RoJenia JudkinsBall State University

Chemistry DepartmentMuncie, 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

Extraction

Soxhlet USE ASE MAE SFE

Cost Low Low High Medium High

Time 6-48h <30 mins <30 mins <30 mins <60 mins

Solvent Use (mL)

200-600 <60 <100 <40 <10

Comparison of various liquid-solid extraction techniques used in the analysis of plant metabolites

USE=ultrasonic extraction

ASE= accelerated solvent extractionMAE= 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% hexane100% dichloromethaneA 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%DCM95%Hexane 100%DCM

TLC

Identification of Attractant

Methanol

Further separation

Positive Selection

Negative Selection

Ρ value

Inflorescences

16 3 .018

Bulk Extract 11 3 .022

95% Hexane 4 2 .23

95% DCM 3 3 .31

100% DCM 8 1 .16

Methanol 1 3 .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 poolRemoval of HexaneIdentify the two compounds using Infrared Spectroscopy, NMR, and/or GC-MS

• Future Researchers

Run bioassays on pooled fractions to obtain statistically sound resultsSeparate 100% DCM pool and run bioassays