Obp56h Modulates Mating Behavior in Drosophila melanogaster

Social interactions in insects are driven by conspecific chemical signals that are detected via olfactory and gustatory neurons. Odorant binding proteins (Obps) transport volatile odorants to chemosensory receptors, but their effects on behaviors remain poorly characterized. Here, we report that RNAi knockdown of Obp56h gene expression in Drosophila melanogaster enhances mating behavior by reducing courtship latency. The change in mating behavior that results from inhibition of Obp56h expression is accompanied by significant alterations in cuticular hydrocarbon (CHC) composition, including reduction in 5-tricosene (5-T), an inhibitory sex pheromone produced by males that increases copulation latency during courtship. Whole genome RNA sequencing confirms that expression of Obp56h is virtually abolished in Drosophila heads. Inhibition of Obp56h expression also affects expression of other chemoreception genes, including upregulation of lush in both sexes and Obp83ef in females, and reduction in expression of Obp19b and Or19b in males. In addition, several genes associated with lipid metabolism, which underlies the production of cuticular hydrocarbons, show altered transcript abundances. Our data show that modulation of mating behavior through reduction of Obp56h is accompanied by altered cuticular hydrocarbon profiles and implicate 5-T as a possible ligand for Obp56h

Genetic Architecture of Abdominal Pigmentation in Drosophila melanogaster.

Pigmentation varies within and between species and is often adaptive. The amount of pigmentation on the abdomen of Drosophila melanogaster is a relatively simple morphological trait, which serves as a model for mapping the genetic basis of variation in complex phenotypes. Here, we assessed natural variation in female abdominal pigmentation in 175 sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel, derived from the Raleigh, NC population. We quantified the proportion of melanization on the two most posterior abdominal segments, tergites 5 and 6 (T5, T6). We found significant genetic variation in the proportion of melanization and high broad-sense heritabilities for each tergite. Genome-wide association studies identified over 150 DNA variants associated with the proportion of melanization on T5 (84), T6 (34), and the difference between T5 and T6 (35). Several of the top variants associated with variation in pigmentation are in tan, ebony, and bric-a-brac1, genes known to affect D. melanogaster abdominal pigmentation. Mutational analyses and targeted RNAi-knockdown showed that 17 out of 28 (61%) novel candidate genes implicated by the genome-wide association study affected abdominal pigmentation. Several of these genes are involved in developmental and regulatory pathways, chitin production, cuticle structure, and vesicle formation and transport. These findings show that genetic variation may affect multiple steps in pathways involved in tergite development and melanization. Variation in these novel candidates may serve as targets for adaptive evolution and sexual selection in D. melanogaster

Genetic basis of natural variation in body pigmentation in <i>Drosophila melanogaster</i>

<p>Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at <i>bab1, t</i> and <i>e</i> affect variation in abdominal pigmentation in several populations of <i>Drosophila melanogaster</i>. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the <i>Drosophila</i> Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in <i>bab1, t</i> and <i>e</i>; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including <i>bab1, t</i> and <i>e</i> and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of <i>Drosophila</i> pigmentation is complex, with a few major genes and many other loci with smaller effects.</p

Natural variation in female abdominal pigmentation.

<p>(<b>A</b>) T5 (gray) and T6 (red). DGRP lines are in order from least to most pigmentation on T6. (<b>B</b>) Scatter plot of T5 and T6 line means.</p

Genome-wide association analyses.

<p>Results are depicted for T5, T6, and the T5-T6 difference. A nominal <i>P</i> ≤ 10^–5 is indicated with a red line for each trait. The triangular heat map depicts the degree of LD, <i>r</i>², between variants. The five major chromosome arms are delineated by the black lines. Red corresponds to complete LD and blue to absence of LD. The upper panels show the mixed model significance threshold (-log10(<i>P</i>)) and the effect size in phenotypic standard deviation units (<i>a</i>/<i>σp</i>) for each trait. The minor allele frequency (MAF) is shown on the bottom panel.</p

Pairwise linkage disequilibrium among the associated <i>bab1</i> variants.

<p>Heat map of LD (<i>r</i>²) of the 21 <i>bab1</i> variants. Individual variants, <i>cis</i>-regulatory regions and transcription factor binding sites are labeled on the diagonal.</p

Female abdominal cuticles of with significant effects on pigmentation in lines with targeted RNAi knockdown constructs.

<p><b>A</b>-<b>J</b>: <i>pnr-GAL4</i> x <i>UAS-RNAi</i> genotypes. (<b>A</b>) VDRC KK library control. (<b>B</b>) <i>btn</i>. (<b>C</b>) <i>CG7852</i>. (<b>D</b>) <i>Klp61F</i>. (<b>E</b>) <i>ru</i>. (<b>F</b>) VDRC GD library control. (<b>G</b>) <i>Efa6</i>. (<b>H</b>) <i>klar</i>. (<b>I</b>) <i>sinu</i>. <b>J</b>-<b>Q</b>: <i>tub</i>-<i>GAL4</i> x <i>UAS-RNAi</i> genotypes. (<b>J</b>) VDRC KK library control. (<b>K</b>) <i>btn</i>, (<b>L</b>) <i>CG10625</i>. (<b>M</b>) <i>CG9134</i>. (<b>N</b>) VDRC GD library control. (<b>O</b>) <i>Efa6</i>. (<b>P</b>) <i>loco</i>. <b>Q</b>-<b>T</b>: <i>ubi</i>-<i>GAL4</i> x RNAi-<i>UAS</i> genotypes. (<b>Q</b>) VDRC KK library control. (<b>R</b>) <i>CG1887</i>. (<b>S</b>) VDRC GD control. (<b>T</b>) <i>klar</i>. ns = not significant. ↑ and ↓ indicate significantly increased and decreased proportions of dark melanin, respectively.</p

Summary of candidate gene validation experiments.

<p>NS = not significant,"–" = line not available or RNAi cross not tested. ↑ = increased pigmentation. ↓ = decreased pigmentation.</p><p>Summary of candidate gene validation experiments.</p

Validation of pigmentation candidate genes using Exelixis insertion mutants and RNAi knockdown.

<p>The <i>y</i>-axis in all panels is the deviation of the appropriate control line mean from the experimental line mean. Increases and decreases in the proportion of melanization are given above and below the <i>x</i>-axis, respectively. (<b>A</b>) Exelixis insertion mutants. (<b>B</b>) <i>pnr-GAL4</i> x RNAi-<i>UAS</i> lines. (<b>C</b>) <i>tub</i>-<i>GAL4</i> x RNAi-<i>UAS</i> lines. (<b>D</b>) <i>ubi</i>-<i>GAL4</i> x RNAi-<i>UAS</i>-lines. ***: <i>P</i><0.0001.</p