Characterizing the genetic basis of copper toxicity inDrosophilareveals a complex pattern of allelic, regulatory, and behavioral variation

Abstract

1A range of heavy metals are required for normal cell function and homeostasis. Equally, 2 the anthropogenic release of heavy metals into soil and water sources presents a pervasive 3 health threat. Copper is one such metal; it functions as a critical enzymatic cofactor, but at high 4 concentrations is toxic, and can lead to the production of reactive oxygen species. Using a 5 combination of quantitative trait locus (QTL) mapping and RNA sequencing in the Drosophila 6Synthetic Population Resource (DSPR), we demonstrate that resistance to the toxic effects of 7 ingested copper in D. melanogaster is genetically complex, and influenced by allelic and 8 expression variation at multiple loci. Additionally, we find that copper resistance is impacted by 9 variation in behavioral avoidance of copper and may be subject to life-stage specific regulation. 10Multiple genes with known copper-specific functions, as well as genes that are involved in the 11 regulation of other heavy metals were identified as potential candidates to contribute to 12 variation in adult copper resistance. We demonstrate that nine of 16 candidates tested by RNAi 13 knockdown influence adult copper resistance, a number of which may have pleiotropic effects 14 since they have previously been shown to impact the response to other metals. Our work 15 provides new understanding of the genetic complexity of copper resistance, highlighting the 16 diverse mechanisms through which copper pollution can negatively impact organisms. 17Additionally, we further support the similarities between copper metabolism and that of other 18 essential and nonessential heavy metals. 19 20 Rearing and assay conditions 126Strains from the DSPR were maintained, reared, and tested in the same incubator under 127 a 12:12hr light:dark photoperiod at 25°C and 50% humidity. To obtain female flies for the adult 128 copper response assay, RNA sequencing, and RNAi validation, adults were transferred to 129 cornmeal-molasses-yeast food, allowed to oviposit for two days, then discarded. Experimental 130 female, presumably mated, flies from the following generation were sorted over CO2 and were 131 placed into vials with new cornmeal-molasses-yeast media for 24 hours prior to the start of each 132 assay before they were transferred to copper-supplemented food. All adult assays were 133 performed on 3-5 day old individuals. 134

The genomic basis of copper tolerance inDrosophilais shaped by a complex interplay of regulatory and environmental factors

“…4 and Table 2). Of these seven confirmed genes, CG11594, Cyp6w1, Cyp6a8 , and Jon65Aiv , are novel candidates, whose full role in copper biology is not yet understood, while the other three genes, Sodh-1, CG6910 , and CG32444 have prior links to the phenotype [12, 20, 55]. On the other hand, CG5966 displayed decreased mortality when knocked-down, which was not predicted by its induction on copper (Fig.…”

“…To date, there have been several studies exploring the nature of copper tolerance in natural strains of D. melanogaster, both with regard to individual genes [16,17] and to broader developmental and learning and memory processes [18,19]. Recently, Everman et al (2021) [20] took benefit of a combination of high throughput genomic and transcriptomic approaches to uncover several new copper gene candidates, using recombinant inbred lines. They found that copper resistance is genetically complex and impacted by variation in copper avoidance behavior.…”

“…They found that copper resistance is genetically complex and impacted by variation in copper avoidance behavior. In addition to identifying natural variants involved in response to copper, their pairing of genomic data with transcriptomic data also provided a greater opportunity to identify factors that regulate copper induced changes in expression, beyond the well-known MTF-1 factor [20]. Prior expression analyses on metal exposure also suggest that there are a number of co-regulated gene clusters linked to broader stress and metabolism related pathways in response to heavy metal exposure, independent of MTF-1 [20][21][22].…”

“…Recently, Everman et al . (2021) [20] took benefit of a combination of high throughput genomic and transcriptomic approaches to uncover several new copper gene candidates, using recombinant inbred lines. They found that copper resistance is genetically complex and impacted by variation in copper avoidance behavior.…”

“…To date, genome-wide studies investigating the genetic basis of tolerance to copper and other heavy metals in D. melanogaster have focused on SNP variants or were naïve to the nature of the causal variant [20, 23]. The recent availability of new whole-genome assemblies based on long-read sequencing gives us the unprecedented opportunity to characterize complex forms of sequence variation that may have previously been overlooked [24, 25].…”