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. 2015 Nov 14:4:e09861.
doi: 10.7554/eLife.09861.

Genetic architecture of natural variation in cuticular hydrocarbon composition in Drosophila melanogaster

Lauren M Dembeck  1   2   3 , Katalin Böröczky  2   3   4 , Wen Huang  1   2   3 , Coby Schal  2   3   4 , Robert R H Anholt  1   2   3 , Trudy F C Mackay  1   2   3
Affiliations

Genetic architecture of natural variation in cuticular hydrocarbon composition in Drosophila melanogaster

Lauren M Dembeck et al. Elife. .

Abstract

Insect cuticular hydrocarbons (CHCs) prevent desiccation and serve as chemical signals that mediate social interactions. Drosophila melanogaster CHCs have been studied extensively, but the genetic basis for individual variation in CHC composition is largely unknown. We quantified variation in CHC profiles in the D. melanogaster Genetic Reference Panel (DGRP) and identified novel CHCs. We used principal component (PC) analysis to extract PCs that explain the majority of CHC variation and identified polymorphisms in or near 305 and 173 genes in females and males, respectively, associated with variation in these PCs. In addition, 17 DGRP lines contain the functional Desat2 allele characteristic of African and Caribbean D. melanogaster females (more 5,9-C27:2 and less 7,11-C27:2, female sex pheromone isomers). Disruption of expression of 24 candidate genes affected CHC composition in at least one sex. These genes are associated with fatty acid metabolism and represent mechanistic targets for individual variation in CHC composition.

Keywords: African Drosophila; Drosophila genetic reference Panel; cuticular lipids; drosophila melanogaster; evolutionary biology; genome wide associaiton study; genomics; multivariate analysis.

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Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Figure 1.
Figure 1.. Representative male and female chromatograms from the DGRP.
Male cuticular lipids of DGRP_38 are shown on the top (blue) and female CHCs of DGRP_786 are mirrored below (red). All peaks for both sexes were assigned a unique number based on its corresponding compound determined by GC-MS; thus compounds shared between the sexes carry the same number. See Table 1 for the list of compound names. Compounds not previously described in D. melanogaster are shown in bold typeface. Some CHC isomers were not resolved by conventional GC, so a few chromatogram peaks contain more than one CHC. pA = picoAmperes, c = cis-vaccenyl acetate, * = contaminants from CHC extraction, IS = internal standard (n-C32). DOI: http://dx.doi.org/10.7554/eLife.09861.004
Figure 2.
Figure 2.. DGRP lines segregate for the female African CHC phenotype, Desat2 allele, and In3R(K) inversion status.
(A) Overlaid chromatograms of African D. melanogaster CHCs (Z30 and Z53), a DGRP line with an African-like CHC phenotype (DGRP_235), and a Cosmopolitan DGRP line (DGRP_714). (B) DGRP lines with at least one ancestral Desat2 allele exhibit natural variation in the percentage of each CHC peak for the isomeric sex pheromones 7,11-C27:2 (2-Me-C26 co-elutes with 7,11-C27:2) (gray) and 5,9-C27:2 (9-C27:1 co-elutes with 5,9-C27:2) (red). (C) Box-plots of the proportion of each sex pheromone peak for DGRP and African lines according to Desat2 allele and In3R(K) genotypes. DGRP_105 and DGRP_551, which have more Cosmopolitan-like phenotypes despite having the functional ancestral Desat2 allele, are indicated. DOI: http://dx.doi.org/10.7554/eLife.09861.005
Figure 3.
Figure 3.. MMC modules of DGRP female CHCs based on Spearman's rank correlation coefficients (ρ).
Correlations are color-coded from +1 (dark red) to -1 (dark blue). Correlated CHCs are clustered into groups (modules). Modules (outlined in black) are arranged along the diagonal according to the average strengths of the correlations within each cluster; the most strongly correlated modules are on the top left and the weakly correlated modules are on the bottom right. DOI: http://dx.doi.org/10.7554/eLife.09861.007
Figure 4.
Figure 4.. MMC modules of DGRP male CHCs based on Spearman's rank correlation coefficients (ρ).
Correlations are color-coded +1 (dark red) to -1 (dark blue). Correlated CHCs are clustered into groups (modules). Modules (outlined in black) are arranged along the diagonal according to the average strengths of the correlations within the groups; the most strongly and weakly correlated are on the top left and bottom right, respectively. DOI: http://dx.doi.org/10.7554/eLife.09861.008
Figure 5.
Figure 5.. Principal component biplots for PC1 and PC2 of DGRP CHCs.
(A) Female and (C) male PC1 and PC2. (B) Female and (D) male PC1 and PC2 eigenvectors. The percent of variance explained by each PC is indicated on the x- and y-axes. In (A) and (C) DGRP lines are color-coded (Supplementary file 5). DOI: http://dx.doi.org/10.7554/eLife.09861.009
Figure 6.
Figure 6.. QQ-plots of CHC PCA GWA P-values.
(A–G) Female PCs. (H–L) Male PCs. DOI: http://dx.doi.org/10.7554/eLife.09861.011
Figure 7.
Figure 7.. Summary of RNAi and mutant experiments for female CHCs.
UAS-RNAi target gene and the CG10097e00276 mutant are indicated on the horizontal axis. CHC names and numbers are listed on the y-axis. Data are color coded to represent P-values (P ≤ 0.05) from t-tests for the mean differences of the experimental and the control lines. Black = no significant change; blue = significant decrease; green = significant increase; gray = not applicable (peaks 46 and 57 split into two peaks for the CG10097 mutant). DOI: http://dx.doi.org/10.7554/eLife.09861.012
Figure 7—figure supplement 1.
Figure 7—figure supplement 1.. Functional validation PCA and total CHCs for RNAi-app.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.013
Figure 7—figure supplement 2.
Figure 7—figure supplement 2.. Functional validation PCA and total CHCs for RNAi-CG5599.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.014
Figure 7—figure supplement 3.
Figure 7—figure supplement 3.. Functional validation PCA and total CHCs for RNAi-CG7724.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.015
Figure 7—figure supplement 4.
Figure 7—figure supplement 4.. Functional validation PCA and total CHCs for RNAi-CG8680.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.016
Figure 7—figure supplement 5.
Figure 7—figure supplement 5.. Functional validation PCA and total CHCs for RNAi-CG8814.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.017
Figure 7—figure supplement 6.
Figure 7—figure supplement 6.. Functional validation PCA and total CHCs for RNAi-CG9458.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.018
Figure 7—figure supplement 7.
Figure 7—figure supplement 7.. Functional validation PCA and total CHCs for RNAi-CG9801.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.019
Figure 7—figure supplement 8.
Figure 7—figure supplement 8.. Functional validation PCA and total CHCs for mutant CG10097.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.020
Figure 7—figure supplement 9.
Figure 7—figure supplement 9.. Functional validation PCA and total CHCs for RNAi-CG13091.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.021
Figure 7—figure supplement 10.
Figure 7—figure supplement 10.. Functional validation PCA and total CHCs for RNAi-CG14688.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.022
Figure 7—figure supplement 11.
Figure 7—figure supplement 11.. Functional validation PCA and total CHCs for RNAi-CG16979.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.023
Figure 7—figure supplement 12.
Figure 7—figure supplement 12.. Functional validation PCA and total CHCs for RNAi-CG18609.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.024
Figure 7—figure supplement 13.
Figure 7—figure supplement 13.. Functional validation PCA and total CHCs for RNAi-CG30008.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.025
Figure 7—figure supplement 14.
Figure 7—figure supplement 14.. Functional validation PCA and total CHCs for RNAi-Cyp4s3.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.026
Figure 7—figure supplement 15.
Figure 7—figure supplement 15.. Functional validation PCA and total CHCs for RNAi-Cyp9f2.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.027
Figure 7—figure supplement 16.
Figure 7—figure supplement 16.. Functional validation PCA and total CHCs for RNAi-Cyp49a1.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.028
Figure 7—figure supplement 17.
Figure 7—figure supplement 17.. Functional validation PCA and total CHCs for RNAi-Desi.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.029
Figure 7—figure supplement 18.
Figure 7—figure supplement 18.. Functional validation PCA and total CHCs for RNAi-Irc.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.030
Figure 7—figure supplement 19.
Figure 7—figure supplement 19.. Functional validation PCA and total CHCs for RNAi-Lip2.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.031
Figure 7—figure supplement 20.
Figure 7—figure supplement 20.. Functional validation PCA and total CHCs for RNAi-Nrt.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.032
Figure 7—figure supplement 21.
Figure 7—figure supplement 21.. Functional validation PCA and total CHCs for RNAi-PHGPx.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.033
Figure 7—figure supplement 22.
Figure 7—figure supplement 22.. Functional validation PCA and total CHCs for RNAi-Prx6005.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.034
Figure 7—figure supplement 23.
Figure 7—figure supplement 23.. Functional validation PCA and total CHCs for RNAi-Pxd.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.035
Figure 7—figure supplement 24.
Figure 7—figure supplement 24.. Functional validation PCA and total CHCs for RNAi-pxn.
(A) PCA biplots for females and males, ○ = female, ○ = male, and ● = control samples. (B) PC1 and PC2 eigenvectors. (C) Box-plots of female and male total amount of CHCs (µg/fly). P-values are reported for the Satterthwaite test, *: P < 0.05, **: P < 0.01, ***: P < 0.001. DOI: http://dx.doi.org/10.7554/eLife.09861.036
Figure 8.
Figure 8.. Summary of RNAi and mutant experiments for male CHCs.
UAS-RNAi target gene and the CG10097 e00276 mutant are indicated on the horizontal axis. CHC names and numbers are listed on the y-axis. Data are color coded to represent P-values (P ≤ 0.05) from t-tests for the mean differences of the experimental and the control lines. Black = no significant change; blue = significant decrease; green = significant increase; gray = not applicable (peaks 46 and 57 split into two peaks for the CG10097 mutant).* DOI: http://dx.doi.org/10.7554/eLife.09861.037
Figure 9.
Figure 9.. Example chromatograms of oenocyte-specific RNAi knockdowns and mutants – CG13091 and CG10097.
(A) and (B) PromE(800)-GAL4 x UAS-CG13091. (C) and (D) Exelixis mutant CG10097 e00276. pA = picoAmperes, IS = internal standard, ↑ CHCs significantly increased or ↓ decreased according to the individual t-tests. DOI: http://dx.doi.org/10.7554/eLife.09861.038
Figure 10.
Figure 10.. Example chromatograms of oenocyte-specific RNAi knockdowns – CG8680 and CG30008.
(A) and (B) PromE(800)-GAL4 x UAS-CG8680. (C) and (D) PromE(800)-GAL4 x UAS-CG30008. pA = picoAmperes, IS = internal standard, ↑ CHCs significantly increased or ↓ decreased according to the individual t-tests. DOI: http://dx.doi.org/10.7554/eLife.09861.039
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