Insulin Signaling Mediates Sexual Attractiveness in Drosophila

Sexually attractive characteristics are often thought to reflect an individual's condition or reproductive potential, but the underlying molecular mechanisms through which they do so are generally unknown. Insulin/insulin-like growth factor signaling (IIS) is known to modulate aging, reproduction, and stress resistance in several species and to contribute to variability of these traits in natural populations. Here we show that IIS determines sexual attractiveness in Drosophila through transcriptional regulation of genes involved in the production of cuticular hydrocarbons (CHC), many of which function as pheromones. Using traditional gas chromatography/mass spectrometry (GC/MS) together with newly introduced laser desorption/ionization orthogonal time-of-flight mass spectrometry (LDI-MS) we establish that CHC profiles are significantly affected by genetic manipulations that target IIS. Manipulations that reduce IIS also reduce attractiveness, while females with increased IIS are significantly more attractive than wild-type animals. IIS effects on attractiveness are mediated by changes in CHC profiles. Insulin signaling influences CHC through pathways that are likely independent of dFOXO and that may involve the nutrient-sensing Target of Rapamycin (TOR) pathway. These results suggest that the activity of conserved molecular regulators of longevity and reproductive output may manifest in different species as external characteristics that are perceived as honest indicators of fitness potential

Tissue-specific insulin signaling mediates female sexual attractiveness

<div><p>Individuals choose their mates so as to maximize reproductive success, and one important component of this choice is assessment of traits reflecting mate quality. Little is known about why specific traits are used for mate quality assessment nor about how they reflect it. We have previously shown that global manipulation of insulin signaling, a nutrient-sensing pathway governing investment in survival versus reproduction, affects female sexual attractiveness in the fruit fly, <i>Drosophila melanogaster</i>. Here we demonstrate that these effects on attractiveness derive from insulin signaling in the fat body and ovarian follicle cells, whose signals are integrated by pheromone-producing cells called oenocytes. Functional ovaries were required for global insulin signaling effects on attractiveness, and manipulations of insulin signaling specifically in late follicle cells recapitulated effects of global manipulations. Interestingly, modulation of insulin signaling in the fat body produced opposite effects on attractiveness, suggesting a competitive relationship with the ovary. Furthermore, all investigated tissue-specific insulin signaling manipulations that changed attractiveness also changed fecundity in the corresponding direction, pointing to insulin pathway activity as a reliable link between fecundity and attractiveness cues. The cues themselves, cuticular hydrocarbons, responded distinctly to fat body and follicle cell manipulations, indicating independent readouts of the pathway activity from these two tissues. Thus, here we describe a system in which female attractiveness results from an apparent connection between attractiveness cues and an organismal state of high fecundity, both of which are created by lowered insulin signaling in the fat body and increased insulin signaling in late follicle cells.</p></div

Comparison of diet, age, and insulin signaling effects on CHCs.

<p>Arrows indicate an increase or decrease in the relative abundance of individual CHCs: 1) for aging - from young to old age on balanced diets <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049799#pone.0049799-Kuo1" target="_blank">[18]</a>, 2) for protein – based on change from S5Y5 to S5Y20, and for sugar – based on change from S5Y5 to S20Y5, 3) for insulin signaling (IIS) - change in transgenic flies with increased insulin signaling (via insulin receptor overexpression - InR^OX) or decreased insulin signaling (insulin substrate mutants <i>chico</i>) compared to controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049799#pone.0049799-Kuo2" target="_blank">[19]</a>. Yellow and blue background indicates statistically significant (at α = 0.05) decrease and increase, respectively. Our previous studies determined that increased insulin signaling or young age makes females more attractive to males and that decreased insulin signaling or old age makes them less attractive <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049799#pone.0049799-Antony1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049799#pone.0049799-Antony2" target="_blank">[15]</a>.</p

Ovarian function influences insulin-dependent attractiveness.

<p>(A) Removal of vitellogenic ovaries reverses attractiveness of flies with globally manipulated insulin signaling. Introducing a single copy of <i>ovo</i>^<i>D1</i> into transgenic flies blocks egg development at early previtellogenic stages, and in flies with global (<i>gsTub5-Gal4</i>) insulin signaling manipulations, it reverses the effects on attractiveness. (B) Manipulation of insulin signaling in late follicle cells (<i>C204-Gal4</i>,<i>TubP-Gal80</i>^<i>ts</i>) is sufficient to affect attractiveness. Opposing insulin signaling manipulations in the germline (Nos-Gal4,TubP-Gal80^ts, first panel) or early follicle cells (C587-Gal4, TubP-Gal80^ts, middle panel) did not produce opposing effects on attractiveness, while manipulations in late follicle cells recapitulated effects of global insulin signaling manipulations (right panel).</p

Effects of dietary sugar and yeast on <i>D. melanogaster</i> female CHCs.

<p>Normalized intensities of individual CHCs are shown across 4 ages (7, 23, 49, and 65 days) for females fed balanced S5Y5 diet (control) as opposed to high-sugar S20Y5 diet (A) or high-protein S5Y20 diet (B). Stars indicate significant effect based on 2-factorial ANOVA models of diet and/or diet by age interaction after sequential Bonferroni correction.</p

Total amounts of CHCs determined by GC-MS analysis for females <i>D. melanogaster</i> maintained on different diets.

<p>ANOVA on log-transformed data yielded highly significant effects of diet (P<0.0001), age (P = 0.0038), and diet by age interaction (P = 0.0003) on total amount of CHC. Based on Tukey HSD post-hoc tests total amounts of CHC in S5Y5 and S20Y5 females are not different from each other and are both different from S5Y20 and S20Y20 females. This indicates that protein in the diet is the main determinant of the total CHC amount.</p

Attractiveness is opposed by fat body insulin signaling, and ovaries are required for the effect.

<p>(A) Female attractiveness is increased by downregulation of insulin signaling in the fat body (<i>Yolk-Gal4</i>), and decreased by upregulation of the pathway in this tissue. (B) The increased attractiveness of females with reduced insulin signaling in the fat body is abolished in females lacking vitellogenic ovaries due to <i>ovo</i>^<i>D1</i>. (C) <i>Dilp6</i> gene expression in the fat body is significantly increased in females following upregulation of insulin signaling in late follicle cells with <i>C204-Gal4</i>,<i>TubP-Gal80</i>^<i>ts</i> (no difference found between control and females with downregulated IS, P = 0.86). Comparisons associated with increases in female attractiveness are indicated by green background shading, while those associated with decreased attractiveness are indicated by red background shading.</p

Insulin signaling in oenocytes does not affect attractiveness.

<p>An oenocyte-specific geneswitch Gal4 driver, <i>PromE800G</i>.<i>S</i>.<i>-Gal4</i>, was used to manipulate insulin signaling in these cells by causing RU486-dependent expression of insulin receptor (InR) for activation or of the phosphatase Pten for inhibition. The driver crossed to a standard laboratory strain (w^-) served as a control. Two-choice preference trials were then performed comparing females fed RU486 to those fed vehicle. P-values from Wilcoxon tests are reported. Box plot boundaries here and in other plots represent 99% confidence intervals around the mean.</p

Cuticular hydrocarbon profiles are influenced by tissue-specific manipulations of insulin signaling.

<p>(A) Increased insulin signaling in late follicle cells (<i>C204</i>,<i>TubP-Gal80</i>^<i>ts</i><i>>InR</i>) induces changes in CHC composition that are distinct from those caused by decreases in insulin signaling (<i>C204</i>,<i>TubP-Gal80</i>^<i>ts</i> ><i>Pten)</i>. CHCs indicated by a yellow outline respond differently to the two manipulations. Comparisons associated with increases in female attractiveness are indicated by green background shading, while those associated with decreased attractiveness are indicated by red background shading. (B) CHC changes in response to global IS upregulation (gsTub5><i>InR</i>) oppose those changes in response to downregulation of IS (gsTub5><i>Pten</i>, Binomial sign test P = 0.0001 for 24 out of 28 CHCs showing opposite response). Saturated CHCs uniformly increased in response to the latter treatment (Binomial sign test P = 0.0078 for 7 out of 7 saturated CHCs responded in the same fashion). Similar changes in 5-T (5-C23:1), 7-T, and 5-P (5-C23:1) are observed for global fat body manipulations as for follicle cell manipulations. (C) Upregulation of insulin signaling in the fat body (Yolk><i>InR</i>) decreased CHC saturation (P = 0.0078 for saturated CHCs), and the response for the whole CHC profile is reversed by opposing IS manipulation (Yolk><i>Pten</i>; p = 0.0004 for 23/28 CHCs showing opposing response). CHC responses to fat body IS upregulation were very similar to CHC responses to global IS downregulation (P = 0.006 for 21/28 CHCs changing in the same direction). Similarly, fat body downregulation and global upregulation of IS produced unidirectional changes for 18 out of 28 CHCs (p = 0.092). Individual CHCs are presented in the following order (from bottom to top): nC21; C22:0; 7,11-TD; 9-C23:1; 7-T, 5-C23:1(5-T); nC23; C24:0; 9,13-C25:2; 7,11-PD; 9-P; 7-P; 5-C25:1; nC25; 9,13-HD; 7,11-HD; 5,9-HD; 7-H; 5-H; nC27; unknown #1; 9,13-ND; 7,11-ND; 29MeBr; 5,9-ND; nC29; unknown #2; unknown #3. Changes in each CHC are color coded in accordance with the degree of CHC saturation: white bars represent saturated CHCs, dark grey bars represent CHCs with single double bond (monoenes), and black bars represent CHCs with two double bonds (dienes). Bars representing the three unidentified CHCs, and a single 29MeBr are empty.</p

Principal component analysis of CHCs detected by GC-MS in <i>D. melanogaster</i> females fed four different diets.

<p>Three PCs were retained that explained 56% total variance, with PC1, PC2, and PC3 explaining 22%, 18%, and 16% of variance after Varimax rotation. PC scores are plotted in 3D space (A), and orthogonal best fit lines for PC scores serve as axes of cones with apexes pointing in the direction of increasing fly age. The numbers designate fly ages: 1 = 7d, 2 = 23d, 3 = 49d, 4 = 65d. Colors indicate food treatments: Grey = S5Y5, Red = S5Y20, Blue = S20Y5, Green = S20Y20. The arrows on PC1-PC2 plane represent the projections of each cone’s axis. PC loadings are shown in the table (B) with shading reflecting the strength of each CHC’s load on each PC (as rendered by JMP). For statistical treatment of individual CHCs, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049799#pone.0049799.s002" target="_blank">Fig. S2</a>.</p