Florida Orchid Conservation Conference 2011 THE SCENT OF A GHOST Orchid Floral Fragrances and Their Niche in Conservation James J. Sadler WHAT IS FLORAL FRAGRANCE? It is the blend of chemicals emitted by a flower to attract pollinators. These chemicals sometimes cannot be detected by our nose. 1 Florida Orchid Conservation Conference 2011 WHY STUDY FLORAL FRAGRANCE? Knowing the floral compounds may help improve measures to attract specific pollinators, increasing pollination and fruit-set to augment conservation. Chemical compounds can be introduced to increase pollinator density. FLORAL FRAGRANCE: BACKGROUND INFORMATION 90 plant families worldwide have been studied for floral fragrance, especially the Orchidaceae6 > 97% of orchid species remain unstudied7 Of all plants studied so far, the 5 most prevalent compounds are: limonene (71%), (E)-β-ocimene (71%), myrcene (70%), linalool (70%), and α-pinene (67%)6 2 Florida Orchid Conservation Conference 2011 POSSIBLE POLLINATORS BEES Found to pollinate via mimicry POSSIBLE POLLINATORS WASPS Similar as bees 3 Florida Orchid Conservation Conference 2011 POSSIBLE POLLINATORS FLIES Dracula spp. produce a smell similar to fungus where the eggs are laid POSSIBLE POLLINATORS MOTHS Usually white, large and fragrant at night 4 Florida Orchid Conservation Conference 2011 CASE STUDY The Ghost Orchid State Endangered1 Dendrophylax lindenii THE GHOST ORCHID Range: S Florida to Cuba Epiphyte of large cypress domes and hardwood hammocks Often affixed to pond apple or pop ash Leafless Flowers May-August 5 Florida Orchid Conservation Conference 2011 Leafless at maturity Understanding in situ pollination (Roots are photosynthetic) biology is critical for conservation Seed capsule Most fragrant at night Sweet-smelling (somewhat fruity) White coloration, long nectar spur suggest pollination by hawkmoths Photo: Emily Massey 6 Florida Orchid Conservation Conference 2011 The Giant Hawkmoth, Cocytius antaeus Drury proboscis www.blog.hmns.org DISTRIBUTION The Giant Hawkmoth, Cocytius antaeus Drury www.butterfliesandmoths.org 7 Florida Orchid Conservation Conference 2011 plants.ifas.ufl.edu Larva of Giant Sphinx Moth Larval food source5 Annona glabra L. silkmoths.bizland.com Pond Apple www.youtube.com/watch_popup?v=ca--GgEe2Zg#t=30 8 Florida Orchid Conservation Conference 2011 HOW CAN WE COLLECT A FLOWER’S SCENT? 1. Apparatus sturdy, yet light-weight 2. Contain non-volatile components 3. Prolonged exposure to floral compounds 4. Clean air to replace filtered floral headspace 5. Not harmful to epiphytes 6. Accommodate a range of floral sizes EXTRACTION OF GHOST ORCHID FLORAL FRAGRANCE IN SITU Jaclyn Smith J.J. Sadler Site located in Collier Co., FL 13 ghost orchids in flower at time Population size = ca. 50 individuals Sampling carried out over 2 nights Flowers sampled on 3 plants Photo: Larry Richardson 9 Florida Orchid Conservation Conference 2011 APPARATUS DIAGRAM 1= rubber stopper, 2=glass flask, 3=Parafilm, 4=oven bag, 5=stem enclosure, 6=air exit, 7=filtered incoming air 1. Sturdy, yet light-weight 2 students could carry all materials 2. Apparatus contain non-volatile components Oven bags and glass were used 3. Prolonged exposure to floral compounds At least 2 hours of exposure per unit of study 4. Clean air to replace filtered floral headspace Charcoal filter for incoming air 5. Not harmful to epiphytes Sturdy stand and seal was created with a plastic tie and bag 6. Accommodate a range of floral sizes A large bell flask was used 10 Florida Orchid Conservation Conference 2011 PVC pipe Adsorbent Charcoal filter Battery-powered vacuum pump GC SPECTRAL ANALYSIS solvent background methyl salicylate α-pinene sabinene linalool (E)-α-bergamotene 3-carene (E)-β-ocimene (E,E)-α-farnesene 11 Florida Orchid Conservation Conference 2011 COMPOUND COMPOSITION AS A PERCENTAGE WHY (E,E)-α-FARNESENE? CLUES: Odorless to humans Oddly, often associated with insects and/or insect-damaged plants8 Aphids (Hemiptera: Apidae) known to release trans-β-farnesene as an alarm pheromone when disturbed9 12 Florida Orchid Conservation Conference 2011 Could insects on the ghost orchid at time of sampling explain the prevalence of (E,E)-α-farnesene in the sample? MAYBE SO! HOW CAN WE KNOW THE POLLINATOR? Gas Chromatography- Electroantennographic Detection Clean air and compounds are puffed on insect antennae attached to the opening of an electrode GC/EAD tests what compounds elicit the antennal response of insects Monitors the electrical potential of each compound Results show which compounds are detectable by the insect’s antennae 13 Florida Orchid Conservation Conference 2011 IMPLICATIONS FOR CONSERVATION 1. Determine if a pollinator is being attracted through chemicals 2. Align pollinator surveys with peak emittance 3. Introduction of a synthetic “blend” of compounds (e.g., air pollution11) 4. Compounds may elude to other environmental interactions we do not “see” FUTURE WORK What pollinates this orchid? How is floral fragrance affected by its carbon source (i.e., mycotropy)? What role do insects have on published floral fragrance analyses? 14 Florida Orchid Conservation Conference 2011 Thank you! Questions? WORKS CITED 1. Brown P.M. (2005) Wild Orchids of Florida. University Press of Florida. Gainesville, FL. 2. Faegri K. & van der Pijl L. (1979) The Principles of Pollination Ecology (3rd ed.). Pergamon Press, Oxford, UK. 3. Dressler R.L. (1981) The Orchids-Natural History and Classification. Harvard University Press, Cambridge, UK. 4. Grant V. (1983) The systematic and geographical distribution of hawkmoth flowers in the temperate North American flora. Botanical Gazette 144: 439-449. 5. Covell C.V. (2005) A Field Guide to Moths of North America. Virginia Museum of Natural History, Special Publication Number 12. 6. Knudsen J.T., Eriksson R. & Gershenzon, J. (2006) Diversity and distribution of floral scent. The Botanical Review 72(1): 1-120. 7. Dressler R.L. (1993) Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, OR. 8. Himanen S., Vuorinen T., Tuovinen T. & Holopainen J.K. (2005) Effects of cyclame mite (Phytonemus pallidus) and leaf beetle (Galerucella tenella) damage on volatile emission from strawberry (Fragaria x ananassa Duch.) plants and orientation of predatory mites (Neoseiulus cucumeris, N. californicus, and Euseius finlandicus). J. Agric. Food Chem. 53: 8624-8630. 9. Yang S.L. & Zettler F.W. (1975) Effects of alarm pheromones on aphid probing behavior and virus transmission efficiency. Plant Disease Reporter 59: 902-905. 10. Paduraru, Peggy. M. et al. (2008) Synthesis of Substituted Alkoxy Benzene Minilibraries, for the Discovery of New Insect Olfaction or Gustation Inhibitors. J. Combinator Chem. 10(1): 123-134. 11. McFrederick Q.S. et al. (2008) Air pollution modifies floral scent trails. Atmospheric Environment 42: 2336-2348. 15 .