1. Genetics and Genomics
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Genetic architecture of natural variation in cuticular hydrocarbon composition in Drosophila melanogaster

  1. Lauren M Dembeck
  2. Katalin Böröczky
  3. Wen Huang
  4. Coby Schal
  5. Robert R H Anholt
  6. Trudy F C Mackay  Is a corresponding author
  1. North Carolina State University, United States
Research Article
Cite this article as: eLife 2015;4:e09861 doi: 10.7554/eLife.09861
10 figures, 3 tables and 10 additional files

Figures

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).

https://doi.org/10.7554/eLife.09861.004
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.

https://doi.org/10.7554/eLife.09861.005
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.

https://doi.org/10.7554/eLife.09861.007
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.

https://doi.org/10.7554/eLife.09861.008
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).

https://doi.org/10.7554/eLife.09861.009
QQ-plots of CHC PCA GWA P-values.

(A–G) Female PCs. (H–L) Male PCs.

https://doi.org/10.7554/eLife.09861.011
Figure 7 with 24 supplements
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).

https://doi.org/10.7554/eLife.09861.012
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.

https://doi.org/10.7554/eLife.09861.013
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.

https://doi.org/10.7554/eLife.09861.014
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.

https://doi.org/10.7554/eLife.09861.015
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.

https://doi.org/10.7554/eLife.09861.016
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.

https://doi.org/10.7554/eLife.09861.017
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.

https://doi.org/10.7554/eLife.09861.018
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.

https://doi.org/10.7554/eLife.09861.019
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.

https://doi.org/10.7554/eLife.09861.020
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.

https://doi.org/10.7554/eLife.09861.021
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.

https://doi.org/10.7554/eLife.09861.022
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.

https://doi.org/10.7554/eLife.09861.023
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.

https://doi.org/10.7554/eLife.09861.024
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.

https://doi.org/10.7554/eLife.09861.025
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.

https://doi.org/10.7554/eLife.09861.026
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.

https://doi.org/10.7554/eLife.09861.027
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.

https://doi.org/10.7554/eLife.09861.028
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.

https://doi.org/10.7554/eLife.09861.029
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.

https://doi.org/10.7554/eLife.09861.030
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.

https://doi.org/10.7554/eLife.09861.031
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.

https://doi.org/10.7554/eLife.09861.032
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.

https://doi.org/10.7554/eLife.09861.033
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.

https://doi.org/10.7554/eLife.09861.034
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.

https://doi.org/10.7554/eLife.09861.035
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.

https://doi.org/10.7554/eLife.09861.036
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).*

https://doi.org/10.7554/eLife.09861.037
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.

https://doi.org/10.7554/eLife.09861.038
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.

https://doi.org/10.7554/eLife.09861.039

Tables

Table 1

Cuticular lipids identified by GC-MS in DGRP males and females. NI = not identified; nd = not detected; bold typeface = not previously identified in D. melanogaster.

https://doi.org/10.7554/eLife.09861.003
#Cuticular componentRetention index#Cuticular componentRetention index
1n-C212100210033NI2516nd
2x-C22:1 (quantified only in ♂)2179217934NI2521nd
3x-C22:1nd218435 13-Me-C25
11-Me-C25
2533nd
ccis-vaccenyl acetatend218936 5-Me-C25 2550nd
4n-C222200220037 8,12-C26:2 2555nd
57,11-C23:22259nd387,11-C26:22560nd
62-Me-C2222632263392-Me-C2525622562
7NInd2267406,10-C26:22566nd
89-C23:12273227341 9-C26:1 (only in ♀)
3-Me-C25
25722572
97-C23:122802283427-C26:125772577
106-C23:122852286436-C26:1 + impurity (i)25812581
115-C23:12291229144n-C2626002600
12x-C23:12294nd459,13-C27:22652nd
13n-C2323002300467,11-C27:2 (only in ♀)
2-Me-C26
26642663
14 11-Me-C23
9-Me-C23
23362336475,9-C27:2 (only in ♀)
9-C27:1
26752675
157,11-C24:22355nd487-C27:126822682
16 x,y-C24:2 23632363495-C27:12693nd
173-Me-C23
9-C24:1 (only in ♀)
2373237350n-C2727002700
187-C24:1 (quantified only in ♂)2377237751 8,12-C28:2 2756nd
196-C24:123802380522-Me-C27
7,11-C28:2 (only in ♀)
27612761
20NI (Variable in DGRP lines but not detected in GC-MS samples)ndnd53 6,10-C28:2
3-Me-C27
9-C28:1
2768nd
215-C24:12386238654NI27722772
22n-C242400240055n-C2828002800
239,13-C25:22451nd569,13-C29:22852nd
247,11-C25:22460nd572-Me-C28
7,11-C29:2 (only in ♀)
28642862
252-Me-C2424632463589-C29:1
5,9-C29:1 (only in ♀)
28752875
26 6,10-C25:2 24682468597-C29:128822882
275,9-C25:2 (only in ♀)
9-C25:1
2474247460n-C2929002900
288-C25:12478nd61 8,12-C30:2 (only in ♀)
2-Me-C29
29612961
297-C25:124822483622-Me-C3030603060
30 6-C25:1 2485nd637,11-C31:23065nd
315-C25:12492249264n-C3131003100
32n-C2525002500ISn-C3232003200
Table 2

Phenotypes and In(3R)K genotypes for females from DGRP lines with functional Desat2 alleles. Red text indicates "mismatched" Desat2 genotype ('+' = ancestral; '-' = 16-bp deletion) and inversion status (‘INV’ = In(3R)K; ‘ST’ = Standard karyotype). Blue background indicates "mismatched" Desat2 genotype and phenotype.

https://doi.org/10.7554/eLife.09861.006
DGRP lineDesat 2 genotype% 7,11-C27:2% 5,9-C27:2RatioIn(3R)K status
DGRP_31 + / -23.718.71.27INV / ST
DGRP_38 + / -19.424.90.78INV / ST
DGRP_48 + / -19.125.00.76INV / ST
DGRP_100 + / +22.332.30.69INV / INV
DGRP_136 + / -14.733.70.44INV / ST
DGRP_309 + / -19.117.81.07INV / ST
DGRP_440 + / -20.014.01.43INV / ST
DGRP_559 + / -23.510.52.24INV / ST
DGRP_646 + / +17.324.60.70INV / INV
DGRP_802 + / -18.523.40.79INV / ST
DGRP_732 + / -19.317.31.12INV / ST
DGRP_367 + / +12.636.80.34ST/ ST
DGRP_776 + / +16.78.981.86ST / ST
DGRP_509 + / +17.827.50.65ST / ST
DGRP_235 + / +10.322.20.46ST / ST
DGRP_551 + / -26.64.296.20ST / ST
DGRP_105 + / +21.45.104.20INV / INV
Table 3

Percent of CHC variation in the DGRP explained by PCs.

https://doi.org/10.7554/eLife.09861.010
SexNumberEigenvaluePercentCumulative percent
Female 10.006141.1641.16
20.004329.4770.63
30.002114.5085.13
40.00096.2291.35
50.00053.0794.42
60.00032.3096.72
70.00021.2998.01
Male 10.017075.5275.52
20.003314.5790.10
30.00104.5994.69
40.00052.0496.73
50.00031.3998.12

Additional files

Supplementary file 1

DGRP line means and African samples CHC data.

(A) DGRP females. (B) DGRP males. (C) DGRP and African females. (D) DGRP and African males.

https://doi.org/10.7554/eLife.09861.040
Supplementary file 2

ANOVA and heritabilities of DGRP CHC peak proportions in females and males.

(A) Females. (B) Males. df: degrees of freedom; SS: Sums of squares (Type III); MS: Mean squares; F: F ratio test statistic; P: P-value; σ2: variance component; H2: broad sense heritability.

https://doi.org/10.7554/eLife.09861.041
Supplementary file 3

ANOVA of female sex pheromones in DGRP lines containing the Desat2 insertion (ins/ins or ins/del).

df: degrees of freedom; P: P-value.

https://doi.org/10.7554/eLife.09861.042
Supplementary file 4

CHC module correlations in the DGRP.

(A) Females. (B) Males.

https://doi.org/10.7554/eLife.09861.043
Supplementary file 5

Color and symbol codes for DGRP lines used in Figure 5.

https://doi.org/10.7554/eLife.09861.044
Supplementary file 6

ANOVA of the effects of Wolbachia infection and common polymorphic inversions on CHC PCs.

df: degrees of freedom; SS: Type III sums of squares; F: F statistic; AIC: Akaike information criterion. ***p<0.001; **p<0.01; *p<0.05.

https://doi.org/10.7554/eLife.09861.045
Supplementary file 7

GWA results for DGRP female and male CHC PCs.

Results are given for female PC 1 - 7 and male PC 1 - 5. P-values (≤ 10-5) of association tests are given for a linear mixed model accounting for relatedness of adjusted line mean PCs versus genotypes.

https://doi.org/10.7554/eLife.09861.046
Supplementary file 8

VDRC and Exelixis transgenic line information.

https://doi.org/10.7554/eLife.09861.047
Supplementary file 9

Difference between experimental lines and controls for individual CHCs (ng/fly) and raw data (µg/fly).

(A) Mean difference between RNAi or mutant CHCs and controls, females. (B) Mean difference between RNAi or mutant CHCs and controls, males. (C) Experiment 1 raw data for KK RNAi lines, females. (D) Experiment 2 raw data for KK RNAi lines, females. (E) Raw data for GD RNAi lines, females. (F) Raw data for Exelixis mutation, females. (G) Experiment 1 raw data for KK RNAi lines, males. (H) Experiment 2 raw data for KK RNAi lines, males. (I) Raw data for GD RNAi lines, males. (J) Raw data for Exelixis mutation, males.

https://doi.org/10.7554/eLife.09861.048
Supplementary file 10

Chemical identification of previously unpublished CHCs in Drosophila melanogaster.

https://doi.org/10.7554/eLife.09861.049

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