Instructor materials
Overview
The aspen leaf miner (Phyllocnistis populiella) is a tiny moth that feeds inside the epidermal tissue of aspen leaves. The species has caused widespread damage to aspen trees in Alaska and northern Canada for several decades. This exercise challenges students to use data on the physiology of damaged and undamaged leaves to assess the impact of the leaf miner for aspen photosynthesis and water balance, and more generally to better understand the important role of the leaf epidermis in leaf function.
Key Concepts
The effect of herbivory on plant function is complex and depends on the feeding behavior of the herbivore
The leaf epidermis plays an important role in the regulation of both photosynthesis and water balance
Photosynthesis and plant water balance are functionally linked through the opening and closing of the stomata
Student Learning Outcomes
Access publicly available data and metadata from a data provider
Explain the role of the leaf epidermis in leaf gas exchange
Propose a hypothesis based on observation and assess the hypothesis with data
Construct a graph in order to answer a biological questions
Use data on leaf physiology to infer the effects of herbivory on leaf function
Prior Knowledge
Students should have a basic understanding of
The parts of a plant and their general function
The purpose and general process of photosynthesis
How to work with data in a spreadsheet (e.g. Google Sheets, Microsoft Excel)
Basic descriptive statistics such as the mean and standard deviation, and ideally standard error
How to read a graph
Background
The aspen leaf miner moth (Phyllocnistis populiella) is a tiny herbivorous insect that has caused major damage to aspen trees in Alaska and parts of Canada since around the year 2000. Larvae of the moth feed in an unusual manner. They dwell throughout the juvenile period within the leaf’s epidermal layer - a single cell layer deep - slicing a path through the cells and drinking the cell contents released. The damaged portion of the epidermis appears as a white serpentine mine on either the top or the bottom of the leaf. This targeted damage to one leaf tissue provides an opportunity to better understand the function and importance of the leaf epidermis.
To disentangle the effects of leaf mining on the top and bottom of the leaf, researchers at the University of Alaska Fairbanks removed leaf miner eggs from the top, bottom, or both sides of aspen leaves. When the damage was at its maximum in mid-summer, they measured gas exchange on the leaves, including photosynthesis - the rate at which CO2 was assimilated by leaves - and stomatal conductance - the rate at which water vapor was lost from leaves through stomata.
To disentangle the effects of leaf mining on the top and bottom of the leaf, researchers at the University of Alaska Fairbanks removed leaf miner eggs from the top, bottom, or both sides of aspen leaves. When the damage was at its maximum in mid-summer, they measured gas exchange on the leaves, including photosynthesis - the rate at which CO2 was assimilated by leaves - and stomatal conductance - the rate at which water vapor was lost from leaves through stomata.
Materials
The data set (available as a Google Sheet through this link)
Access to either Google Sheets or Microsoft Excel. If using Microsoft Excel, it should be the installed version, not the web based version. Students with the web-based version of Microsoft Excel should use Google Sheets instead.
The student worksheet (Google Doc format or pdf format), which includes
written directions for the calculations and graph construction in Google Sheets or Microsoft Excel
a walk-through video that goes over carrying out the calculations and constructing the graph in Google Sheets or Microsoft Excel
If you prefer to use a Google Form to collect your students' responses and work, a copyable Google Form is available here.
To copy it for your own use, locate the "Copyable Student Worksheet..." Click on the three dots at the far right and choose "Make a copy."
When your copy has been made, click on the three dots at the far right of its name and choose "Move to." You must move your copy to your own Google Drive before giving your students access in order to protect their personal information and responses. Check that the form settings on your copy match your preferences. To give students access, share the link to the form with them.
You will need to enable file uploads before your students can use the form. To do this, locate your copy of the form in your Google Drive. Double-click on it to open it. You should get the error message "Missing File Upload Folders." Click "Restore" to fix this issue. You are then ready to share the responder link to the form with your students. You can also delete the file upload questions from the form if you do not need students to upload files to the form.
Teaching Tips
Working with the data
Students will work with either Google Sheets or Microsoft Excel in this activity. If you have a preference for which one students use, direct them to the resources for it.
For either program, students will need to make a copy of the Google Sheet or download it as a Microsoft Excel file in order to work with the data.
Running the activity
Students begin by watching four short videos:
After watching the videos, students answer questions that guide them to think about the location of the stomata on aspen leaves. They generate hypotheses about how mining damage to the leaf’s upper and lower epidermis will affect stomatal function in general, then they make specific predictions about the effect of leaf mining on stomatal conductance of water vapor and photosynthesis.
Students will be asked to visit the Bonanza Creek LTER website to find the actual research data set this activity is based on. By visiting this site, they can also learn about other data resources that are available publicly through this site. While looking at the dataset, students have a chance to see the different variables that were measured in this experiment, and identify the two variables this exercise focuses on.
Students will need to go to the Google Sheet that contains the data they will work with from the research data. This sheet has prepared the data by averaging across replicate dates to make it easier for students to work with. If a goal is to have students work with spreadsheets, you could have them work with the original dataset on the Bonanza Creek LTER website and prepare the data for graphing themselves.
Students will either need to create a Google Sheets copy of the file or download the file as an Excel file depending on which program they will use.
Students will need to calculate the mean and standard error for stomatal conductance for the top-mined, bottom-mined, and control leaves. They will graph the means using a column graph and add error bars using the calculated standard error. Written guidelines and a video walk-through are provided to guide students through this process in both programs (see the “Materials” section above).
Students will also calculate the mean and standard error for photosynthesis for the top-mined, bottom-mined, and unmined leaves, graph the means, and add error bars using the calculated standard errors.
Based on the graphs and what they have learned from the videos about leaf structure and function, students will describe the effect of top and bottom leaf mining on stomatal conductance and on photosynthesis.
Students will be asked to evaluate their original hypotheses based on these results. They are asked to consider how leaf miner damage to the top or bottom of the leaf can result in the observed effects even though the miner is not feeding on the photosynthetic cells of the leaf.
Additional Reading
Basic biology of the aspen leaf miner
U.S. Forest Service. 2011. Aspen leaf miner. Forest Health Protection, Rocky Mountain Region. https://www.fs.usda.gov/Internet/FSE_DOCUMENTS/stelprdb5347213.pdf
Impact of the aspen leaf miner on aspen physiology and productivity
Boyd, Melissa A., M. A., T. B. Logan, D. Patricia, G. Scott, R. Brendan, W. Diane, W. Xanthe, and C. M. Michelle. 2019. Impacts of climate and insect herbivory on productivity and physiology of trembling aspen (Populus tremuloides) in Alaskan boreal forests. Environmental Research Letters:085010.https://doi.org/10.1088/1748-9326/ab215f
Wagner, D., L. Defoliart, P. Doak, and J. Schneiderheinze. 2008. Impact of epidermal leaf mining by the aspen leaf miner (Phyllocnistis populiella) on growth, physiology, and leaf longevity of quaking aspen. Oecologia 157:259–267.https://doi.org/10.1007/s00442-008-1067-1
Wagner, D., J. M. Wheeler, and S. J. Burr. 2020. The leaf miner Phyllocnistis populiella negatively impacts water relations in aspen. Tree Physiology 40:580–590. https://doi.org/10.1093/treephys/tpz109
Aspen and the changing boreal forest
Johnstone, J. F., T. N. Hollingsworth, F. S. Chapin, and M. C. Mack. 2010. Changes in fire regime break the legacy lock on successional trajectories in Alaskan boreal forest. Global Change Biology 16:1281–1295. https://doi.org/10.1111/j.1365-2486.2009.02051.x
Mack, M. C., X. J. Walker, J. F. Johnstone, H. D. Alexander, A. M. Melvin, M. Jean, and S. N. Miller. 2021. Carbon loss from boreal forest wildfires offset by increased dominance of deciduous trees. Science 372:280–283. http://doi.org/10.1126/science.abf3903
Extensions to the module
The data set that students work with in this activity contains a large number of variables that are not used in this particular activity, but that could be used to extend the activity for students. Some examples of extension follow.
Leaf mining - Students could explore the extent of mining damage to the leaves; or explore top and bottom leaf mining as a continuous effect on photosynthesis rather than a nominal effect as we do here. Students might also explore the effect of the location of leaf mining on other variables in the dataset, such as the water content of the leaves (% water content).
Leaf water potential - Advanced students could explore the effects of top and bottom epidermal mining on leaf water potential. Water potential is a measure of the free energy of water, reported in units of pressure. Plant water potentials are always negative, because they are measured in relation to pure water. A brief video introducing leaf water potential and how it is measured can be accessed here. A broader video explaining how water moves through plants, including the role of water potential and leaf conductance, can be accessed here. Water moves from high to low water potential. Evaporation from leaves causes a strong negative water potential in the leaf, which draws water up from the roots. The data set reports aspen leaf water potential measured at midday, when we assume the rate of water loss through stomata is relatively high (and leaf water potential strongly negative), and pre-dawn, when no active photosynthesis is occurring and we assume the stomata are closed. The latter allows us to investigate the role of leaf mining on the rate water vapor is lost across the leaf cuticle (called cuticular conductance), rather than through stomata. This information can be used to address the question, does leaf mining weaken the ability of the cuticle to resist water loss? (Whole leaf water content (%) can also be informative in this regard.)
Leaf carbon stable isotope composition - Another advanced investigation could examine the effect of upper and lower epidermal mining on leaf carbon stable isotope composition. There are two stable isotopes of carbon: 12C and the rarer 13C. Relative to the heavier isotope, 12C diffuses more rapidly into the leaf from the atmosphere and enters more rapidly into the reaction that fixes C in photosynthesis. As a result, the ratio of 13C to 12C in leaf tissue is lower (more negative) than that in the atmosphere. The stable isotope composition is reported as 𝛿13C, with units of ‰ (per mil). (More information about delta notation and stable isotopes in general can be found here.) When the stomata are narrowed or closed, there is less opportunity for fractionation and the 𝛿13C of the carbon fixed in photosynthesis is less negative; closer in value to the atmosphere. Leaf 𝛿13C can be used to test the hypothesis that stomata of mined leaves are more often closed and unresponsive relative to unmined leaves.