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 StantonTeam Project Assistant: Bhavik Shah

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

Why are we so concerned?

• Impact on the economy and environment

• Effort put into eradicating the infestation

• Detection and prevention

Overall Direction• We will be testing tree samples for:

– Sugar concentration– Lignin– Protein– Vessel space– Extractives

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

Cellulose and Hemicelluloses• Structure

– Cellulose– Hemicelluloses

• Hydrogen bonding• Digestion by ALB• Hypothesis: Trees with high

concentrations of cellulose and hemicellulose will more likely attract the ALB

Cellulose

Hemicellulose monomers

Reducing Sugars Test

Aldehyde Carboxylate Collected Red

Precipitate• 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

Reducing Sugars Test ResultsOak Maple Pine Hackberry Willow

Sample 1 (g) 0.172 0.049 0.051 0.046 0.177

Sample 2 (g) 0.192_______

0.034________ ________

Average (g) 0.182 0.049 0.042 0.046 0.177

Concentration (g/L) 4.810 1.286 1.114 1.205 4.668

• Hypothesized results:

Maple > Willow > Hackberry > Oak > Pine

• Actual results:

Oak ≈ Willow > Maple ≈ Hackberry > Pine

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

Lignin• 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

Example of lignin molecule

Lignin and Cellulose Isolation

• Cellulase enzyme digests cellulose/hemicellulose

• Optimal conditions:Acetate buffer of pH 4.5Heated at 40°C for 48 hours

• Lignin boiled with HCl to break any remaining bonds

• Lignin 92% pureLignin isolation setup

Lignin ResultsOak Maple Pine Hackberry Willow

Sample 1 (g) 4.154 5.150 4.142 4.570 4.814

Sample 2 (g) 3.775 5.001 3.869 3.894 3.917

Average (g) 3.965 5.076 4.006 4.232 4.365

• Hypothesized results:

Pine > Oak > Hackberry > Willow > Maple

• Actual results:

Maple > Willow ≈ Hackberry ≈ Pine ≈ Oak

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

ALB Pupa

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

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

• Absorbance of tree samples compared to the calibration curve to determine protein concentration

UV/Vis Spectrophotometer

Protein Content Results

• Hypothesized results:

Maple > Willow > Hackberry > Oak > Pine

• Actual results:

Willow > Maple ≈ Pine > Hackberry > Oak

Oak Maple Pine Hackberry Willow

Sample 1 .488 .813 .508 .849 1.123

Sample 2 .466 .867 .500 .908 1.101

Average .477 .840 .504 .879 1.112

Concentration (µg/mL) 8.865 20.629 20.274 13.086 41.365

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

• 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

SEM Image

Scanning Electron Microscope

SEM ResultsOak Maple Pine Hackberry Willow

Total Vessel Area (µm2) 35169.5 24871.4 19143.9 14760.4 13661.5

Total Area (µm2) 90982.5 88196.5 66151.9 91285.9 79620.2

Percentage Vessel Area 38.7 28.2 28.9 16.2 17.2

• Hypothesized results:

Pine > Oak > Hackberry > Willow > Maple

• Actual results:

Oak > Pine ≈ Maple > Willow ≈ Hackberry

• Tree structure may have minimal impact on ALB selectivity

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

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

Extractives in Cyclohexane

Extractives in Ethyl Acetate

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

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

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)