Genetic and transcriptomic basis of copper tolerance across natural European Drosophila melanogaster populations

Resistance to oxidative stress plays a vital role in animal physiology, where it influences both life history traits and the ability to tolerate the effects of a myriad of environmental stressors. While stress resistance has previously been shown to share a role in shaping an organism's response to traits as varied as desiccation, thermal tolerance and xenobiotic resistance, heavy metal tolerance presents a particular challenge with regards to adaptation to stress. Heavy metals contamination can result in robust and persistent selection pressure: not only is acute exposure highly toxic, but metals can accumulate in the environment over long periods, prolonging this exposure. However, many heavy metals, such as copper and zinc, are also essential micronutrients, which may constrain adaption in high copper conditions. To determine the genetic basis of copper tolerance in European Drosophila melanogaster, we phenotyped 76 inbred lines sampled from nine locations across Western Europe on copper sulphate, demonstrating that that copper tolerance is a highly variable trait. A combination of long-read nanopore sequencing and high-throughput RNA-seq analysis carried out before and after copper exposure shows that while copper tolerance is a highly heterogenous trait, affected by changes in expression across multiple loci, the greatest changes in expression a seen in the digestive tract. In addition, a large proportion of genes found differentially expressed upon copper exposure have previously shown to be regulated by a number of transcription factors with known roles in a broad range of metabolic processes, indicating that while the initial response may be tissue restricted, long term affects of copper expose are more likely to be systemic. Overall design: Of 76 lines phenotyped, three for the more sensitive tail of the distribution (COR-018, JUT-008, AKA-018) and three from the more resistant end (MUN-008, GIM-012, MUN-020) were selected for further analysis. Briefly, 3- to 7-day-old females from the six lines were exposed to 20mM copper sulfate in a 5% solution for 24 hours. Exposure involved both contact and ingestion. 20 females were used in each biological repeat, for a total of three repeats. Controls were conducted under the same conditions, without the use of copper.