Identification and Composition of Clasper Scent Gland Components of the Butterfly Heliconius erato and Its Relation to Mimicry.

The butterfly Heliconius erato occurs in various mimetic morphs. The male clasper scent gland releases an anti-aphrodisiac pheromone and additionally contains a complex mixture of up to 350 components, varying between individuals. In 114 samples of five different mimicry groups and their hybrids 750 different compounds were detected by gas chromatography/mass spectrometry (GC/MS). Many unknown components occurred, which were identified using their mass spectra, gas chromatography/infrared spectroscopy (GC/IR)-analyses, derivatization, and synthesis. Key compounds proved to be various esters of 3-oxohexan-1-ol and (Z)-3-hexen-1-ol with (S)-2,3-dihydrofarnesoic acid, accompanied by a large variety of other esters with longer terpene acids, fatty acids, and various alcohols. In addition, linear terpenes with up to seven uniformly connected isoprene units occur, e. g. farnesylfarnesol. A large number of the compounds have not been reported before from nature. Discriminant analyses of principal components of the gland contents showed that the iridescent mimicry group differs strongly from the other, mostly also separated, mimicry groups. Comparison with data from other species indicated that Heliconius recruits different biosynthetic pathways in a species-specific manner for semiochemical formation

Mating strategy does not affect the diversification of abdominal chemicals in Heliconiini butterflies

Antiaphrodisiacs are chemical bouquets physically delivered from male to female individuals upon copulation which discourage further mating and reduce sperm competition by rendering the female less attractive. Since antiaphrodisiacs may not offer an honest signal of female receptivity, in polyandrous species they may undergo faster diversification resulting from sexual conflict. The Heliconiini tribe of butterflies includes a polyandrous (free-mating) and a monandrous (pupal-mating) clade, both known to produce diverse antiaphrodisiac mixtures as part of their abdominal blends. Using multivariate phylogenetic comparative methods, we analyzed the genital blends of 36 Heliconiini species to test the hypothesis that blend diversity results from male-male competition in polyandry. We found no evidence for shifts in blend diversification rate corresponding to changes in mating strategy, implying male-male competition may have a weaker effect on pheromone diversification in this group than previously thought. The genital blends of most species are dominated by one of four highly volatile compounds; (E)-β-ocimene, octen-3-one, sulcatone and 4-hydroxycyclopent-2-en-1-one. Based on the function of (E)-β-ocimene as the behaviourally active antiaphrodisiac in H. melpomene, we propose a similar role in other species for the other volatiles. We test this hypothesis by investigating 4-hydroxycyclopent-2-en-1-one occurrence in Heliconius sara. While we detect no sex-based differences on its presence, we find the compound is undetectable when larvae are not fed their preferred host plant, providing an intriguing potential link between host plant and reproductive cues. This in turn shows that captive-bred samples do not always provide realistic results and this awareness is important for future experiments

A novel terpene synthase controls differences in anti-aphrodisiac pheromone production between closely related Heliconius butterflies

Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-beta-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-beta-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-beta-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-beta-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.Peer reviewe

A novel terpene synthase controls differences in anti-aphrodisiac pheromone production between closely related Heliconius butterflies

Funder: Deutsche Forschungsgemeinschaft; Grant(s): Emmy Noether Fellowship GZ:ME4845/1-1Funder: Jane ja Aatos Erkon Säätiö; funder-id: http://dx.doi.org/10.13039/501100004012Funder: Smithsonian Tropical Research Institute; funder-id: http://dx.doi.org/10.13039/100009201Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-β-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-β-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-β-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-β-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins

Exploitation of an ancestral pheromone biosynthetic pathway contributes to diversification in Heliconius butterflies

Peer reviewed: TrueFunder: Natural Environment Research Council (NERC)Funder: Smithsonian Tropical Research Institute (STRI)During courtship, male butterflies of many species produce androconial secretions containing male sex pheromones (MSPs) that communicate species identity and affect female choice. MSPs are thus likely candidates as reproductive barriers, yet their role in speciation remains poorly studied. Although Heliconius butterflies are a model system in speciation, their MSPs have not been investigated from a macroevolutionary perspective. We use GC/MS to characterize male androconial secretions in 33 of the 69 species in the Heliconiini tribe. We found these blends to be species-specific, consistent with a role in reproductive isolation. We detected a burst in blend diversification rate at the most speciose genus, Heliconius; a consequence of Heliconius and Eueides species using a fatty acid (FA) metabolic pathway to unlock more complex blends than basal Heliconiini species, whose secretions are dominated by plant-like metabolites. A comparison of 10 sister species pairs demonstrates a striking positive correlation between blend dissimilarity and range overlap, consistent with character displacement or reinforcement in sympatry. These results demonstrate for the first time that MSP diversification can promote reproductive isolation across this group of butterflies, showcasing how implementation of an ancestral trait, the co-option of the FA metabolic pathway for pheromone production, can facilitate rapid speciation