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• W2985890668 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Pheromones play an important role in the behavior, ecology, and evolution of many organisms. The structure of many insect pheromones typically consists of a hydrocarbon backbone, occasionally modified with various functional oxygen groups. Here we show that sex-specific triacylclyerides (TAGs) are broadly conserved across the subgenus Drosophila in 11 species and represent a novel class of pheromones that has been largely overlooked. In desert-adapted drosophilids, 13 different TAGs are secreted exclusively by males from the ejaculatory bulb, transferred to females during mating, and function synergistically to inhibit courtship from other males. Sex-specific TAGs are comprised of at least one short branched tiglic acid and a long linear fatty acyl component, an unusual structural motif that has not been reported before in other natural products. The diversification of chemical cues used by desert-adapted Drosophila as pheromones may be related to their specialized diet of fermenting cacti. https://doi.org/10.7554/eLife.01751.001 eLife digest For animals, the ultimate purpose of life is to have sex, as nothing is more important than passing down your genes to future generations. A wide range of strategies are therefore employed throughout nature to maximize the chances of sexual success, from ostentatious courtship rituals to the subtle subliminal signals sent out using chemicals called pheromones. Plants and animals release pheromones to influence the behavior of other plants and animals, often without the recipient being aware of it. Hundreds of different insect pheromones have been discovered. Fruit flies release a number of different pheromones, all with similar chemical structures. Now, Chin et al. have discovered that male flies belonging to several species of fruit fly that live in the desert release chemicals called triacylglycerides (TAGs), which are commonly used for energy storage by many organisms as pheromones. During sex, the male fly rubs the TAGs onto the body of the female, which makes her less attractive to other male flies for several hours, thus increasing his chances of parenthood and passing his genes to future generations. TAGs are also found in other insect species, but have been largely overlooked as pheromones. Moreover, the TAGs discovered by Chin et al. have an unusual structure, not previously seen in nature, which may result from the diet of fermenting cacti the desert-dwelling fruit flies enjoy. https://doi.org/10.7554/eLife.01751.002 Introduction Chemical communication significantly influences many complex social behaviors, including aggression, kin recognition, and courtship (Wyatt, 2003). The chemical structures and functions of insect pheromones have been intensely studied because of the fascinating diversity of behavioral properties and relevance to questions of speciation, reproductive isolation, and applications to pest control (Witzgall et al., 2010). Since the discovery of Bombykol in 1959 (Butenandt et al., 1959), hundreds of insect pheromones have been identified, including straight chain and branched alkanes and alkenes, oxygen-containing derivatives such as wax esters, fatty alcohols, and aldehydes, sterols, and isoprene-based compounds (Tillman et al., 1999; El-Sayed, 2012). In Drosophila, pheromones are produced by oenocytes (specialized epithelial cells in both males and females) and the male ejaculatory bulb and subsequently secreted onto the cuticular surface and anogenital region, respectively (Billeter et al., 2009; Yew et al., 2009). Previous studies of Sophophora and Drosophila flies identified alkanes, alkenes, and oxygen-modified hydrocarbons as the major lipids used as pheromones (Jallon and David, 1987; Greenspan and Ferveur, 2000). Recently, triacylglycerides (TAGs), which are normally found in the fat bodies and used for energy storage, were observed on the cuticles of flies from the Drosophila repleta and Drosophila quinaria groups (Yew et al., 2011; Curtis et al., 2013). However, almost nothing is known about the structure, chemical diversity, conserved expression, and functional roles of these exogenously secreted TAGs. To explore the role of TAGs as pheromones and the ubiquity of their expression in Drosophila, we used ultraviolet laser desorption/ionization mass spectrometry (UV-LDI MS) to analyze the cuticular profiles of flies from seven major Sophophora and Drosophila groups. We also investigated the chemical structures of sex-specific TAGs and their role as sex pheromones in species from the D. repleta group. Our studies indicate that TAGs are a broadly conserved, structurally atypical class of Drosophila pheromones that has been overlooked. Results Sex-specific triacylglycerides are conserved in other drosophilids We used UV-LDI MS to perform a broad survey of cuticular lipid profiles of flies from the Drosophila and Sophophora subgenera. UV-LDI MS provides spatially resolved chemical profiling from single, intact insects by probing the cuticular surface with a 200 μm laser (Yew et al., 2009, 2011). Chemical signatures consistent with TAG structures were found to be largely conserved across 3 different Drosophila groups: the repleta radiation (including Drosophila hydei, Drosophila buzzatii, Drosophila navojoa, Drosophila wheeleri, and Drosophila aldrichi), the virilis group (Drosophila americana, Drosophila virilis, and Drosophila montana), and within the robusta group (Drosophila robusta) (Figure 1; Figure 1—figure supplement 1). The TAGs were expressed only in the ejaculatory bulb of males. In contrast, sex-specific TAGs were not detected in any of the species tested from the Sophophora subgenus. Many of the TAG-producing species are capable of feeding and reproducing on cacti, fungi (mushroom), and tree sap or slime fluxes, substrates that contain high levels of toxins, plant defensive compounds, or bacteria, a characteristic that may be related to their ability to produce sex-specific TAGs. Figure 1 with 1 supplement see all Download asset Open asset Male-specific TAG expression is broadly conserved across the Drosophila subgenus and not found in species from Sophophora. The primary diets of each species are indicated, based on the previous studies. Branches for TAG-producing species are colored in red. Branch lengths are proportional to evolutionary time. *Evidence for TAG-expression is based on Curtis et al., 2013. https://doi.org/10.7554/eLife.01751.003 Sex-specific TAGs are correlated with age and synthesized in the ejaculatory bulb To characterize the structures and functions of sex-specific TAGs, we focused on desert-adapted drosophilids from the D. repleta group, Drosophila arizonae and Drosophila mojavensis, two well-characterized models for speciation, reproductive isolation, and ecological studies (Ruiz et al., 1990; Markow, 1996; Etges and Jackson, 2001). Analysis by UV-LDI MS detected 13 TAGs and several long-chain acetyldienyl acetates, 30 or 32 carbons in length (referred to as long OAcs) exclusively in the anogenital region of D. arizonae and D. mojavensis males and not on virgin females of either species (Figure 2A,B; Figure 2—figure supplement 1). Figure 2 with 1 supplement see all Download asset Open asset Pheromone profiles and age-related increase in sex-specific TAGs. (A and B) UV-LDI MS allows spatially resolved detection of high molecular weight lipids directly from intact insects, with minimal damage to the cuticle. Representative mass spectra from the anogenital region (inset) of D. arizonae and D. mojavensis males show signals corresponding to triglycerides (TAGs, red) and long chain alkadienyl acetates (long OAcs, blue). The hydrocarbon C35:2 (number of carbons: number of double bonds) is found on cuticles of males and females. Labeled signals correspond to potassiated molecules [M + K]+. Scale bar: 1 mm. (C and D) Relative intensity of TAGs and long OAcs on male D. arizonae and D. mojavensis, respectively. TAGs and long OAcs increase with age, with trace quantities first appearing at 4 day old. The signal intensity for all detected TAGs or long OAcs was normalized to the signal intensity of C35:2. https://doi.org/10.7554/eLife.01751.005 We next tested whether sex-specific TAG expression is correlated with male sexual maturity. Chemical profiling of D. arizonae from 0 to 15 days old indicated that the expression of the TAGs and long OAcs increased in abundance as males get matured, with little or no expression in the first 4 days after eclosion and higher expression towards the age of maturity at approximately 8 day old, the age when males exhibit full courtship behaviors (Markow, 1981) (Figure 2C). D. mojavensis followed a similar maturation profile (Figure 2D). Analysis of dissected male reproductive organs by UV-LDI MS revealed qualitatively similar chemical profiles exclusively in the ejaculatory bulbs (Figure 2—figure supplement 1). In addition, no predicted precursors of these compounds such as diacylglycerol or glycerol-3-phosphate were detected from the accessory glands or other reproductive organs. These results indicate that the TAGs and long OAcs are synthesized in the ejaculatory bulb. Sex-specific TAGs exhibit unusual structural features and are expressed as a complex blend of isomers From D. arizonae and D. mojavensis, we isolated both TAG and long OAc lipid classes using thin layer chromatography (TLC; Figure 3—figure supplement 1). Chemical derivatization of the long OAc fraction confirmed the presence of an acetyl group (Figure 3—figure supplement 2). Gas chromatography MS (GCMS) analysis of transesterified TLC fractions indicated tiglic acid, a 5-carbon branched unsaturated acid, as one of the fatty acyl moieties (Figure 3—figure supplement 3). No other 5-carbon fatty acid methyl esters were detected. Tandem MS with low energy collision-induced dissociation (CID) analysis provided the chain length and degree of unsaturation of each of the acyl chains present in each TAG. A similar motif was revealed among each of the molecules: a single long-chain fatty acyl component, 16–18 carbons in length, together with 2 short-chain fatty acyl side chains, each 2–5 carbons in length (Figure 3A; Figure 3—figure supplement 4). For several of the more abundant TAG molecules, the position of the acyl chains on the glycerol backbone could be deduced based on the relative abundance of the product ions in the CID spectra. As described by Hsu and Turk (1999 and 2010), fragments reflecting the loss of substituent at the sn-2 carbon (middle of the glycerol backbone) are less abundant than ions reflecting losses at either the sn-1 or sn-3 carbons. Based on this observation, long-chain fatty acids are predominantly located at either sn-1 or sn-3 for the major TAGs at [M + Na]+ 543 and 541 (Figure 3A; Figure 3—figure supplement 4) and [M + Li]+ 499, 501, and 527 (Figure 3—figure supplement 4). It was not possible to distinguish between sn-1 and sn-3 positions. The position assignments on the backbone are supported by analysis of synthetic standards in which a long-chain fatty acid is placed at sn-1. The relative abundances of fragment ions are similar to those observed from crude extract (Figure 3—figure supplement 6–8). Notably, CID analysis of a fourth major TAG at [M + Li]+ 487 resulted in low-intensity signals corresponding to loss of the C18:1 fatty acyl substituent, suggesting that this component is likely to reside at the sn-2 position (Figure 3—figure supplement 4). Low-abundance signals for isobaric TAGs containing C18:2 and C18:1 fatty acids were also observed at [M + Li]+ 501, 499, and 487 and are likely to represent minor components. In these cases, it was not possible to assign substituent positions. Figure 3 with 8 supplements see all Download asset Open asset Structural elucidation of sex-specific TAGs. (A) The low energy collision-induced dissociation (CID) mass spectrum of a TAG-related signal from crude D. arizonae extract ([M + Na]+ 543) shows fragments corresponding to losses of a 5 carbon fatty acid with a single double bond (C5:1) and an 18 carbon fatty acid with a single double bond (C18:1). Both sodiated (major peak) and protonated chain side losses are observed. The schematic rationalizes the product ions formed during CID of mass-selected [M + Na]+ of unsaturated lipids. (B) Ozone-induced dissociation (OzID) of a TAG-related signal (shown in A) indicates isomers with variant double bond positions. The fragments at m/z 461 and m/z 433 are aldehyde products consistent with double bonds (db) at positions n-7 and n-9, respectively. The fragment at m/z 531 confirms the n-2 double bond position found in the tiglic acyl component. The corresponding Criegee product ions (m/z 477 and m/z 449, respectively) are also observed. The schematic rationalizes the product ions formed during OzID of mass-selected [M + Na]+ of unsaturated lipids. Product ions are assigned as outlined by Thomas et al., 2008 and Brown et al., 2011. (C) CID and OzID MS analyses of the most abundant sex-specific TAGs reveal significant combinatorial complexity. A generic TAG molecule consisting of a glycerol backbone and 3 fatty acyl (FA) side chains, R1, R2, and R3, is shown. Each TAG species is comprised of 2 short chain and 1 long chain FA component. Shaded boxes indicate the composite side chains of each TAG species. The glycerol backbone positions for several TAGs are assigned based on the comparison with synthetic standards and ion product abundance patterns (dark gray boxes). Ambiguous backbone positions are in light gray. https://doi.org/10.7554/eLife.01751.007 To determine the double bond positions within each fatty acid, we used ozone-induced dissociation (OzID) mass spectrometry (Thomas et al., 2008; Brown et al., 2011). Individual TAG species were mass-selected within an ion-trap mass spectrometer where they were exposed to ozone vapor. The resulting gas-phase ion–molecule reaction facilitates targeted oxidative dissociation of carbon–carbon double bonds present in the acyl chains. Fragmentation of the ozonide leads to formation of characteristic aldehyde and Criegee ions with a mass indicative of the positions of each double bond. OzID analysis of the TAG fraction revealed numerous positional isomers, with double bond positions between C9-C10 (n-9) and C11-C12 (n-7), indicating oleic and palmitoleic acid side chains, and between C2-C3 (n-2), consistent with tiglic acid (Figure 3B,C; Figure 3—figure supplement 5). Spectra from OzID analysis of TAG standards synthesized with oleic acid (C18:1, n-9) or linoleic acid (C18:2, n-6,9) support the double bond position assignments (Figure 3—figure supplement 6 and 8). Double-bond geometry could also be deduced for two of the more abundant TAGs. cis- and trans-alkenes exhibit differential reactivity to ozone, resulting in differences in the overall abundances of the fragment ions and the relative abundance of the Criegee and aldehyde product ions (Poad et al., 2010). The relative abundance of the aldehyde and Criegee ions for the molecules at [M + Na]+ 543 and 541 are consistent with those of synthetic TAG standards synthesized with oleic acid and linoleic acid, both of which contain cis- double bonds (Figure 3—figure supplement 6 and 8). In summary, MS analysis revealed considerable variation in the carbon chain length, degree of unsaturation, positions of fatty acyl chains, and double bond positions of both the short chain and long chain fatty acyl components (Figure 3C). All of the analyzed TAGs contained tiglic acid. The unusual combination of short odd-branched chain fatty acids with a single linear long-chain component has not been reported before in natural products. Diet-related effects on sex-specific TAGs To determine the contribution of diet to TAG production, we compared TAG levels between males raised on standard fly media for 2 generations vs media supplemented with cactus powder and banana. Thin layer chromatography of the lipid contents of ejaculatory bulbs indicated that males raised on standard media produced a significantly lower amount of some of the sex-specific TAGs, including [M + K]+ 559, one of the most abundant molecules (Figure 4). The results show that although a specialized diet is not essential for sex-specific TAG production, precursors derived from food can influence the quantity of several of the TAGs. Figure 4 Download asset Open asset Diet changes the quantity but not composition of sex-specific TAGs. (A) TAGs from individual ejaculatory bulbs of males raised on standard fly food (n = 10) or cactus-banana supplemented food (n = 9) were quantified using direct tissue thin layer chromatography. Each lane contains a single bulb. c: control band (point of origin) used for normalization; f2 and f3: fractions containing sex-specific TAGs. (B) The amount of TAGs in f3 from ejaculatory bulbs of males raised on standard food is significantly lower than supplemented food conditions (Student’s t-test, two-tailed, p=0.0016). TAGs found in f2 were not significantly different (p=0.062). Error bars indicate SEM. **: p<0.005; ns: not significant; a.u.: arbitrary units. https://doi.org/10.7554/eLife.01751.016 Sex-specific lipids are transferred to females during mating and are correlated with loss of female attractiveness In some species of insects, males anoint the females with anti-aphrodisiacs during mating to suppress subsequent courtship from other males (Zawistowski and Richmond, 1986; Bownes and Partridge, 1987; Wigby et al., 2009; Yew et al., 2009). We hypothesized that sex-specific TAGs may play a similar role based on the sexually dimorphic pattern of expression and localization to a male sex organ. To test this prediction, a mate choice assay was used in which a naïve male was given a choice to court either a virgin female or a recently mated female (Figure 5A). Male Drosophila courtship behavior consists of a sequence of stereotyped, quantifiable features, including wing vibration (‘singing’), foreleg tapping, proboscis extension, and copulation (Spieth, 1974). Courtship initiation and copulation preferences were measured since both indicate male choice while the latter is also influenced by female rejection behavior. Males from D. arizonae and D. mojavensis were significantly more attracted to virgin females than recently mated females (Figure 5B). Notably, significant levels of both TAGs and long OAcs were found on the anogenital regions of D. arizonae females shortly after mating but decreased by approximately 80% at 2–4 hr post-mating and were almost negligible at 8 hr post-mating (Figure 5C; Figure 5—figure supplement 1). Mated D. arizonae females became increasingly attractive over time, correlating with a decrease of the levels of TAGs and long OAcs on the cuticle (Figure 5D). From 4 hr onwards, males showed no significant preference between mated and virgin females. Female remating was observed starting only at 8 hr post-mating (Figure 5—figure supplement 2). Taken together, these results show that the presence of male-transferred lipids on female cuticles is accompanied by a concomitant decrease in female attractiveness. Figure 5 with 6 supplements see all Download asset Open asset Sex-specific lipids suppress male mating behavior. (A) To measure male courtship behavior, one male fly is placed with 2 females, one mated (M), and one virgin (V). (B) D. arizonae (Dari) and D. mojavensis (Dmoj) prefer to court virgin females over recently mated females (n = 20, Fisher’s exact test, p=0.00123; n = 31, p=0.0105). **: p<0.01; ns: not significant. A preference score of 1 indicates all males initiate courtship first with the virgin female; −1 indicates all males initiated courtship first with the mated female. (C) Levels of male-transferred TAGs and long OAcs on the female cuticle after first mating decreases by 2 hr post-mating. (D) Females are significantly less attractive for up to 2 hr after mating. By 4 hr, males do not exhibit significant preference for courting mated vs virgin females. **: Fisher’s exact test, p=0.00123; *: p=0.0256. (E) D. arizonae males are more reluctant to initiate courtship with females perfumed with the contents of [1.25] ejaculatory bulb (eb) (n = 27, Fisher’s exact test, p=0.000624) or [0.5] eb (n = 28, p<0.0001) but not [0.25] eb (n = 28, p=0.176). Extracts from immature male ebs were ineffective at inhibiting male courtship (n = 28, p=0.000389). D. mojavensis and D. navojoa (Dnav) males also avoided virgin females perfumed with eb contents (Dmoj: n = 21, p=0.00160; Dnav: n = 21, p<0.0002). **: p<0.002; ***: p<0.0001. C: solvent control; P: perfumed. (F) Suppression of D. arizonae courtship initiation is elicited only when TAGs and long OAcs are combined (n = 28, Fisher’s exact test, p<0.0001). TAGs alone are ineffective (n = 28, p=0.593). Long OAcs on their own could be attractive to males (n = 28, p=0.006). *: p<0.05; ***: p<0.0001. (G) D. arizonae copulation is suppressed in the presence of TAGs alone (n = 28; Fisher’s exact probability test, p=0.0287) or TAGs combined with long OAcs (n = 28; p<0.0001), but not long OAcs alone (n = 28; p=0.0543). *: p<0.05; ***: p<0.0001. A copulation choice score of 1 indicates all males copulated with solvent-perfumed females; −1 indicates all males copulated with TAG-perfumed females. (H) Perfuming with synthetic TAGs recapitulates copulation suppression. Oleic acid (C18:1)-containing TAGs produced significant effects at high and low doses (750 ng: n = 21, Fisher’s exact test, p<0.0001; 75 ng: n = 21, p=0.00167). Only the (R)-18:1 stereoisomer was bioactive (75 ng: n = 21, p=0.00480); the (S)-18:1 stereoisomer did not elicit a significant behavioral response (75 ng: n = 21, p=1). *: p<0.01; **: p<0.002; ***: p<0.0001. (I) Two combinations of TAGs produced synergistic effects on copulation suppression: oleic acid-TAG paired with stearic acid-TAG (n = 21, p=0.000139) and stearic acid-TAG paired with linoleic acid-TAG (n = 19, p=0.022). The oleic acid-containing TAG is not bioactive at a dose of 37.5 ng/fly (n = 20, Fisher’s exact test, p=0.751). *: p<0.05; ***: p<0.0005. (J) UV-LDI MS spectra of females perfumed with TLC fractions or synthesized TAGs. Signals for TAGs and long OAcs are indicated in red and blue, respectively. *: C35:2 Pentatriacontadiene reference peak, m/z 527.5 [M + K]+. https://doi.org/10.7554/eLife.01751.017 Sex-specific lipids are anti-aphrodisiacs The reluctance of males to court mated females could be the result of female rejection behavior or the presence of additional transferred compounds. To determine whether male-transferred lipids account solely for the loss of female attractiveness, we tested the preference of males from D. arizonae, D. mojavensis, and D. navojoa when presented with a choice between virgin females perfumed with evaporated solvent or extracts from ejaculatory bulbs. In these species, fewer males chose to initiate courtship with extract-perfumed females (Figure 5E). D. arizonae was also significantly less likely to copulate with bulb-perfumed females, whereas D. mojavensis showed a tendency to avoid perfumed females (Figure 5—figure supplement 3). D. navojoa only rarely copulated under these experimental conditions, likely because males prefer to court in the presence of more flies. However, the few males that copulated preferred to do so with control females. These results suggest that the use of these sex-specific TAGs as anti-aphrodisiacs is conserved across several species. Further analysis of D. arizonae indicated that the higher the concentration of the extract, the greater the aversion exhibited by males. A minimum dose of extract from approximately 0.5 ejaculatory bulbs was needed to achieve a significant behavioral effect (Figure 5E). Copulation choice was similarly affected (Figure 5—figure supplement 3). Ejaculatory bulb extract from immature males (and therefore, containing negligible amounts of TAGs and long OAcs) did not suppress male courtship (Figure 5E; Figure 5—figure supplement 3). Thus, courtship inhibition is not a generalized aversion to other compounds extracted from ejaculatory bulb tissue. Since virgin females were used, female rejection behavior was not a major contributing factor to copulation choice. Furthermore, it is unlikely that female rejection behavior was triggered by females’ sensory feedback from perfuming. D. arizonae males continued to avoid mating with perfumed females from which the major olfactory and gustatory organs have been removed (Figure 5—figure supplement 4). Taken together, the presence of male-specific long OAcs and TAGs on females was sufficient to fully recapitulate the loss of attractiveness observed in recently mated females. Sex-specific TAGs function synergistically with other lipids and exhibit stereospecificity To determine whether TAGs and long OAcs function synergistically with each other, we examined male response to females perfumed with these two classes of compounds separately and together (Figure 3—figure supplement 1 shows TLC separation). Perfuming with both long OAcs and TAGs strongly suppressed courtship initiation and copulation (Figure 5F,G). The presence of TAGs alone significantly reduced the likelihood of copulation (Figure 5G). Interestingly, long OAcs had an attractive effect when used alone (Figure 5F). These results suggest that both TAGs and long OAcs are needed to suppress courtship initiation, whereas TAGs are important for discouraging later stages of courtship. We next tested whether individual TAG species plays a role in suppressing courtship. We synthesized four of the postulated TAGs along with a TAG containing stearic acid as racemic mixtures (Table 1). Additionally, individual (R)- and (S)-enantiomers were synthesized for a TAG species containing oleic acid, the most abundant of the sex-specific TAGs (Mori, 2012). Males were given a choice of solvent-perfumed females or females perfumed with a single TAG species together with long OAcs. Under these conditions, TAGs containing oleic acid or linoleic acid significantly suppressed courtship initiation (Figure 5—figure supplement 5). However, only the former was capable of suppressing copulation as well and at a dose of 75 ng per female (Figure 5H). Moreover, only the (R) configuration of this TAG was bioactive (Figure 5H; Figure 5—figure supplement 5). Perfuming females with the (S)-enantiomer showed no significant effect on male choice, signifying that courtship aversion is specific to the stereochemistry of the TAG and not due to general avoidance of a foreign molecule or masking of female aphrodisiacs. Table 1 Synthetic TAGs used in this study https://doi.org/10.7554/eLife.01751.024 Calculated [M + K]+Fatty acyl components*,†Long chain fatty acid531.31C16:1 (n-7)-C5:1-C5:1Palmitoleic acid533.32C16:0-C5:1-C5:1Palmitic acid557.32C18:2 (n-6)-C5:1-C5:1Linoleic acid559.34C18:1 (n-9)-C5:1-C5:1Oleic acid561.36C18:0-C5:1-C5:1Stearic acid559.34C18:1 (n-9)-C5:1-C5:1 (R isomer)Oleic acid559.34C18:1 (n-9)-C5:1-C5:1 (S isomer)Oleic acid * Synthesized as racemic mixtures unless otherwise noted. † Notation indicates number of carbons followed by number of double bonds for each fatty acyl component; double bond position indicated in brackets. To examine the possibility that TAGs function synergistically with each other, we paired several combinations of synthetic TAGs together with long OAcs. At a dose of 37.5 ng per fly, none of the TAGs were effective by themselves. However, two combinations of TAGs reduced copulation: 1-steroyl-2,3-ditigloyl glycerol together with either 1-oleoyl-2,3-ditigloyl glycerol or 1-linoleyl-2,3-ditiglyoyl glycerol (Figure 5I). Both combinations also inhibited courtship initiation (Figure 5—figure supplement 6). Additionally, courtship initiation was affected by the combination of long OAcs with 1-palmitoleyl-2,3-ditiglyol and 1-oleyl-2,3-ditiglyol (Figure 5—figure supplement 6). Thus, low amounts of several TAG species, inactive on their own, can act in synergy with each other to suppress male courtship and copulation. It is notable that none of the TAG combinations tested were effective without long OAcs despite our finding that a mixture of TAGs purified from extract was by itself sufficient to deter male attraction. It may be the case that a combination of several different TAG species is needed for courtship inhibition without the presence of long OAcs. Discussion Lipid and protein compounds transferred from male to female Drosophila during copulation are known to inhibit courtship from other males, trigger rejection behaviors in mated females, and serve as nutrition to aid in fertilization and oogenesis in the female (Zawistowski and Richmond, 1986; Bownes and Partridge, 1987; Wigby et al., 2009; Yew et al., 2009). Our results show that TAGs are a novel class of mating-related pheromones that are used by males to manipulate the post-mating attractiveness of females. It remains to be determined whether sex-specific TAGs or their hydrolyzed side chains serve other functions such as nuptial gifts (Gwynne, 2008) or defensive compounds (Will et al., 2000). Earlier studies of D. arizonae and D. mojavensis cuticular lipids using GCMS found mostly linea" @default.
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• W2985890668 title "Author response: Sex-specific triacylglycerides are widely conserved in Drosophila and mediate mating behavior" @default.
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