J Chem Ecol (2016) 42:17–23 DOI 10.1007/s10886-015-0660-0 Parapheromones for Thynnine Wasps Björn Bohman1,2,3 & Amir Karton3 & Ruby C. M. Dixon1 & Russell A. Barrow1 & Rod Peakall2,3 Received: 3 August 2015 /Revised: 17 November 2015 /Accepted: 2 December 2015 /Published online: 14 December 2015 # Springer Science+Business Media New York 2015 Abstract Sexually deceptive orchids produce floral volatiles 5-dimethylpyrazine. All blends tested stimulated approaches by that attract male insect pollinators. This interaction between male wasps, with some also eliciting landing and attempted flower and pollinator normally is highly specific. In the few copulation. High-level calculations (G4(MP2)) showed the cases where the chemical composition of the volatiles is energy differences between the structural isomers were known, the compounds have been found to be identical to small, although the degree of sexual attraction varied, those that comprise the sex pheromone of the female wasp. indicating the importance of structural factors for activity. In this study, we investigated whether there is potential for One of the parapheromones, 2-hydroxymethyl-3,5-dimethyl- flexibility in the molecular structure of the chemical cues used 6-ethylpyrazine, elicited similar proportions of approaches, to mediate these specific interactions. Specifically, we asked landings, and attempted copulations as the sex pheromone at whether strong sexual attraction can be maintained with struc- the ratio and dose tested. The findings suggest that there is tural modifications of sex pheromone components. Our study potential for chemical flexibility in the evolution of sexual focused on the orchid, Drakaea glyptodon, which is pollinated deception. by males of the thynnine wasp, Zaspilothynnus trilobatus. Three alkylpyrazines and a unique hydroxymethylpyrazine Keywords Drakaea . Sexual deception . Pyrazine . are components of the female produced sex pheromone of Z. Parapheromone . Specificity trilobatus, and also the semiochemicals produced by the or- chid that lures the males as pollinators. A blend of 2-butyl-3,5- dimethylpyrazine and 2-hydroxymethyl-3,5-diethyl-6- Introduction methylpyrazine (3:1) is as attractive as the full blend of four compounds. Therefore, in this study we substituted 2-hy- Sex pheromones mediate chemical communication between droxymethyl-3,5-diethyl-6-methylpyrazine with one of five the sexes of a species in order to assist reproduction. At a local structurally related parapheromones in a blend with 2-butyl-3, scale, sex pheromone signals typically are highly specific. At the chemical level, this specificity is achieved either by using unique chemicals or by specific blends of more common chemicals (Roelofs and Comeau 1969). In Lepidoptera, the * Björn Bohman [email protected] group that has been the subject of the great majority of studies of insect chemical communication, variable blends of struc- turally similar compounds are common as female sex phero- mones (Francke and Schulz 2010). However, some exceptions 1 Research School of Chemistry, The Australian National University, are known, such as the case of the single compound Canberra, ACT 0200, Australia disparlure, which is the sex pheromone of the gypsy moth, 2 Research School of Biology, The Australian National University, Lymantria dispar (Bierl et al. 1970). Given the usually high Canberra, ACT 0200, Australia specificity of sex pheromones or sex pheromone blends, it is 3 School of Chemistry and Biochemistry, The University of Western of interest to know whether some components can be Australia, Crawley, WA 6009, Australia substituted with different but structurally similar compounds. 18 J Chem Ecol (2016) 42:17–23 The possibility that sex pheromone components could be the Asteraceae and Iridaceae (Ayasse et al. 2011; Borg- substituted appears not to have been widely explored, at least Karlson 1985;Frankeetal.2009;Gaskett2011; Phillips et in part because in the few cases reported the authentic phero- al. 2014; Schiestl et al. 2003). Multiple unrelated orchid gen- mones show by far the strongest activity (Cardé 1990; Renou era have independently evolved this pollination strategy and Guerrero 2000; Schneider et al. 1974). on at least four continents (Ayasse et al. 2011; Gaskett Despite the specific nature of sex pheromones, it some- 2011; Phillips et al. 2014). In sexually-deceptive orchids, pol- times is possible to attract several species with a common lination occurs during either a pre-copulatory routine, or mixture of compounds. For example, 52 species of moths attempted copulation with the flower (Paulus and Gack were attracted to a two- or three-component blend of alkenals, 1990; Peakall 1990). Despite comprehensive early studies alkenols, and alkenol acetates in a screening experiment on the pollination of sexually-deceptive orchids in Europe by Ando et al. (1981). Cis-7,8-epoxy-3-methyloctadecane, a and Australia (Coleman 1929, Kullenberg 1950), the chemis- parapheromone with close structural resemblance to try of these interactions is only now beginning to be elucidat- disparlure (cis-7,8-epoxy-2-methyloctadecane), effectively ed, with progress spanning several different orchid/pollination attracted male gypsy moths, as well as male Lymantria systems on both continents (Borg-Karlson 1987, 1990; monarcha in field experiments (Schneider et al. 1974). Schiestl et al. 2003). There also are examples where authentic sex pheromones Although the number of fully investigated cases is limited, have been successfully modified to ease preparation, reduce so far the orchid semiochemical components identified match cost, or improve applicability. For example, as early as 1968 it closely the components of the sex pheromone of the pollina- was demonstrated that when various synthetic compounds tors. For example, in European Ophrys orchids, the bee- wereaddedto(E)-11-tetradecenyl acetate, the female sex pollinated O. sphegodes utilises a similar blend of alkanes pheromone of the red-banded leaf roller, Argyrotaenia and alkenes as the pollinator Andrena nigroaenea (Schiestl velutinana, different effects were observed (Roelofs and et al. 1999, 2000), and the scoliid wasp pollinated O. Comeau 1968). The Z-isomer of the sex pheromone strongly speculum shares the attractive oxycarboxylic acids and inhibited the attraction of males, while the analogue dodecyl hydroxycarboxylic acids with its pollinator Campsoscolia acetate exhibited synergistic effects, increasing male attraction ciliata (Ayasse et al. 2003). In Australia, Chiloglottis to traps (Roelofs and Comeau 1968). trapeziformis shares the same cyclohexane-1,3-dione with its In this study, we focused on an example drawn from thynnine wasp pollinator, Neozeleboria cryptoides (Peakall et Hymenoptera (ants, bees, and wasps). Despite possessing a al. 2010;Schiestletal.2003). Drakaea glyptodon uses the similar number of species to Lepidoptera, the sex pheromones same pyrazine compounds to lure male Zaspilothynnus of Hymenoptera are considerably less well known. Ayasse et trilobatus wasps as the females of the corresponding species. al. (2001) noted that Hymenopteran sex attractants, which are To date, all these orchid species are believed to utilize the typically emitted by females, normally consist of just one or a same compound(s) found in the female sex pheromones of few compounds, although they also queried whether that was their pollinators. because more complex systems had not been extensively stud- In the present study, our aim was to investigate whether it is ied at the time (Ayasse et al. 2001). However, with the inclu- possible for a critical component of the sex pheromone of Z. sion of more recent studies (e.g., Bohman et al. 2014b; trilobatus to be substituted, and to determine if there is a Niehuis et al. 2013), this trend still appears to hold, even if relationship between the structural properties of the investigat- exceptions are known where male-emitted pheromones and ed parapheromones and biological activity. We conclude by complex blends are used (e.g., Stökl et al. 2014). Species of discussing the implications of the findings for understanding ants and bees, which exhibit complex social interactions, con- the evolution of sexual deception. stitute one group of Hymenoptera where sex pheromone com- plexity could be expected. In these species, it is common for both males and females to emit semiochemicals, and the iden- Methods and Materials tification of specific sex pheromones has proven difficult (Beani et al. 2014). In a study of sex pheromone specificity Test Compounds We previously identified the sex phero- in a semi-social Hymenopteran species of Vespa in Japan, mone blend for Z. trilobatus as consisting of three cross attraction among all six species was observed when trialkylpyrazines (1–3) and 2-hydroxymethyl-3,6-diethyl-5- sexually mature males were exposed to queens or queen ex- methylpyrazine (4) (Bohman et al. 2014b)(Fig.1). tracts from other species (Ono and Sasaki 1987). However, a blend of 3 and 4 (ratio 3:1) was as attractive as In sexual deception, potential insect pollinators such as the full blend of all four compounds, i.e., compounds 1 and 2 bees and wasps are sexually lured to flowers by sex phero- were not absolutely necessary. In this study, we substituted 4 mone mimicry. Pollination by sexual deception is most prev- with one of five structural analogues, while holding 3 constant alent in the Orchidaceae, although it has been documented in and at the same proportion (ratio 3:1) as in the