Determining the Cuticular Hydrocarbons Involved in Chemosensory Systems of the Parasitoid Wasp, Dasymutilla occidentalis Deep Sangani and Bradley Vaughn December 6th, 2013 Dasymutilla occidentalis Dasymutilla occidentalis Copyright © 2004 Sara A. Tangren Copyright © 2011 Roy Brown Female Male Ovipositor Also Known As: Velvet Ant, Cow Ant, & Cow Killer Nests of Bombus colonies Brood Chamber © Peter O'Toole / gettyimages.com Brood Chambers of the Bombus Host Larvae Ovipositor Parasitic D. occidentalis larva feeding on a host larva © Ernie Hunter How does the velvet ant find the underground nest of its host? Search Discovery? Key Hypothesis: During excavation of their nest, the host inadvertently impregnate soil with hydrocarbons from the hairs and outer layers of cuticle. Dasymutilla occidentalis uses these chemosensory cues to localize nest opening. C0 – coconut husk C1 –house cricket HB –honeybee BB – bumblebee Dried substrates were ground with HB mortise and pestle, then filtered to particles less than 1mm. Equivalent masses were glued to filter paper in 11.3 cm2 circular areas. C0 C1 Relative position of four substrates in arena were random for each run. Each female D. occidentalis was allowed to roam for 6 minutes in the arena. The total time the head was BB above each substrate was recorded to calculate percent occupancy time. Full 6 min First 2min B time time B occupancy occupancy A A A A A A Percent Percent Substrate Substrate Red line represents expected random occupancy time for given area of a substrate (8%). The percent time spent on bumblebee substrate was significantly greater than the other substrates (ANOVA, n = 15, α = 0.05, p < 0.0001). Which cuticle hydrocarbons from the Bombus host are chemosensory signals for the velvet ant? What chemosensory compounds are we looking for? The cuticular hydrocarbons excreted by bees. H3C CH3 C25H52 C15H31CO2C25H51 CH3 O O H3C CH3 C25H50 H3C Table of hydrocarbon compounds in wasps Extracting hydrocarbons Protocol: 1. Place bees in sample vial with 1 mL hexane (C6H14). 2. Swirl 3 minutes to extract hydrocarbons. Let rest for 2 minutes .. for a total of 5 minutes. 3. Pipet off hexane‐ hydrocarbon solution into GC/MS vial. 4. Inject 1 L into GC/MS. Gas chromatography (GC): The laboratory separation technique Wofford GC/MS Capillary Helium carrier Column gas Chromatogram of bumble bee hydrocarbons Time (minutes) Each peak in the spectrum is an individual hydrocarbon. Identifying the compound is the difficult work. The mass spectrum provides a good guess at the compound Library spectrum Bumble bee honey bee comparison reveals target compounds Red –Honey bee hydrocarbons Black – Bumble bee hydrocarbons Target compounds Matching compounds on the gas chromatogram We purchased pure pentacosane (C25H52). C25H52 Red – pure pentacosane (C25H52) Black – Bumble bee hydrocarbons Something handy in the lab helped identify several hydrocarbons Bumblebee C H 25 52 hydrocarbons C27 C26H54 C28 C H Parafilm wax 25 52 C29 C30 C24H50 C31 C23H48 C32 C33 C34 Bees preferentially produce the odd numbered hydrocarbons. What are our target compounds? Red –Honey bee hydrocarbons Black – Bumble bee hydrocarbons Target compounds Library matches have been a big help. To confirm our target molecules we would like to purchase pure tricosene and pentacosene. Continuing research Our big goal is to develop a separations scheme that will allow us to isolate our two target compounds. Try target compounds in the velvet ant study . Develop a method to determine the amounts of compounds present on the bees. Questions ? time occupancy Percent Substrate Figure 3. Plot of individuals from Mutillid species that do not use bumble bees as hosts..