Multiple phenotypic traits as triggers of host attacks towards ant symbionts: body size, morphological gestalt, and chemical mimicry accuracy.

BACKGROUND: Ant colonies are plagued by a diversity of arthropod guests, which adopt various strategies to avoid or to withstand host attacks. Chemical mimicry of host recognition cues is, for example, a common integration strategy of ant guests. The morphological gestalt and body size of ant guests have long been argued to also affect host hostility, but quantitative studies testing these predictions are largely missing. We here evaluated three guest traits as triggers of host aggression-body size, morphological gestalt, and accuracy in chemical mimicry-in a community of six Eciton army ant species and 29 guest species. We quantified ant aggression towards 314 guests in behavioral assays and, for the same individuals, determined their body size and their accuracy in mimicking ant cuticular hydrocarbon (CHC) profiles. We classified guests into the following gestalts: protective, myrmecoid, staphylinid-like, phorid-like, and larval-shaped. We expected that (1) guests with lower CHC mimicry accuracy are more frequently attacked; (2) larger guests are more frequently attacked; (3) guests of different morphological gestalt receive differing host aggression levels. RESULTS: Army ant species had distinct CHC profiles and accuracy of mimicking these profiles was variable among guests, with many species showing high mimicry accuracy. Unexpectedly, we did not find a clear relationship between chemical host similarity and host aggression, suggesting that other symbiont traits need to be considered. We detected a relationship between the guests' body size and the received host aggression, in that diminutive forms were rarely attacked. Our data also indicated that morphological gestalt might be a valuable predictor of host aggression. While most ant-guest encounters remained peaceful, host behavior still differed towards guests in that ant aggression was primarily directed towards those guests possessing a protective or a staphylinid-like gestalt. CONCLUSION: We demonstrate that CHC mimicry accuracy does not necessarily predict host aggression towards ant symbionts. Exploitation mechanisms are diverse, and we conclude that, besides chemical mimicry, other factors such as the guests' morphological gestalt and especially their body size might be important, yet underrated traits shaping the level of host hostility against social insect symbionts.

Chemical convergence between plants and insects: biosynthetic origins and functions of common secondary metabolites.

Despite the phylogenetic distance between plants and insects, these two groups of organisms produce some secondary metabolites in common. Identical structures belonging to chemical classes such as the simple monoterpenes and sesquiterpenes, iridoid monoterpenes, cyanogenic glycosides, benzoic acid derivatives, benzoquinones and naphthoquinones are sometimes found in both plants and insects. In addition, very similar glucohydrolases involved in activating two-component defenses, such as glucosinolates and cyanogenic glycosides, occur in both plants and insects. Although this trend was first noted many years ago, researchers have long struggled to find convincing explanations for such co-occurrence. In some cases, identical compounds may be produced by plants to interfere with their function in insects. In others, plant and insect compounds may simply have parallel functions, probably in defense or attraction, and their co-occurrence is a coincidence. The biosynthetic origin of such co-occurring metabolites may be very different in insects as compared to plants. Plants and insects may have different pathways to the same metabolite, or similar sequences of intermediates, but different enzymes. Further knowledge of the ecological roles and biosynthetic pathways of secondary metabolites may shed more light on why plants and insects produce identical substances.

The chemical and visual bases of the pollination of the Neotropical sexually deceptive orchid Telipogon peruvianus.

Deception of floral visitors in pollination systems is widely distributed among flowering plants. In deceptive systems, the flower (or part of it) or inflorescence mimics either a specific or an unspecific model to attract pollinators. A previous study showed that Telipogon peruvianus flowers developed sexual deception for pollination. However, it was unknown which stimuli were playing a role in pollination. Therefore, we aim to throw some light onto these questions using colour and chemical analysis and biotests. Interestingly, using spectral reflectance, we show here that the flowers present high contrast similar to that produced by a female tachinid fly sitting on a daisy inflorescence, which is used as food resource. We also tested the role of chemical signals in pollinator attraction by collecting floral and female extracts for chemical and electrophysiological analyses, and carried out behavioural tests. For biotests, various treatments, including synthetic mixtures of the electrophysiologically active compounds found in common in females and flowers, have demonstrated that T. peruvianus flowers mimic the sexual pheromone of their pollinator's females. Thus, we give evidence that T. peruvianus flowers mimic a model composed of two organisms. Our study contributes to the understanding of the evolution of deceptive pollination.

Chemical Deception and Structural Adaptation in Microdon (Diptera, Syrphidae, Microdontinae), a Genus of Hoverflies Parasitic on Social Insects.

Various organisms, especially arthropods, are able to live as parasites in ant nests and to prey upon ant broods without eliciting any aggressive behaviour in the hosts. Understanding how these intruders are able to break the ants' communication codes in their favour represents a challenging and intriguing evolutionary question. We studied the chemical strategies of three European hoverfly species, Microdon mutabilis (parasitic on Formica cunicularia), M. analis (parasitic on Lasius emarginatus) and M. devius (parasitic on L. distinguendus). The peculiar slug-like larvae of these three species live inside ant nests feeding upon their broods. Gas chromatography-mass spectrometry analyses show that: 1) these parasites mimic the host brood rather than the ant workers, although each differs distinctly in the extent of chemical mimicry; 2) isolation experiments indicate that after 14 days the responsible cuticular hydrocarbons (CHCs) are not passively acquired but synthesized by the fly larvae. Additionally, Microdon larvae show an array of protective structural features, such as a thick and multi-layered cuticle, retractable head, dome-shaped tergum and a flat and strongly adhesive "foot" (sternum). This combination of protective chemical and structural features represents a successful key innovation by Microdon species, and one that may facilitate host switching. The results of a preliminary adoption analysis confirm that Microdon larvae of at least some species can readily be accepted by different species of ants.

Chemical and behavioral integration of army ant-associated rove beetles - a comparison between specialists and generalists.

Host-symbiont interactions are embedded in ecological communities and range from unspecific to highly specific relationships. Army ants and their arthropod guests represent a fascinating example of species-rich host-symbiont associations where host specificity ranges across the entire generalist - specialist continuum. In the present study, we compared the behavioral and chemical integration mechanisms of two extremes of the generalist - specialist continuum: generalist ant-predators in the genus Tetradonia (Staphylinidae: Aleocharinae: Athetini), and specialist ant-mimics in the genera Ecitomorpha and Ecitophya (Staphylinidae: Aleocharinae: Ecitocharini). Similar to a previous study of Tetradonia beetles, we combined DNA barcoding with morphological studies to define species boundaries in ant-mimicking beetles. This approach found four ant-mimicking species at our study site at La Selva Biological Station in Costa Rica. Community sampling of Eciton army ant parasites revealed that ant-mimicking beetles were perfect host specialists, each beetle species being associated with a single Eciton species. These specialists were seamlessly integrated into the host colony, while generalists avoided physical contact to host ants in behavioral assays. Analysis of the ants' nestmate recognition cues, i.e. cuticular hydrocarbons (CHCs), showed close similarity in CHC composition and CHC concentration between specialists and Eciton burchellii foreli host ants. On the contrary, the chemical profiles of generalists matched host profiles less well, indicating that high accuracy in chemical host resemblance is only accomplished by socially integrated species. Considering the interplay between behavior, morphology, and cuticular chemistry, specialists but not generalists have cracked the ants' social code with respect to various sensory modalities. Our results support the long-standing idea that the evolution of host-specialization in parasites is a trade-off between the range of potential host species and the level of specialization on any particular host.

Arthropods Associate with their Red Wood ant Host without Matching Nestmate Recognition Cues.

Social insect colonies provide a valuable resource that attracts and offers shelter to a large community of arthropods. Previous research has suggested that many specialist parasites of social insects chemically mimic their host in order to evade aggression. In the present study, we carry out a systematic study to test how common such chemical deception is across a group of 22 arthropods that are associated with red wood ants (Formica rufa group). In contrast to the examples of chemical mimicry documented in some highly specialized parasites in previous studies, we find that most of the rather unspecialized red wood ant associates surveyed did not use mimicry of the cuticular hydrocarbon recognition cues to evade host detection. Instead, we found that myrmecophiles with lower cuticular hydrocarbon concentrations provoked less host aggression. Therefore, some myrmecophiles with low hydrocarbon concentrations appear to evade host detection via a strategy known as chemical insignificance. Others showed no chemical disguise at all and, instead, relied on behavioral adaptations such as particular defense or evasion tactics, in order to evade host aggression. Overall, this study indicates that unspecialized myrmecophiles do not require the matching of host recognition cues and advanced strategies of chemical mimicry, but can integrate in a hostile ant nest via either chemical insignificance or specific behavioral adaptations.

Evidence that Cerambycid Beetles Mimic Vespid Wasps in Odor as well as Appearance.

We present evidence that cerambycid species that are supposed mimics of vespid wasps also mimic their model's odor by producing spiroacetals, common constituents of vespid alarm pheromones. Adults of the North American cerambycids Megacyllene caryae (Gahan) and Megacyllene robiniae (Forster) are conspicuously patterned yellow and black, and are believed to be mimics of aculeate Hymenoptera, such as species of Vespula and Polistes. Adult males of M. caryae produce an aggregation-sex pheromone, but both sexes produce a pungent odor when handled, which has been assumed to be a defensive response. Headspace aerations of agitated females of M. caryae contained 16 compounds with mass spectra characteristic of spiroacetals of eight distinct chemical structures, with the dominant compound being (7E,2E)-7-ethyl-2-methyl-1,6-dioxaspiro[4.5]decane. Headspace samples of agitated males of M. caryae contained five of the same components, with the same dominant compound. Females of M. robiniae produced six different spiroacetals, one of which was not produced by M. caryae, (2E,7E)-2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane, and five that were shared with M. caryae, including the dominant (2E,8E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. The latter compound is the sole spiroacetal produced by both males and females of a South American cerambycid species, Callisphyris apicicornis (Fairmaire & Germain), which is also thought to be a wasp mimic. Preliminary work also identified spiroacetals of similar or identical structure released by vespid wasps that co-occur with the Megacyllene species.

Sexual Deception in the Eucera-Pollinated Ophrys leochroma: A Chemical Intermediate between Wasp- and Andrena-Pollinated Species.

Ophrys flowers mimic sex pheromones of attractive females of their pollinators and attract males, which attempt to copulate with the flower and thereby pollinate it. Virgin females and orchid flowers are known to use the same chemical compounds in order to attract males. The composition of the sex pheromone and its floral analogue, however, vary between pollinator genera. Wasp-pollinated Ophrys species attract their pollinators by using polar hydroxy acids, whereas Andrena-pollinated species use a mixture of non-polar hydrocarbons. The phylogeny of Ophrys shows that its evolution was marked by episodes of rapid diversification coinciding with shifts to different pollinator groups: from wasps to Eucera and consequently to Andrena and other bees. To gain further insights, we studied pollinator attraction in O. leochroma in the context of intra- and inter-generic pollinator shifts, radiation, and diversification in the genus Ophrys. Our model species, O. leochroma, is pollinated by Eucera kullenbergi males and lies in the phylogeny between the wasp and Andrena-pollinated species; therefore, it is a remarkable point to understand pollinator shifts. We collected surface extracts of attractive E. kullenbergi females and labellum extracts of O. leochroma and analyzed them by using gas chromatography with electroantennographic detection (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS). We also performed field bioassays. Our results show that O. leochroma mimics the sex pheromone of its pollinator's female by using aldehydes, alcohols, fatty acids, and non-polar compounds (hydrocarbons). Therefore, in terms of the chemistry of pollinator attraction, Eucera-pollinated Ophrys species might represent an intermediate stage between wasp- and Andrena-pollinated orchid species.

Efficacy of Chemical Mimicry by Aphid Predators Depends on Aphid-Learning by Ants.

Chemical mimicry is an effective strategy when signal receivers recognize and discriminate models by relying on chemical cues. Some aphid enemies mimic the cuticular chemicals of aphids through various means thus avoiding detection and attack by aphid-tending ants. However, because ants have been reported to learn the chemical signatures of aphids in order to distinguish the aphids, the efficacy of chemical mimicry is predicted to depend on the experience of the ants that had tended aphids. The present study tested this hypothesis using two predator species: larvae of the green lacewing Mallada desjardinsi, and larvae of the ladybeetle Scymnus posticalis. Lacewing larvae carry the carcasses of aphids on which they have preyed upon their backs, and these function via chemical camouflage to reduce the aggressiveness of aphid-tending ants toward the larvae. Ladybeetle larvae reportedly produce a covering of wax structures, and their chemicals appear to attenuate ant aggression. We examined whether the behavior of the ant Tetramorium tsushimae toward these predators changed depending on their aphid-tending experience. Ants moderated their aggressiveness toward both predators when they had previously tended aphids, indicating that chemical mimicry by both aphid predators is dependent on previous experience of the ants in tending aphids. Chemical mimicry by the predators of ant-tended aphids is therefore considered to exploit learning-dependent aphid recognition systems of ants.

Striking cuticular hydrocarbon dimorphism in the mason wasp Odynerus spinipes and its possible evolutionary cause (Hymenoptera: Chrysididae, Vespidae).

Cleptoparasitic wasps and bees smuggle their eggs into the nest of a host organism. Here the larvae of the cleptoparasite feed upon the food provision intended for the offspring of the host. As cleptoparasitism incurs a loss of fitness for the host organism (offspring of the host fail to develop), hosts of cleptoparasites are expected to exploit cues that alert them to potential cleptoparasite infestation. Cuticular hydrocarbons (CHCs) could serve as such cues, as insects inevitably leave traces of them behind when entering a nest. By mimicking the host's CHC profile, cleptoparasites can conceal their presence and evade detection by their host. Previous studies have provided evidence of cleptoparasites mimicking their host's CHC profile. However, the impact of this strategy on the evolution of the host's CHC profile has remained unexplored. Here, we present results from our investigation of a host-cleptoparasite system consisting of a single mason wasp species that serves syntopically as the host to three cuckoo wasp species. We found that the spiny mason wasp (Odynerus spinipes) is able to express two substantially different CHC profiles, each of which is seemingly mimicked by a cleptoparasitic cuckoo wasp (i.e. Chrysis mediata and Pseudospinolia neglecta). The CHC profile of the third cuckoo wasp (Chrysis viridula), a species not expected to benefit from mimicking its host's CHC profile because of its particular oviposition strategy, differs from the two CHC profiles of its host. Our results corroborate the idea that the similarity of the CHC profiles between cleptoparasitic cuckoo wasps and their hosts are the result of chemical mimicry. They further suggest that cleptoparasites may represent a hitherto unappreciated force that drives the evolution of their hosts' CHCs.

The betrayed thief - the extraordinary strategy of Aristolochia rotunda to deceive its pollinators.

Pollination of several angiosperms is based on deceit. In such systems, the flowers advertise a reward that ultimately is not provided. We report on a previously unknown pollination/mimicry system discovered in deceptive Aristolochia rotunda (Aristolochiaceae). Pollinators were collected in the natural habitat and identified. Flower scent and the volatiles of insects (models) potentially mimicked were analyzed by chemical analytical techniques. Electrophysiological and behavioral tests on the pollinators identified the components that mediate the plant-pollinator interaction and revealed the model of the mimicry system. The main pollinators of A. rotunda were female Chloropidae. They are food thieves that feed on secretions of true bugs (Miridae) while these are eaten by arthropod predators. Freshly killed mirids and Aristolochia flowers released the same scent components that chloropids use to find their food sources. Aristolochia exploits these components to deceive their chloropid pollinators. Aristolochia and other trap flowers were believed to lure saprophilous flies and mimic brood sites of pollinators. We demonstrate for A. rotunda, and hypothesize for other deceptive angiosperms, the evolution of a different, kleptomyiophilous pollination strategy. It involves scent mimicry and the exploitation of kleptoparasitic flies as pollinators. Our findings suggest a reconsideration of plants assumed to show sapromyiophilous pollination.

Chemical Strategies of the Beetle Metoecus Paradoxus, Social Parasite of the Wasp Vespula Vulgaris.

The parasitoid beetle Metoecus paradoxus frequently parasitizes colonies of the common wasp, Vespula vulgaris. It penetrates a host colony as a larva that attaches itself onto a foraging wasp's body and, once inside the nest, it feeds on a wasp larva inside a brood cell and then pupates. Avoiding detection by the wasp host is crucial when the beetle emerges. Here, we tested whether adult M. paradoxus beetles avoid detection by mimicking the cuticular hydrocarbon profile of their host. The beetles appear to be chemically adapted to their main host species, the common wasp, because they share more hydrocarbon compounds with it than they do with the related German wasp, V. germanica. In addition, aggression tests showed that adult beetles were attacked less by common wasp workers than by German wasp workers. Our results further indicated that the host-specific compounds were, at least partially, produced through recycling of the prey's hydrocarbons, and were not acquired through contact with the adult host. Moreover, the chemical profile of the beetles shows overproduction of the wasp queen pheromone, nonacosane (n-C29), suggesting that beetles might mimic the queen's pheromonal bouquet.

Nectar Attracts Foraging Honey Bees with Components of Their Queen Pheromones.

Floral nectar often contains chemicals that are deterrent to pollinators, presenting potential challenges to outcrossing plant species. Plants may be able to co-opt pollinator chemical signals to mitigate the negative effects of nectar deterrent compounds on pollination services. We found that buckwheat (Fagopyrum esculentum) and Mexican sunflower (Tithonia diversifolia) produce nectar with abundant phenolics, including three components of the Apis honeybee queen mandibular pheromone (QMP). In addition, these nectars contain a non-pheromonal phenolic, chlorogenic acid (CA), which was toxic to honeybees, and T. diversifolia nectar also contained isochlorogenic acid (IA). Fresh nectar or solutions containing nectar phenolics reduced Apis individual feeding compared to sucrose solutions. However, freely foraging bees preferred solutions with QMP components to control solutions, and QMP components over-rode or reversed avoidance of CA and IA. Furthermore, prior exposure to the presence or just the odor of QMP components removed the deterrent effects of CA and IA. By mimicking the honey bee pheromone blend, nectar may maintain pollinator attraction in spite of deterrent nectar compounds.

"Double-trick" visual and chemical mimicry by the juvenile orchid mantis hymenopus coronatus used in predation of the oriental honeybee apis cerana.

It has long been hypothesized that the flower-like appearance of the juvenile orchid mantis is used as visual camouflage to capture flower-visiting insects, although it is doubtful whether such morphological resemblance alone could increase their success in hunting. We confirmed that juvenile female orchid mantes often succeed in capturing oriental honeybees, while adult females often fail. Since most of the honeybees approached the juveniles from the front, we hypothesized that juvenile orchid mantes might attract honeybees by emitting some volatile chemical cues. Gas chromatography-mass spectrometry analyses revealed that the mantes' mandibular adducts contained 3-hydroxyoctanoic acid (3HOA) and 10-hydroxy-(E)-2-decenoic acid (10HDA), both of which are also features of the pheromone communication of the oriental honeybee. We also successfully detected 3HOA emitted in the head space air only at the time when the juvenile mantes were attempting to capture their prey. Field bioassay showed that the Oriental Honeybee predominantly preferred to visit dummies impregnated with a mixture of the appropriate amounts and ratios of 3HOA and 10HDA. We therefore conclude that the juvenile mantes utilize these as allelochemicals to trick and attract oriental honeybees.

Chemical imitation of yeast fermentation by the drosophilid-pollinated deceptive trap-flower Aristolochia baetica (Aristolochiaceae).

Deceptive flowers, unlike in mutualistic pollination systems, mislead their pollinators by advertising rewards which ultimately are not provided. Although our understanding of deceptive pollination systems increased in recent years, the attractive signals and deceptive strategies in the majority of species remain unknown. This is also true for the genus Aristolochia, famous for its deceptive and fly-pollinated trap flowers. Representatives of this genus were generally assumed to be oviposition-site mimics, imitating vertebrate carrion or mushrooms. However, recent studies found a broader spectrum of strategies, including kleptomyiophily and imitation of invertebrate carrion. A different deceptive strategy is presented here for the western Mediterranean Aristolochia baetica L. We found that this species is mostly pollinated by drosophilid flies (Drosophilidae, mostly Drosophila spp.), which typically feed on fermenting fruit infested by yeasts. The flowers of A. baetica emitted mostly typical yeast volatiles, predominantly the aliphatic compounds acetoin and 2,3-butandiol, and derived acetates, as well as the aromatic compound 2-phenylethanol. Analyses of the absolute configurations of the chiral volatiles revealed weakly (acetoin, 2,3-butanediol) to strongly (mono- and diacetates) biased stereoisomer-ratios. Electrophysiological (GC-EAD) experiments and lab bioassays demonstrated that most of the floral volatiles, although not all stereoisomers of chiral compounds, were physiologically active and attractive in drosophilid pollinators; a synthetic mixture thereof successfully attracted them in field and lab bioassays. We conclude that A. baetica chemically mimics yeast fermentation to deceive its pollinators. This deceptive strategy (scent chemistry, pollinators, trapping function) is also known from more distantly related plants, such as Arum palaestinum Boiss. (Araceae) and Ceropegia spp. (Apocynaceae), suggesting convergent evolution. In contrast to other studies working on floral scents in plants imitating breeding sites, the present study considered the absolute configuration of chiral compounds.

Pollination biology of Aristolochia bianorii Sennen & Pau: promoting cross-pollination but assuring reproductive success in island ecosystems.

Deceptive pollination has been reported in the genus Aristolochia, but the floral biology and pollination strategy of A. bianorii, an endemic of the Balearic Islands, have not yet been studied. Here, we investigated floral anthesis, mating system, pollinators and volatile organic compounds (VOCs) emitted by its flowers. Flower buds were marked and monitored daily to define floral stages and their duration. Experimental bagging and hand-pollination were performed to test for autonomous self-pollination, induced self-pollination and cross-pollination. Flowers were collected to analyse the presence of entrapped pollinators. VOCs emitted by flowers were evaluated by means of solid phase microextraction followed by immediate GC-MS. Anthesis lasted between 63 and 96 h, and the species exhibited autonomous self-pollination with moderate inbreeding depression. Pollinators were mainly females of Oscinomorpha longirostris (Diptera; Chloropidae). The number of pollinators inside flowers was affected by floral stage and time of flowering. The most common VOCs were alkanes, oximes, esters, alkenes, cyclic unsaturated hydrocarbons, isocyanates, amides and carboxylic acids. Aristolochia bianorii can set seed by autonomous self-pollination, in contrast to other Aristolochia species, in which both protogyny and herkogamy prevent autonomous self-pollination. However, the species may encourage cross-pollination by attracting female chloropid flies though emission of floral scents that may mimic an oviposition site and, possibly, freshly killed true bugs (i.e. Heteroptera). In conclusion, A. bianorii promotes cross-pollination, but delayed autonomous self-pollination assures reproductive success in the putative absence of pollinators.

Evidence for a chemical arms race between cuckoo wasps of the genus Hedychrum and their distantly related host apoid wasps.

BACKGROUND: Brood parasites can exert strong selection pressure on their hosts. Many brood parasites escape their detection by mimicking sensory cues of their hosts. However, there is little evidence whether or not the hosts are able to escape the parasites' mimicry by changing these cues. We addressed this question by analyzing cuticular hydrocarbon (CHC) profiles of Cerceris and Philanthus wasps and their brood parasites, cuckoo wasps mimicking the CHC profiles of their hosts. Some of these hosts use hydrocarbons to preserve their prey against fungal infestation and thus, they cannot significantly change their CHC composition in response to chemical mimicry by Hedychrum brood parasites. RESULTS: We found that the CHC overlap between brood parasites and their hosts was lower in case of host wasps not preserving their prey than in case of prey-preserving host wasps, whose CHC evolution is constrained. Furthermore, the CHC profiles in non-preserving host wasps is more strongly diversified in females than in males, thus in the sex that is chemically mimicked by brood parasites. CONCLUSION: Our results provide evidence for a chemical arms race between those hosts that are liberated from stabilizing selection on their chemical template and their parasites.

Inter- and Intrasexual Variation in Cuticular Hydrocarbons in Trichrysis cyanea (Linnaeus, 1758) (Hymenoptera: Chrysididae).

Cuckoo wasps (Chrysididae, Hymenoptera) are known for their parasitoid or cleptoparasitic life histories. Indeed, the biology of only a few species has been studied in detail and often only little more is known than the host species. By mimicking their hosts' cuticular hydrocarbon (CHC) profiles, species that parasitize single (or a few closely related) host species manage to deceive their hosts. However, the variability of the CHC profile in generalist cuckoo-wasp species is still unknown. Here, we used gas chromatography-mass spectrometry (GC-MS) and DNA barcoding to study intraspecific variation in cuticular hydrocarbons of one less host-specific species of cuckoo wasps, Trichrysis cyanea. Cuticular hydrocarbon (CHC) patterns were found to differ between males and females. Additionally, we found chemical polymorphism among females, which formed three distinct chemical subgroups characterized by different alkene patterns. A lack of divergence in the DNA barcoding region suggests that these different chemotypes do not represent cryptic species. Whether this intrasexual CHC-profile variation is an adaptation (mimicry) to different host species, or simply signaling the reproductive status, remains unclear.

An unusual tricosatriene is crucial for male fungus gnat attraction and exploitation by sexually deceptive Pterostylis orchids.

Cross-kingdom mimicry of female insect sex pheromones by sexually deceptive orchids has evolved multiple times.1 Fungus gnats (Diptera) are predicted to be pollinators of hundreds of sexually deceptive orchids,2-4 yet unlike orchids that sexually attract bees and wasps (Hymenoptera),5-11 the chemistry of fungus gnat-pollinated orchids remains unknown. Furthermore, despite the importance of fungus gnats as pollinators, pests, and decomposers of organic material, and evidence for sex pheromones since 1971,12-17 no structure of any fungus gnat sex pheromone has to date been confirmed. In this study, we found a mixture of five hydrocarbons shared between Pterostylis orbiculata orchids and female Mycomya sp. (Mycetophilidae) fungus gnats, which included three alkanes, a C23 diene, and a C23 triene. The triene was an undescribed natural product, which we synthesized and confirmed to be (6Z,9Z)-1,6,9-tricosatriene. Field bioassays with a synthetic blend of the five hydrocarbons elicited attraction and sexual behavior from male gnats. The triene alone elicited attraction and low levels of sexual behavior, but the blend without it was unattractive, suggesting that this compound is a key component of orchid pollinator attraction and the female fungus gnat sex pheromone. In two closely related Pterostylis species, we found related C23 trienes, but not (6Z,9Z)-1,6,9-tricosatriene. These results suggest that unusual long-chain unsaturated hydrocarbons hold the key to sexual deception in Pterostylis orchids, and are an important step toward deciphering female fungus gnat sex pheromones.