RoJenia Judkins Ball State University Chemistry Department Muncie, IN 47306

We aim to identify the volatile compounds in inflorescences of Rudbeckia hirta, that may be responsible for the olfactory attraction of the crab spider, Misumenoides formosipes, to this plant.

Sub-adult females commonly forage from the inflorescences of R. hirta. In order for copulation to occur adult males search their vegetatively complex habitat for the foraging locations of these females and ultimately locate the females.

Males will locate inflorescences despite the absence of a female Males were able to locate inflorescences without the presence of visual cues Thus, these studies provide strong evidence that males detect and navigate towards inflorescences primarily based on floral chemistry

ExtractionSoxhletUSEASEMAESFE CostLow HighMediumHigh Time6-48h<30 mins <60 mins Solvent Use (mL) <60<100<40<10 Comparison of various liquid-solid extraction techniques used in the analysis of plant metabolites USE=ultrasonic extraction ASE= accelerated solvent extraction MAE= microwave-assisted extraction SFE= supercritical fluid extraction

 60 ml of 1:2 solution of hexane:diethyl ether  10g of cut up inflorescences  Sonicated for 30 minutes in the dark  Approximately.4g of extract was collected

100% Dichloromethane – Heavy spot on solvent line as well as spot at application line (as well as other spots between) 95% Dichloromethane – Separated components near the application line but not at the solvent line 95% Hexane – Separated components at the solvent line, but not at the application line

We used solvent mixtures of: 95% hexane:5% dichloromethane 95% dichloromethane:5% hexane 100% dichloromethane A methanol wash of the column A total of 33 15ml fractions were collected

 95% Hexane fractions No components were observed Pooled together based on color  95% Dichloromethane fractions All were pooled together due to similar color  100% Dichloromethane fractions Pooled together based on similar Rf values Remaining were pooled together based on color

Bioassay Ultrasonic Extract (hexane:diethyl ether) Bioassay Flash Chromatography (Silica Column) 95%DCM 95%Hexane 100%DCM TLC Identification of Attractant Methanol Further separation

Positive Selection Negative Selection Ρ value Inflorescences Bulk Extract % Hexane % DCM % DCM81.16 Methanol13.25 An exact binomial calculation was used for each trial. This P value represents the probability of getting the results we obtained in comparison to chance.

 Further separated using flash chromatography  TLC results reveal 2 potentially different compounds

Myself Separate components in the 100% DCM pool Removal of Hexane Identify the two compounds using Infrared Spectroscopy, NMR, and/or GC-MS Future Researchers Run bioassays on pooled fractions to obtain statistically sound results Separate 100% DCM pool and run bioassays