The winged insects of the order Diptera are colloquially named for their most recognizable phenotype: flight. These insects rely on flight for a number of important life history traits, such as dispersal, foraging, and courtship. Despite the importance of flight, relatively little is known about the genetic architecture of flight performance. Accordingly, we sought to uncover the genetic modifiers of flight using a measure of flies' reaction and response to an abrupt drop in a vertical flight column. We conducted a genome wide association study (GWAS) using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, and identified a combination of additive and marginal variants, epistatic interactions, whole genes, and enrichment across interaction networks. Egfr, a highly pleiotropic developmental gene, was among the most significant additive variants identified. We functionally validated 13 of the additive candidate genes' (Adgf-A/Adgf-A2/CG32181, bru1, CadN, flapper (CG11073), CG15236, flippy (CG9766), CREG, Dscam4, form3, fry, Lasp/CG9692, Pde6, Snoo), and introduce a novel approach to whole gene significance screens: PEGASUS_flies. Additionally, we identified ppk23, an Acid Sensing Ion Channel (ASIC) homolog, as an important hub for epistatic interactions. We propose a model that suggests genetic modifiers of wing and muscle morphology, nervous system development and function, BMP signaling, sexually dimorphic neural wiring, and gene regulation are all important for the observed differences flight performance in a natural population. Additionally, these results represent a snapshot of the genetic modifiers affecting drop-response flight performance in Drosophila, with implications for other insects. Author summary Insect flight is a widely recognizable phenotype of many winged insects, hence the name: flies. While fruit flies, or Drosophila melanogaster, are a genetically tractable model, flight performance is a highly integrative phenotype, and therefore challenging to identify comprehensively which genetic modifiers contribute to its genetic architecture. Accordingly, we screened 197 Drosophila Genetic Reference Panel lines for their ability to react and respond to an abrupt drop. Using several computational approaches, we identified additive, marginal, and epistatic variants, as well as whole genes and altered sub-networks of gene-gene and protein-protein interaction networks that contribute to variation in flight performance. More generally, we demonstrate the benefits of employing multiple methodologies to elucidate the genetic architecture of complex traits. Many variants and genes mapped to regions of the genome that affect neurodevelopment, wing and muscle development, and regulation of gene expression. We also introduce PEGASUS_flies, a Drosophila-adapted version of the PEGASUS platform first used in human studies, to infer gene-level significance of association based on the gene's distribution of individual variant P-values. Our results contribute to the debate over the relative importance of individual, additive factors and epistatic, or higher order, interactions, in the mapping of genotype to phenotype.
