Chemical Analysis of Trees Infested by the Asian Longhorned Beetle

1. Chemical Analysis of Trees Infested by the Asian Longhorned Beetle R. Bruce, D. Cherla, P. Duran, J. Li, T. Rastogi, A. Sin, G. Springsted, A. Yang, D. Yerramilli, E. Yoo, A. Zozula Team Project Leader: Jeremy Stanton Team Project Assistant: Bhavik Shah

2. Introduction to the ALB • Where did it come from? • Description • What do they attack? • How do they attack? • Possible treatments for ALB infestations

3. Why are we so concerned? • Impact on the economy and environment • Effort put into eradicating the infestation • Detection and prevention

4. Overall Direction • We will be testing tree samples for: • Sugar concentration • Lignin • Protein • Vessel space • Extractives

5. The Sample Pool • Trees tested (in order of ALB’s preference): • Sugar Maple (Acer sacrum) • White Willow (Salix alba) • Hackberry (Celtis spp.) • Northern Red Oak (Quercus rubra) • Eastern White Pine (Pinus strobus) • Grinding trees increases surface area for further reactions

6. Cellulose and Hemicelluloses Cellulose • Structure • Cellulose • Hemicelluloses • Hydrogen bonding • Digestion by ALB • Hypothesis: Trees with high concentrations of cellulose and hemicellulose will more likely attract the ALB Hemicellulose monomers

7. Reducing Sugars Test Collected Red Precipitate Aldehyde Carboxylate • Benedict’s solution • Cu2O formed, collected, and massed • Created calibration curve using standard dextrose solutions: 10.0 g/L, 7.5 g/L, 5.0 g/L, 2.5 g/L, 1.0 g/L • Performed same procedure using tree pulp • Determined sugar concentration of each tree sample using calibration curve

8. Reducing Sugars Test Results • Hypothesized results: • Maple > Willow > Hackberry > Oak > Pine • Actual results: • Oak ≈ Willow > Maple ≈ Hackberry > Pine

9. Reducing Sugars Analysis • Sugar concentration may not be a factor • Trees may have enough sugars for beetle survival • Sugar preference of Benedict’s reaction as compared to that of ALB Buchner filtration setup

10. Lignin Example of lignin molecule • Derived from sugar through removal of water • Comprises 25-35% mass of tree • Complex, phenolic molecular formula • Lignin covalently bonded to hemicellulose, provides strength of tree cell walls • Hypothesis: More lignin inside tree, less likely ALB will infest that tree

11. Lignin and Cellulose Isolation • Cellulase enzyme digests cellulose/hemicellulose • Optimal conditions: Acetate buffer of pH 4.5 Heated at 40°C for 48 hours • Lignin boiled with HCl to break any remaining bonds • Lignin 92% pure Lignin isolation setup

12. Lignin Results • Hypothesized results: • Pine >Oak > Hackberry > Willow > Maple • Actual results: • Maple > Willow ≈ Hackberry ≈ Pine ≈ Oak

13. Lignin Isolation Analysis • Theoretically correct procedure • Lignin may provide better habitat for ALB ALB Pupa

14. Proteins • ALB may degrade proteins contained in wood to gain nutrients • Protein less than 1% of the mass of wood • Common proteins in trees: hydroxyproline-rich glycoproteins, glycine-rich proteins, and proline-rich proteins • Hypothesis: ALB is attracted to trees with higher protein content

15. Protein Content Analysis • Materials used: bovine serum albumin (BSA), Bradford reagent • Instruments used: UV/Vis Spectrophotometer • Beer’s Law: A = Єbc • Standard solutions of BSA used with Bradford reagent to create calibration curve at 595 nm UV/Vis Spectrophotometer • Absorbance of tree samples compared to the calibration curve to determine protein concentration

16. Protein Content Results • Hypothesized results: • Maple > Willow > Hackberry > Oak > Pine • Actual results: • Willow > Maple ≈ Pine > Hackberry > Oak

17. Protein Content Analysis • Proteins may not be critical to ALB • Values may not be relevant if all trees have values above the threshold required • Bradford reagent only reacts with certain amino acids • Incubation time differences Bradford reagent

18. SEM Image Scanning Electron Microscope • Tree samples cut, mounted, and coated • Images recorded from core, inner, middle, and outer sections of the sample at various magnifications • Percentage of vessel space for each tree calculated through ImageJ • Hypothesis: Trees with small percentage of vessel space are more likely to attract the ALB

19. SEM Results • Hypothesized results: • Pine >Oak > Hackberry > Willow > Maple • Actual results: • Oak > Pine ≈ Maple > Willow ≈ Hackberry • Tree structure may have minimal impact on ALB selectivity

20. Extractives • Consist of organic and inorganic compounds • Protect wood from decay and pests • Boost the structural integrity of the tree • Provide distinct colors and odors three to five percent by weight of wood material • Hypothesis: ALB is potentially attracted or repelled by specific extractives present Cis-3-hexen-1-ol

21. Gas Chromatographic/Mass Spectrum (GCMS) Analysis • Gas chromatography (GC) • Separates chemical mixtures by polarity and boiling point • Non-polar solvent: cyclohexane • Polar solvent: ethyl acetate • Mass spectrometer (MS) • identifies and quantifies the chemicals separated by the GC based on their masses • Added anthracene as reference point

22. Extractives in Cyclohexane

23. Extractives in Ethyl Acetate

24. Overall Analysis • Limited number of trials • Incomplete reactions and extractions • Tree branches versus tree • Multiple factors possibly involved for host selection • Interdependency of variables • Hierarchy of variables

25. Future Experiments • Work with beetles • Test variables together • Further GCMS tests, including leaf extractives • Confirm effectiveness of benzenemethanol and dibutyl phthalate as potential repellents • Investigation of the beetle’s host preference is complex but important to understand

26. Acknowledgements • Dr. Miyamoto, Drew University, Madison, New Jersey (for his ninja-like sputtering skills) • Dr. Fukunaga, Drew University, Madison, New Jersey (for his drugs, we needed something to get through our “headaches”) • Michelle Yap, McNair Academic High School, Jersey City, New Jersey (for getting DEAD trees)

27. Questions?