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Forschungsarbeit

By Annemarie Heiduk (05.06.2012)

To advertise their flowers, plants use both visual (e.g. shape and colour) and olfactory (scent) cues [1], however, the specific cues (e.g. scent compounds) used for successful attraction of pollinators are understood for just a few pollination systems [2,3]. In general, the olfactory display of flowers is considered to be more specific than the visual one [4] (Dobson, 1994) and is therefore most important in specialized pollination systems [5].

For three years now, I am investigating the importance of f
loral scent for pollinator attraction in Ceropegia L. (Apocynaceae, Asclepiadoideae), a plant genus which comprises more than 180 species, all restricted to the Old World. The plants are characterized by their floral “Bauplan” of so called pitfall flowers with astonishing forms and functions (Fig. 1). The corolla of Ceropegia flowers is highly fused and three main functional sections can be distinguished from top to bottom (Fig. 1): corolla lobes, a narrow tube, and a basal inflation. The corolla lobes are often fused at their tips forming a cage like structure with five orifices through which insects enter the flower [6].

Figure 1: Flowers of Ceropegia dolichophylla, C. sandersonii and C. denticulata (drawings by H.-J. Thorwarth)[Bildunterschrift / Subline]: Figure 1: Flowers of Ceropegia dolichophylla, C. sandersonii and C. denticulata (drawings by H.-J. Thorwarth)

The pollination process of Ceropegia, which has been described in detail by Vogel [6], includes a time-limited trapping of the insect pollinator in the corolla inflation. Therefore, the flower possesses various trapping devices (e.g. sliding areas, trapping trichomes) to catch, temporarily trap and finally release the pollinator [6,7]. Ceropegia pollinators identified thus far are small dipterans (<3 mm in length), mainly females, which belong to at least 26 genera in 20 families [8]. Only in one Ceropegia species larger flies were just recently described as pollinators [9].

 

Flowers of Ceropegia are considered to be deceptive flowers [6,10,11], and three possibilities of mimicry are discussed:

  • 1) mimicry of male sex pheromones, because the attracted flies are mainly female [6,8],
  • 2) mimicry of rotting plant material, because it is used as food substrate by larvae of some pollinating flies, and
  • 3) mimicry of animal related odours, because the majority of fly species that visit flowers of Ceropegia feed either in the larval or adult stage on animals or animal secretions/excrement to which they are attracted by odours [6,10].

Detailed analyses on the composition of floral scent and the identification of compounds attracting the pollinators are essential to prove these hypotheses.

 

I started analysing floral scent in Ceropegia by gas chromatography coupled to mass spectrometry (GC-MS) to learn more about the compounds emitted by flowers of these plants. Before these studies, the identity of compounds emitted by Ceropegia was not known in any species. I included C. dolichophylla [12], C. denticulata, and C. sandersonii (see Fig. 1) in my analyses, which all are visited/pollinated by Desmometopa flies (Milichiidae). These flies have the noteworthy trait of being kleptoparasites – they steal food from other animals, generally from predatory arthropods (e.g. spiders), by feeding on haemolymph or other secretions released by their prey items [13-18]. While Milichiidae in general feed on different kinds of true bugs and other insects, Desmometopa flies have a strong liking for bees; they often can be observed in great numbers when feeding on fluids coating the exterior of honey bees caught by spiders [19]. So far, however, the chemicals responsible for attraction of these flies to bees are not known.

 

I wanted to know whether C. dolichophylla, C. denticulata, and C. sandersonii mimic the scent of the preferred food of their pollinating flies, i.e. preyed upon honey bees.

I specifically asked: a) are the flies attracted to their preferred food (i.e. honey bees caught by spiders) by the alarm pheromone of bees, which might be released after spider attack?, and b) do the Ceropegia species mimic the alarm pheromone of honey bees in order to attract Desmometopa flies? To get an answer to these questions, I used a multifaceted approach combining chemical analytical and electrophysiological techniques (Gas chromatography – Electroantennographic detection, GC-EAD) with behavioural assays.

a) Are Desmometopa flies attracted to the alarm pheromone of honey bees (Apis mellifera)?

The alarm pheromone of honey bees is a multi-component blend and released when a bee extrudes its sting and from stings left by bees in a target [20]. Following spider attack, honey bees repeatedly extrude their sting and Desmometopa flies may use the thereby released components to find appropriate feeding sites. To test this hypothesis, flower-visiting honey bees were caught in the Botanical Garden in Bayreuth and they were hold within gauze using fingers. Bees repeatedly extruded the sting, and the pheromone thereby released was found to be highly attractive for Desmometopa. The first fly typically approached the honey bee against the wind within less than a minute, and in four replicates, in total 17 Desmometopa flies were attracted (unpubl. data). No flies were attracted to honeybees, which also were within gauze but which were not hold with fingers and therefore did not extrude the sting. This experiment demonstrates that Desmometopa flies are indeed attracted to volatiles, most likely the alarm pheromone, released from threatened honey bees.

b) Does Ceropegia mimic the alarm pheromone of honey bees for pollinator attraction?

After knowing that the alarm pheromone of honey bees is highly attractive for kleptoparasitic Desmometopa, the components released by honey bees when extruding the sting as well as the components released by C. sandersonii, C. denticulata and C. dolichophylla were collected by standard dynamic headspace techniques. The scent samples were then tested on antennae of flies (GC-EAD) in order to determine the bee’s and Ceropegia’s compounds which can be detected by the flies.

The GC-MS analysis revealed that C. sandersonii and C. denticulata are similarly scented and interestingly, both emitted compounds also found in the samples collected from honey bees. Of these compounds six elicited physiological responses in GC-EAD measurements with Desmometopa flies (Fig. 2) and are already identified. Five of these compounds were tested in behavioural assays in the Ecological Botanical Garden and the mixture turned out to be highly attractive for Desmometopa. In three experiments, each of which lasted an hour, a total of 49 flies were attracted. No fly responded to the paired negative controls consisting of solvent (acetone) only. This experiment unequivocally demonstrates that one or several compounds, Ceropegia and honey bees (alarm pheromone) have in common, effectively attract these kleptoparasitic flies. Biotests in the natural habitat are needed to confirm these preliminary data obtained in a non-native range of the plants.

Figure 2: Coupled gas chromatographic (FID) and electroantennographic (EAD) analyses of dynamic headspace scent samples of Ceropegia sandersonii (red line) and Apis mellifera (black line) tested each on antennae of Desmometopa flies.[Bildunterschrift / Subline]: Figure 2: Coupled gas chromatographic (FID) and electroantennographic (EAD) analyses of dynamic headspace scent samples of Ceropegia sandersonii (red line) and Apis mellifera (black line) tested each on antennae of Desmometopa flies. 1-6: compounds which elicited physiological responses.

 

In contrast to C. sandersonii and C. denticulata, C. dolichophylla emits a scent, which has no compound in common with the alarm pheromone of the honey bee. Even more, in the scent of C. dolichophylla not a single compound was found which also occurs in the other two Ceropegia species. Instead, C. dolichophylla emits, besides a few other compounds, mainly spiroacetals, alkanes as well as alkenes [12]. In preliminary GC-EAD analyses with antennae of Desmometopa flies, prominent responses were elicited by the most abundant compound, and also by a minor nitrogen-bearing compound (unpubl. data). These two compounds were not described as plant volatile in any other species before and are therefore very unusual flower scents. However, they are well known as glandular secretion from different insects (wasps, fruit flies, and lacewings).

Overall, an EAD-active compound common to all three Ceropegia species does not seem to exist, suggesting that Desmometopa flies can be attracted by quite different compounds. However, the three species have in common that their scent contains compounds which occur in pheromones and/or secretions of insects. While C. sandersonii and C. denticulata seem to mimic the alarm pheromone of honey bees, C. dolichophylla might imitate pheromones of Hymenoptera other than honey bees or other insects to attract their pollinating flies. This also suggests that Desmometopa flies not only feed on preyed upon honey bees but also on other insects. These three case studies support the hypothesis of Vogel [6], that Ceropegia fools flies into pollinating its flowers through food source mimicry. However, with exception of the three species studied, the models of other Ceropegia species are unknown, which especially has to do with the lack of data on their scent composition. More species need to be studied in order to be able to draw a general conclusion on deceptive pollination in this fascinating plant group.

 References

1. Chittka, L. & Thompson, J.D., 2001. Cognitive ecology of pollination. Cambridge University Press, Cambridge. Daly, H.V., Doyen, J.T., Purcell III, A.H., 1998. Introduction to Insect Biology and Diversity, 2nd ed. Oxford University Press, New York.

2. Dötterl, S., Jürgens, A., Seifert, K., Laube, T., Weißbecker, B., Schütz, S., 2006. Nursery pollination by a moth in Silene latifolia: the role of odours in eliciting antennal and behavioural responses. New Phytologist 169, 707‐718.

3. Schiestl, F.P., Ayasse, M., Paulus, H.F., Löfstedt, C., Hansson, B.S., Ibarra, F. & Francke, W., 1999. Orchid pollination by sexual swindle. Nature 399, 421‐422.

4. Dobson, H.E.M., 1994. Floral volatiles in insect biology. In: Insect‐plant Interactions (Bernays, E.A., ed). London, Tokyo: CRC Press, 47‐81.

5. Raguso, R.A., 2008. Wake up and smell the roses: the ecology and evolution of floral scent. Annual Review of Ecology, Evolution, and Systematics 39, 549‐569.

6. Vogel, S., 1961. Die Bestäubung der Kesselfallen‐Blüten von Ceropegia. Beiträge zur Biologie der Pflanzen 36, 159‐237.

7. Mueller, L., 1926. Zur biologischen Anatomie der Blüte von Ceropegia woodii Schlechter. Biologia Generalis 2, 799‐814.

8. Ollerton, J., Masinde, S., Meve, U., Picker, M. & Whittington, A., 2009. Fly pollination in Ceropegia (Apocynaceae: Asclepiadoideae): biogeographic and phylogenetic perspectives. Annals of Botany 103, 1501‐1514.

9. Coombs, G., Dold, A.P. & Peter, C.I., 2011. Generalized fly‐pollination in Ceropegia ampliata (Apocynaceae–Asclepiadoideae): the role of trapping hairs in pollen export and receipt. Plant Systematics and Evolution, DOI 10.1007/s00606‐011‐0483‐6.

10. Vogel, S., 1993. Betrug bei Pflanzen: Die Täuschblumen. Akademie der Wissenschaften und der Literatur, Mainz. Abhandlungen der mathematisch‐naturwissenschaftlichen Klasse, pp. 5‐48.

11. Masinde, P.S., 2007. A revision of Brachystelma Sims (Apocynaceae: Asclepiadoideae ‐ Ceropegieae in East Africa. Kew Bulletin 62, 37‐84.

12. Heiduk, A., Brake, I., Tolasch, T., Frank, J., Jürgens, A., Meve, U., & Dötterl, S., 2010. Scent chemistry and pollinator attraction in the deceptive trap flowers of Ceropegia dolichophylla. South African Journal of Botany 76, 762‐769.

13. Eisner, T., Eisner, M., Deyrup, M., 1991. Chemical attraction of kleptoparasitic flies to heteropteran insects caught by orb‐weaving spiders. Proceedings of the National Academy of Sciences of the United States of America 88, 8194‐8197.

14. Robinson, M.H. & Robinson, B., 1977. Associations between flies and spiders: Bibiocommensalism and Dipsoparasitism? Psyche 84, 150‐157.

15. Sabrosky, C.W., 1983. A synopsis of the world species of Desmometopa Loew (Diptera, Milichiidae). Contributions of the American Entomological Institute 19, 1‐69.

16. Sivinski, J., 1985. Mating by kleptoparasitic flies (Diptera: Chloropidae) on a spider host. The Florida Entomologist 68, 216‐222.

17. Sivinski, J., Marshall, S. & Petersson, E., 1999. Kleptoparasitism and phoresy in the diptera. The Florida Entomologist 82, 179‐197.

18. Sivinski, J. & Stowe, S., 1980. A kleptoparasitic cecidomyiid and other flies associated with spiders. Psyche 87, 337‐348.

19. Landau, G.D. & Gaylor, M.J., 1987. Observations on commensal Diptera (Milichiidae and Chloropidae) associated with spiders in Alabama. Journal of Arachnology 15, 270‐272.

20. Breed, M.D., Guzmán‐Novoa, E. & Hunt, G.J., 2004. Defensive behavior of honey bees: Organization, genetics and comparisons with other bees. Annual Review of Entomology 49, 271‐298.


Stations
  • Current
  • PhD candidate in Pollination Ecology (granted by the Bavarian Excellence Program (Bayerisches Eliteförderungsgesetz))
  • Since 10/2008
  • University of Bayreuth: Elite study program Macromolecular Science
  • 10/2008 - 04/2011
  • University of Bayreuth: Master of Molecular Ecology
  • 03/2010 - 04/2011
  • Master thesis: “Scent chemistry and pollinator attraction in Ceropegia spp.”
  • 10/2005 - 09/2008
  • University of Bayreuth: Bachelor of Biology
  • 05/2008 - 08/2008
  • Bachelor thesis: “Ceropegia dolichophylla (Apocynaceae): Flower scent as attractant for pollinating flies”
  • 10/2009 - 03/2011
  • Membership in the Max Weber Program of the Bavarian Excellence Program (Bayerisches Eliteförderungsgesetz)
  • 10/2007 - 03/2011
  • Scholar of the German National Academic Foundation (Studienstiftung des deutschen Volkes)

Work as Student Research Assistant
  • 06/2009 - 07/2009
  • Research on plant-pollinator interactions between Lysimachia and the oligolectic bee Macropis
  • 06/2008
  • Supervising practical courses in animal physiology for students
  • 04/2008 - 06/2008
  • Expression and functional analysis of metal transporter genes in contrasting ecotypes of Arabidopsis thaliana and A. halleri
  • 10/2007 - 11/2007
  • Supervising practical courses in plant physiology for students
  • 07/2006 - 10/2006
  • Research on the microfauna of different restored castle walls

Internships
  • 03/2009 - 04/2009
  • Max Planck Institute for Chemical Ecology (Jena, Germany) Research on chemical defence secretions in the dorsal glands of different leaf beetle larvae (Dep. of Bio-Organic Chemistry)
  • 08/2007 - 09/2007
  • Lajuma Research Station (Soutpansberg, South Africa) Setting up a collection of ants (Formicidae) of the region and collecting data about habitats, behaviour and population density; trapping, radio-collaring and GPS-tracking of leopards; studies on primate
  • 02/2005 - 04/2005
  • ‘Ventanas en Corcovado’ butterfly farm (Costa Rica) Organizing and managing the production of eggs, larvae and pupae; collecting data about the different instars, cultivating of the appropriate food plants
  • 11/2004 - 12/2004
  • ‘Idea’ Tropical Park (Neuenmarkt/Wirsberg, Germany) Laying out enclosures and maintaining the rainforest hall; taking care of a variety of tropical vertebrates and invertebrates
  • 09/2004
  • ‘Austrop’ field station (Cape Tribulation, Queensland, Australia) Taking care of spectacled flying foxes; control of neophyte spreading along several beaches; rainforest regeneration
  • 07/2004 - 08/2004
  • University of New South Wales (Cowan Field station, Australia) Behavioural studies (foot-thumping, predator response, flight-distance) on kangaroos and taking care of the animals (kangaroos, potoroos)
  • 1999 - 2004
  • Zoology (Germany) Planning enclosures for zoo animals and designing animal signs; organizing workshops

Publications
  • 04/2012
  • Heiduk, A, Meve, U. & Dötterl, S. (2012): Ceropegia sandersonii und Ceropegia denticulata – Der Trick mit dem „Sonntagsbraten“. Caralluma 14 (1): 8-11.
  • 08/2010
  • Heiduk, A. (2010): Ceropegia - Alles Lug und Trug. Bestäubungserfolg durch Vortäuschen falscher Tatsachen. Caralluma 12 (2): 40-43.
  • 05/2010
  • Heiduk, A., Brake, I., Tolasch, T., Frank, J., Jürgens, A., Meve, U., & Dötterl, S. (2010): Scent chemistry and pollinator attraction in the deceptive trap flowers of Ceropegia dolichophylla. South African Journal of Botany 76: 762-769.