Cross-Study Comparison Reveals Common Genomic, Network, and Functional Signatures of Desiccation Resistance in Drosophila melanogaster Marina Telonis-Scott,*,1 Carla M. Sgro, 1 Ary A. Hoffmann,2 and Philippa C. Griffin2 1School of Biological Sciences, Monash University, Clayton, Melbourne, VIC, Australia 2School of BioSciences, Bio21 Institute, University of Melbourne, Parkville, Melbourne, VIC, Australia *Corresponding author: E-mail: [email protected]. Associate editor: Ilya Ruvinsky Abstract Repeated attempts to map the genomic basis of complex traits often yield different outcomes because of the influence of genetic background, gene-by-environment interactions, and/or statistical limitations. However, where repeatability is low at the level of individual genes, overlap often occurs in gene ontology categories, genetic pathways, and interaction networks. Here we report on the genomic overlap for natural desiccation resistance from a Pool-genome-wide association study experiment and a selection experimentinfliescollectedfromthesameregioninsoutheasternAustraliaindifferent years. We identified over 600 single nucleotide polymorphisms associated with desiccation resistance in flies derived from almost 1,000 wild-caught genotypes, a similar number of loci to that observed in our previous genomic study of selected lines, demonstrating the genetic complexity of this ecologically important trait. By harnessing the power of cross-study comparison, we narrowed the candidates from almost 400 genes in each study to a core set of 45 genes, enriched for stimulus, stress, and defense responses. In addition to gene-level overlap, there was higher order congruence at the network and functional levels, suggesting genetic redundancy in key stress sensing, stress response, immunity, signaling, and gene expression pathways. We also identified variants linked to different molecular aspects of desiccation physiology previously verified from functional experiments. Our approach provides insight into the genomic basis of a complex and ecologically important trait and predicts candidate genetic pathways to explore in multiple genetic backgrounds and related species within a functional framework. Key words: desiccation, Drosophila, GWAS, gene overlap. Introduction In climate change research there is increasing interest to con- commonly tolerating water loss (Gibbs and Matzkin 2001; sider not only the obvious impact of changing temperatures Gibbs et al. 2003). Water retention appears to be the primary Article on biodiversity, but also fluctuations in rainfall and humidity mechanism for withstanding desiccation in highly resistant (Bonebrake and Mastrandrea 2010; Clusella-Trullas et al. 2011; cactophilic Drosophila (Gibbs and Matzkin 2001), as well as in Chown 2012). Changes in water availability pose specific chal- Drosophila melanogaster (Telonis-Scott et al. 2006). How lenges to terrestrial ectotherms such as insects, impacting water is preserved, however, is highly variable depending on activity, range distributions, species richness, and disease the species and/or genetic background. Cactophilic species vector populations (reviewed in Chown et al. 2011,andref- have repeatedly colonized arid habitats via reduced metabolic erences therein). Efforts to understand the responses of in- rates that stem respiratory and cuticular water loss and sects to water availability are underway, given that extend energy utilization (Gibbs and Matzkin 2001; Marron physiological responses are an integral component of predict- et al. 2003), while in the laboratory, water retention and se- ing species responses to climate change (Chown et al. 2011; questration arise from multiple evolutionary pathways less Hoffmann and Sgro2011 ). Insect water balance physiology is clearly related to metabolic rate (Hoffmann and Parsons relatively well elucidated (Hadley 1994; Chown and Nicolson 1989a), often acting in concert (Hoffmann and Parsons 2004; Bradley 2009), and the field is undergoing a crucial shift 1989a; Djawdan et al. 1998; Folk et al. 2001; Telonis-Scott toward understanding the molecular underpinnings, largely et al. 2006, 2012). Less resolved is the role of respiratory rel- achieved through high-throughput and transgenic technolo- ative to cuticular water loss via discontinuous gas exchange, gies in Drosophila. although this might be an important water budget strategy Insects can lose water from the epicuticle, through respi- for insects in general (Chown et al. 2011). ration via the spiracles or across the gut epithelia (Hadley Recent candidate gene-based approaches have seen new 1994). Drosophila balance water via three main mechanisms: developments in understanding specific molecular aspects of Altering water content, slowing water loss rate, and less this variable ecological trait in Drosophila. Diuretic peptide signaling in ion and water balance regulated by excretion via 2009). Accordingly, we sampled natural genetic variation the Malpighian tubules (MTs) and gut absorption has been from almost 1,000 inseminated wild-caught females, and shown to impact desiccation resistance in D. melanogaster compared the upper 5% desiccation resistant tail from (Kahsai et al. 2010; Terhzaz et al. 2012, 2014, 2015). Specifically, almost 10,000 F1 progeny with a control sample chosen ran- knockdown of the Capability (Capa) neuropeptide gene en- domly from the same progeny set. Harnessing the power of hanced desiccation resistance by reductions in respiratory, Pool-GWAS, we sequenced a pool of 500 natural “resistant” cuticular, and excretory water loss, while also cross-conferring genomes and compared allele frequencies with a pool of 500 cold tolerance (Terhzaz et al. 2015). Cuticular hydrocarbon “random control” genomes. (CHC) levels are also implicated; a single gene encoding a fatty We employed a novel comparative approach with the re- acid synthase mFAS was shown to impact both desiccation sulting set of candidate loci and those detected in our previ- resistance and mate choice in Australian rainforest endemics ous artificial selection study of flies collected several seasons Drosophila serrata and Drosophila birchii (Chung et al. 2014). earlier from the same region of southeastern Australia In D. melanogaster,knockdownofCYP4G1 also reduced CHC (Telonis-Scott et al. 2012). Cross-study repeatability of loci production and impaired survival under low humidity (Qiu associated with complex phenotypes is often low (Sarup et al. 2012). Metabolic signaling has also been shown to et al. 2011), and can be influenced by genetic background, impact desiccation resistance, and includes components of selection intensity, epistatic effects, and gene-by-environment the insulin signaling pathways (Soderberg€ et al. 2011; Liu et al. interactions (Mackay et al. 2009; Civelek and Lusis 2014). 2015). Several other single gene studies highlight different Multiple genetic solutions appear to underlie the array of mechanisms such as desiccation avoidance in larvae via no- desiccation responses, and we further explored this by inves- ciceptors encoded by members of the transient receptor tigating overlap at the level of individual genes, functional potential (TRP) aquaporin family (Johnson and Carder gene ontology (GO) categories, and protein–protein interac- 2012), cyclic adenosine monophosphate (cAMP)-dependent tion (PPI) networks. Genetic variants contribute to final phe- signaling protein kinase desi (Kawano et al. 2010), and poten- notypes by way of “intermediate phenotypes” (transcript, tial tissue protection via trehalose sugar accumulation protein, and metabolite abundances) and correlations (Thorat et al. 2012). should theoretically occur across these biological scales Collectively, these functional approaches highlight the (Civelek and Lusis 2014). complexity of insect water balance strategies in controlled Here we report on a screen of natural nucleotide vari- backgrounds, but do not explain the variation observed in ants associated with desiccation resistance and, using a pow- natural populations and species. Resistance evolves rapidly erful analysis approach, demonstrate common cross-study and is highly heritable (up to 60%) in the cosmopolitan and signatures across different hierarchical levels from gene, to unusually resistant species D. melanogaster, whereas heritabil- network, and function. We found evidence that some func- ity is much lower in less tolerant range-restricted species tional desiccation candidates may be important in wild (Hoffmann and Parsons 1989b; Kellermann et al. 2009). populations, and discovered variants linked to multiple phys- Multiple, genome-wide quantitative trait loci were identified iological responses, consistent with the trait’s complex under- from mapping lines constructed from a natural D. melanoga- pinnings at both the physiological and molecular levels. ster population, suggesting a polygenetic architecture (Foley and Telonis-Scott 2011). Transcriptomics revealed differential Results regulation of thousands of genes in response to desiccation in Drosophila mojavensis (Matzkin and Markow 2009), while Genome Wide Differentiation for Natural Desiccation artificial selection for desiccation resistance altered the basal Resistance expression of over 200 genes in D. melanogaster (Sørensen We collected over 1,000 D. melanogaster isofemale lines et al. 2007). from southern Australia, and after one generation of lab- Previously, we used microarray-based genomic hybridiza- oratory culture screened over 9,000 female progeny for tion to survey allele frequency