A naturally-occurring 22-bp coding deletion in Ugt86Dd reduces nicotine resistance in Drosophila melanogaster

This work is licensed under a Creative Commons Attribution 4.0 International License.Objective Segregating genetic variants contribute to the response to toxic, xenobiotic compounds, and identifying these causative sites can help describe the mechanisms underlying metabolism of toxic compounds. In previous work we implicated the detoxification gene Ugt86Dd in the genetic control of larval nicotine resistance in Drosophila melanogaster. Furthermore, we suggested that a naturally-occurring 22-bp deletion that leads to a stop codon in exon 2 of the gene markedly reduces resistance. Here we use homology directed CRISPR/Cas9 gene editing to specifically test this hypothesis. Results We edited chromosome three from an inbred strain named A4 which carries the insertion allele at Ugt86Dd, successfully generated four alleles carrying the 22-bp Ugt86Dd deletion, and substituted edited chromosomes back into the A4 background. The original A4 strain, and an un-edited control strain in the same A4 background, show no significant difference in egg-to-adult or larva-to-adult viability on either control media or nicotine-supplemented media, and only slightly delayed development in nicotine media. However, strains carrying the 22-bp deletion showed reduced viability in nicotine conditions, and significantly longer development. Our data strongly suggest that the naturally-occurring 22-bp insertion/deletion event in Ugt86Dd directly impacts variation in nicotine resistance in D. melanogaster

Genetics of cocaine and methamphetamine consumption and preference in Drosophila melanogaster.

Illicit use of psychostimulants, such as cocaine and methamphetamine, constitutes a significant public health problem. Whereas neural mechanisms that mediate the effects of these drugs are well-characterized, genetic factors that account for individual variation in susceptibility to substance abuse and addiction remain largely unknown. Drosophila melanogaster can serve as a translational model for studies on substance abuse, since flies have a dopamine transporter that can bind cocaine and methamphetamine, and exposure to these compounds elicits effects similar to those observed in people, suggesting conserved evolutionary mechanisms underlying drug responses. Here, we used the D. melanogaster Genetic Reference Panel to investigate the genetic basis for variation in psychostimulant drug consumption, to determine whether similar or distinct genetic networks underlie variation in consumption of cocaine and methamphetamine, and to assess the extent of sexual dimorphism and effect of genetic context on variation in voluntary drug consumption. Quantification of natural genetic variation in voluntary consumption, preference, and change in consumption and preference over time for cocaine and methamphetamine uncovered significant genetic variation for all traits, including sex-, exposure- and drug-specific genetic variation. Genome wide association analyses identified both shared and drug-specific candidate genes, which could be integrated in genetic interaction networks. We assessed the effects of ubiquitous RNA interference (RNAi) on consumption behaviors for 34 candidate genes: all affected at least one behavior. Finally, we utilized RNAi knockdown in the nervous system to implicate dopaminergic neurons and the mushroom bodies as part of the neural circuitry underlying experience-dependent development of drug preference

Identifying Loci Contributing to Natural Variation in Xenobiotic Resistance in Drosophila.

Natural populations exhibit a great deal of interindividual genetic variation in the response to toxins, exemplified by the variable clinical efficacy of pharmaceutical drugs in humans, and the evolution of pesticide resistant insects. Such variation can result from several phenomena, including variable metabolic detoxification of the xenobiotic, and differential sensitivity of the molecular target of the toxin. Our goal is to genetically dissect variation in the response to xenobiotics, and characterize naturally-segregating polymorphisms that modulate toxicity. Here, we use the Drosophila Synthetic Population Resource (DSPR), a multiparent advanced intercross panel of recombinant inbred lines, to identify QTL (Quantitative Trait Loci) underlying xenobiotic resistance, and employ caffeine as a model toxic compound. Phenotyping over 1,700 genotypes led to the identification of ten QTL, each explaining 4.5-14.4% of the broad-sense heritability for caffeine resistance. Four QTL harbor members of the cytochrome P450 family of detoxification enzymes, which represent strong a priori candidate genes. The case is especially strong for Cyp12d1, with multiple lines of evidence indicating the gene causally impacts caffeine resistance. Cyp12d1 is implicated by QTL mapped in both panels of DSPR RILs, is significantly upregulated in the presence of caffeine, and RNAi knockdown robustly decreases caffeine tolerance. Furthermore, copy number variation at Cyp12d1 is strongly associated with phenotype in the DSPR, with a trend in the same direction observed in the DGRP (Drosophila Genetic Reference Panel). No additional plausible causative polymorphisms were observed in a full genomewide association study in the DGRP, or in analyses restricted to QTL regions mapped in the DSPR. Just as in human populations, replicating modest-effect, naturally-segregating causative variants in an association study framework in flies will likely require very large sample sizes

Identifying Loci Contributing to Natural Variation in Xenobiotic Resistance in Drosophila

Natural populations exhibit a great deal of interindividual genetic variation in the response to toxins, exemplified by the variable clinical efficacy of pharmaceutical drugs in humans, and the evolution of pesticide resistant insects. Such variation can result from several phenomena, including variable metabolic detoxification of the xenobiotic, and differential sensitivity of the molecular target of the toxin. Our goal is to genetically dissect variation in the response to xenobiotics, and characterize naturally-segregating polymorphisms that modulate toxicity. Here, we use the Drosophila Synthetic Population Resource (DSPR), a multiparent advanced intercross panel of recombinant inbred lines, to identify QTL (Quantitative Trait Loci) underlying xenobiotic resistance, and employ caffeine as a model toxic compound. Phenotyping over 1,700 genotypes led to the identification of ten QTL, each explaining 4.5–14.4% of the broad-sense heritability for caffeine resistance. Four QTL harbor members of the cytochrome P450 family of detoxification enzymes, which represent strong a priori candidate genes. The case is especially strong for Cyp12d1, with multiple lines of evidence indicating the gene causally impacts caffeine resistance. Cyp12d1 is implicated by QTL mapped in both panels of DSPR RILs, is significantly upregulated in the presence of caffeine, and RNAi knockdown robustly decreases caffeine tolerance. Furthermore, copy number variation at Cyp12d1 is strongly associated with phenotype in the DSPR, with a trend in the same direction observed in the DGRP (Drosophila Genetic Reference Panel). No additional plausible causative polymorphisms were observed in a full genomewide association study in the DGRP, or in analyses restricted to QTL regions mapped in the DSPR. Just as in human populations, replicating modest-effect, naturally-segregating causative variants in an association study framework in flies will likely require very large sample sizes

Founder haplotype means and 1-SDs for the three QTL mapped in both panels of DSPR RILs.

<p>Means are presented for both pA (light gray bars) and pB (dark gray bars), and the number of RILs for which we assign a founder genotype (probability > 0.95) is listed at the bottom of each bar. Only founder means associated with at least 5 observations are presented. Since line AB8 was used to found both synthetic populations, and the haplotype mean was independently estimated from pA and pB RILs, the line is presented twice in each plot.</p

Effects of single gene RNAi knockdown experiments.

<p>Gal4-UAS-RNAi female progeny of several genotypes were tested in our caffeine resistance assay against control strains ("Con"). The genes tested were <i>Cyp12d1-d</i> and <i>Cyp12d1-d</i> under QTL Q2, <i>Cyp301a1</i> under Q3, and <i>Cyp6d5</i> and under Q9. Each bar represents the mean lifespan (± 1-SD) in the assay across a number of genetically-identical individuals (sample size is in the bottom right corner of each bar), and asterisks represent the significance of Welch's <i>t</i>-test comparing each RNAi genotype to its respective control (^ns = not significant, *<i>p</i> < 0.01, **<i>p</i> < 0.001, ***<i>p</i> < 10⁻¹⁰). (<b>A</b>) Driving Gal4 using a ubiquitous promoter with KK-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60100, 109248, 109256, 109771, and 107641. (<b>B</b>) Driving Gal4 using a ubiquitous promoter with GD-UAS lines. Left-to-right the VDRC stock numbers of the test genotypes are 60000, 50507, 21235, and 49269. (<b>C</b> and <b>D</b>) Driving Gal4 ubiquitously in adults using an RU486-inducible promoter in two independent trials, the first (<b>C</b>) with flies on RU486 for 24 hours prior to the assay and throughout, and the second (<b>D</b>) with flies on RU486 for 48 hours prior to the assay and throughout. Genotypes tested are the same as those depicted in (<b>A</b>).</p

Details of mapped caffeine resistance QTL.

<p>^a 2-LOD CI indicates the 2-LOD support interval of the QTL. Physical positions are given based on release 5 of the <i>Drosophila</i> reference genome.</p><p>^b The number of protein-coding genes in the 2-LOD support interval.</p><p>^c The proportion of the phenotypic variance due to each QTL comes directly from the linear model used for mapping (page 77 of [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005663#pgen.1005663.ref076" target="_blank">76</a>]). The percentage of the broad-sense heritability (<i>H</i>²) is simply this estimate divided by the broad-sense heritability of the mean measure of caffeine resistance.</p><p>Details of mapped caffeine resistance QTL.</p

Genome scan for caffeine resistance QTL.

<p>Scans for population pA and pB are shown in blue and red curves, respectively, and the horizontal dotted lines represent population-specific genomewide 5% permutation thresholds (pA, LOD = 8.1; pB, LOD = 7.4). Genetic distances along the chromosomes are indicated along the <i>x</i>-axis. The centromeres are at positions 54 and 47 on chromosomes 2 and 3, respectively. The positions of the ten QTL we describe in the text are indicated on the plot for ease of reference. Intervals implicated by these QTL are highlighted as vertical bars, with pA-specific QTL in light blue, pB-specific QTL in pink, and QTL identified in both panels in yellow.</p