. This will generate testable predictions about differences in metabolic fluxes underlying malnutrition tolerance and identify nodes of the metabolic network critical for differential fluxes and resulting allocation patterns (question 1 and 2). It will also be used to evaluate the roles of nutrient supply versus demand on metabolic output in shaping the patterns of metabolic flux (question 4).Second, we will verify the contribution of a cis-regulatory variant in an ecdysone oxidase gene fiz to enhanced tolerance to undernutrition, and test whether the adaptive value of this variant is contingent on the presence of other elements of this complex adaptation. fiz emerged as a candidate with potential large effects on this adaptation (questions 3); it is thought to regulate growth by deactivating ecdysone, but this hypothesis appears incompatible with the direction of its effects on growth. To elucidate the effect of variation in fiz on ecdysteroid signaling we will study its effect on the abundance of different ecdysteroid species, identifying those most likely to mediate its growth-regulating effect. Third, we will combine the two above threads by investigating how fiz expression affects the rate of nutrient acquisition, metabolite abundance and the allocation of metabolic resources. Given that fiz is thought to act by modulating the demand for key metabolites, we will be able to ascertain to what degree increased demand for biomass building blocks has effects that propagate through metabolism and affect resource allocation question 4). To achieve these aims, we will combine experimental evolution with state-of-the art approaches, including genome editing, metabolomics, isotope tracing, high resolution mass spectroscopy and genome-scale flux balance analysis. This project will advance our understanding of a poorly understood and ecologically important evolutionary adaptation. It will also throw light on the broader fundamental question of how changes indifferent metabolic and regulatory elements interact to generate complex adaptations that enhance Darwinian fitness under conditions of environmental stress. Through novel application of recent experimental techniques and system-scale computational models the project will expand the boundaries of dissecting this complexity.