Chemical Analysis of Trees Infested by the Asian Longhorned Beetle
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Transcript of Chemical Analysis of Trees Infested by the Asian Longhorned Beetle
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)