Due to differences in genetic makeup and exposure to environmental factors (such as soil moisture and nutrient levels and exposure to plant pathogens and herbivores), plants vary in their chemical and physical traits. This can cause differences in susceptibility to herbivory or differences in nutritional quality that attract herbivores. Therefore, one might expect to find differences among plants in the number of herbivores that feed on them, the ways herbivores select feeding and oviposition sites, and the success of these herbivores. In this observational experiment, students will conduct investigations of sawfly galls (Hymenoptera) on willow (Salix) trees to examine some of the ecological and possible evolutionary consequences of plant-herbivore relationships to each of the interacting species. Galls make great sampling units for investigating herbivory because they are discrete (each gall contains one herbivorous larva), quantifiable (easy to see and count), and indicative of insect preference (since each gall represents one successful oviposition by a female sawfly) and performance (successful larval development is indicated by an emergence hole). During a single lab period, students will become familiar with the plant-herbivore system and work in teams to collect data to test a general hypothesis proposed by the instructor. Teams then choose a second instructor-generated hypothesis or develop their own to test. Examples of these are (1) The level of herbivory by sawflies varies among willow trees, and (2) Galls on leaves with competing galls are less successful than single galls on leaves. Outside of lab time, students will analyze their data statistically, and prepare a formal oral report on their investigation.
One 3-hour lab period, plus approximately an hour of another lab or lecture period for oral reports. Additional lab time for data entry into a spreadsheet (requires computer access), and statistical analysis.
OUTSIDE OF CLASS TIME
Two to three hours for statistical analysis and preparation of oral report.
The field work is conducted at any site with several willow trees that have galls on the leaves. This investigation works only in the fall, when galls are fully developed and easily visible on the leaves.
I use this lab activity in a junior-level general ecology course for all biology majors. I have 20-24 students per lab section.
Public, primarily undergraduate university of 8500 students, with a small master’s program.
This lab should be transferable to other types of institutions. It may be used for sophomore to senior levels, primarily for biology majors. Both plant and herbivore have broad distributions and are speciose. More than 200 species of sawflies form galls on willow (Salix) species (Nyman et al. 1999). Salix occurs in every state of the U.S. (see USDA map for Salix: http://plants.usda.gov/cgi_bin/topics.cgi? earl=plant_profile.cgi&symbol=SALIX), and Pontania sawflies have a broad distribution in North America as well as Europe and Asia. However, Pontania galls may not be present or common at all sites where willows grow. Euura is another sawfly genus that forms galls on willow, but on petioles or stems rather than leaves. The hypotheses could be adjusted to address the distribution of galls on stems or branches. Alternate host plant-herbivore systems are available in most sites, but it may take a bit of searching to find an appropriate system. Consider galls on maple (Acer) leaves (commonly caused by eriophyid mites), goldenrod (Solidago) stems (caused by Epiblema caterpillars or the tephritid fruit fly, Eurosta) or leaves (Asteromyia gall midges), oak (Quercus) stems and leaves (primarily caused by cynipid wasps), hackberry (Celtis) leaves (by jumping plant lice, psyllids), or creosotebush (Larrea) stems and buds (20+ species of the cecidomyid genus Asphondylia)..
Synopsis of the Experiment
Students observe galls on willow leaves, and begin their investigations in teams by collecting data to test the instructor-posed hypothesis that the number of galls per leaf varies among willow trees. This step gives students more guidance, allows them to practice sampling on a question they will discuss in class but won’t include in their graded assignment, and gives them a chance to view a number of galls and leaves to get a better sense of the study system and the typical pattern that herbivory varies among leaves and among plants (due to differences in plant chemistry, physical traits, environmental traits, etc.). Student teams then choose among several instructor-directed questions (such as whether female sawflies oviposit independently of other oviposition events, whether galls on leaves with other galls are less successful than single galls on leaves, and whether leaf-chewing herbivores select leaves independently of galls), or pose their own hypothesis. Instructors may assign these randomly to ensure that each hypothesis is tested by at least one team, or briefly discuss why each hypothesis might be interesting to test. More motivated teams might be challenged to formulate their own question based on their preliminary observations at the site. For example, they may notice that trees vary in size/age, or in distance to surface water, or that not all galls are the same size. Instructors can capitalize on these observations by encouraging students to ask how these variations might influence gall distribution or success. Once teams select a hypothesis, they then collect data to test the hypothesis, analyze their data, and prepare a formal oral report on their investigation.
At the conclusion of this lab, students will be able to:
Summary of What is Due
Description of the Experiment
Introduction (written for students)
The interactions between herbivores and their host plants are often complex, involving plant chemical and physical defenses, herbivore foraging behaviors, and many other factors. Most plants are attacked by several to many different types of herbivores. Each herbivore may feed in a different manner or on different plant tissues, causing different types of feeding damage. One of the more unique plant-herbivore interactions is the formation of galls. Galls are modified plant tissue stimulated by the oviposition and feeding activities of certain insects and spider mites. They result when the cells around the damaged area grow larger or divide more often than normal cells. As the insect feeds on the plant, it becomes surrounded by this abnormal plant growth. The insect continues to feed from within the gall, which protects it from many (but not all!) of its natural enemies. Other organisms, including viruses, bacteria, fungi, nematodes, and mites, may induce plant galls, but insects are the most common gall formers.
Galls can be used to test a number of interesting ecological and evolutionary questions about plant-herbivore interactions. The hypothesis that host plant quality affects herbivore densities and community structure was tested by Fritz et al. (1987b). As predicted, both densities of individual sawfly species and the relative abundances of these species varied among clones of arroyo willow. Additional data showed that shoot size is an important plant trait affecting gall densities: larger shoots have higher sawfly densities (Fritz et al. 1987a.) Since galls act as nutrient sinks (Nakamura, et al. 2003, Price et al. 1987), larger galls should provide more nutrients and therefore increase the success rate of the galling insect. Investigating the relationship between gall size and gall success (e.g., percent emergence) would provide a test of the generally supported hypothesis that plant galls are adaptive for the galling insect. The mechanisms through which habitat affects the density of galling insects were investigated by Fernandes and Price (1992). Lower rates of parasitism and fungal attack of galls may be at least partly responsible for higher gall densities in xeric (dry) environments compared with mesic (moist) habitats.
Willow trees (genus Salix) are attacked by several gall-forming herbivores. Gall midges form galls on buds, and sawflies form galls on leaves and shoots. Studies for this lab will be conducted at Engelhorn Pond on the Central Washington University campus where many of the willows have leaves with elongate, reddish capsules emerging from the leaf surface (gall – upper surface [left] and lower surface [right] – photos © K.A. Ernest).
These galls are caused by sawflies of the genus Pontania. Sawflies are not actually flies but relatives of bees and wasps (Order Hymenoptera). Adult females oviposit (lay eggs) into the leaf tissue. The egg hatches into a larva, which feeds on the leaf tissue while enclosed in the gall. When the larva has completed its development, it chews a hole in the gall and departs. See weblink in References to “Forest and Timber Insects in New Zealand” for pictures of egg, larva, pupa, and adult Pontania.
Willows are also eaten by a variety of free-feeding invertebrate herbivores. Lace bugs suck sap from leaves, spider mites chew leaves, and flea weevils chew on leaves and new shoots. You may find other insects feeding on the willows at the study site.
During this lab, you will (collectively) test a number of hypotheses about the gall-forming sawflies on willows. Particular questions chosen by student groups, in consultation with the instructor, may include:
In the process, perhaps you will become expert cecidologists (students of plant galls)!
Materials and Methods (written for faculty)
We are fortunate in being able to walk to our study site, a very small reserve (<1 ha) across the street from the Biology Building at the western edge of Central Washington University’s campus. Engelhorn Pond formed as a borrow pit in the 1920’s when gravel was excavated for use in the construction of the Interstate 90. The pit filled with water from runoff and groundwater and vegetation (including willows) colonized the site. Although very small, and nearly surrounded by university buildings, this site offers an urban refuge for ducks and other wildlife. As a wetland it garnered the attention of The Nature Conservancy, which purchased the site and donated it to the Biology Department. The pond is the dominant feature of the site and is surrounded by willow trees (Pacific Willow, Salix lasiandra). Gall densities vary from year to year, but usually galls are fairly abundant on the leaves. Numerous other sites are possible inside the city limits where willows grow along streams and irrigation canals. At your location, any site where willows grow and you can easily find galls would be appropriate. You might also consider sites with other plant species that harbor galls (such as poplars, goldenrod, maple trees).
Overview of Data Collection and Analysis Methods:
Introduction to the Study System
When we first get to the field site, I show students galls on the willows. Have a few students carefully open the galls (with a pocket knife or thumbnails) to find a sawfly larva. You could collect a few larvae in advance of the lab, and let students view them under a microscope during the lab introduction, or take a portable dissecting scope or simple hand lens with you to the field.
Hypothesis 1: all teams
In addition, each team will choose one of the other hypotheses described below, or can design a new hypothesis, to test. Read through these hypotheses to see which interests you most, or discuss ideas with your instructor. You may come up with interesting hypotheses based on your initial observations of the trees and galls, or even by reading the titles of some of the journal articles in the References section. Alternatively, your instructor may assign a hypothesis to each team to be sure each hypothesis gets tested by your class. See the Appendix for suggestions on random sampling, descriptive statistics, and statistical tests. When you “sample” leaves, please avoid removing them from the branch so that other teams may sample the same plants and we leave the willows as undisturbed as possible.
Analysis: This test is sensitive to small expected frequencies in a cell (ˆf < 5), so you may need to group together cells (e.g., 5-6 galls/leaf) to make sure ˆf ≥ 5. Then see how closely f matches ˆf. Are there more or fewer leaves with just one gall than expected? Are the galls distributed independently of one another on leaves? See Appendix.
Analysis: G-test of independence. See Appendix. Are single galls more successful?
Analysis: G-test of independence. See Appendix. Is gall presence independent of chewing damage?
Questions for Further Thought and Discussion
Conclusions about hypothesis 1:
For additional hypotheses tested by individual teams:
References and Links
Tools for Assessment of Student Learning Outcomes
While students are deciding which hypothesis to test, I wander among groups and ask them some simple questions to assess whether they are grasping the main concepts and hypotheses.
While they are collecting and analyzing data, I informally walk among teams of students and ask them simple questions such as:
We discuss the results of hypothesis 1 as a class, after putting the means and standard deviations on the board. I ask students whether trees vary in the extent of herbivory by galling sawflies.
I assess student learning primarily by requiring a formal oral report (similar in style to paper presentations at ESA meetings). I give students a guideline (below) for what material they should include in the talk, and give them the grade sheet I use to grade their oral report (see Oral Report Grade Sheet). Assessment of their data analysis is via written data summary and analysis they hand in at the time of the oral report. Students also evaluate oral presentations by other teams, providing me an opportunity to see if they understand what other teams found.
Your team will present a short, concise (5-10 min.), well organized oral presentation (in PowerPoint) based on the additional hypothesis you tested.
Use the following outline to prepare for your talk:
Your grade will be based on the criteria listed in the Oral Report Grade Sheet.
Tools for Formative Evaluation of this Experiment
NOTE: An extensive discussion on Evaluation appears in the Teaching section of TIEE.
NOTES TO FACULTY
Challenges to Anticipate and Solve
Comments on the Lab Description
Introducing the Lab to Your Students:
I usually try to introduce herbivory as a type of species interaction in the lecture part of the course. I cover the various ways plants defend themselves from herbivores, and how herbivores both respond to and are affected by chemical and physical defenses. During the lab, I give a brief overview of galls as a particular type of herbivory, and show some different types of galls. The Detailed Description of the Experiment provides introductory information.
Activities in the Lab:
Potential modifications of this lab activity:
Questions for Further Thought
Conclusions about hypothesis 1:
For additional hypotheses tested by individual teams:
Assessment of Student Learning Outcomes
I find that students often have difficulty with statistical tests. I try to make sure I visit each team several times while they are analyzing their data to make sure they properly summarize their data before beginning analysis, understand the basic idea of what the statistic is testing, and understand how to interpret P-values and test statistic values. Preparation for the oral report should be pretty straight forward if the students read through the Oral Report Gradesheet in advance.
Translating the Activity to Other Scales
Student Collected Data from this Experiment
Hypothesis 2: Females select leaves for oviposition independently of whether the leaf has other galls. Sample data provided in the Appendix.
Hypothesis 4: Leaves with galls are more likely to have leaf damage from chewing herbivores than are leaves without galls. Sample data provided in the attached Hypothesis 4 Data document and are reproduced below.
Example of student-collected data for hypothesis 4:
G-test of independence:
Compare with critical value (alpha .05) of 3.841
Conclusion: do not reject null hypothesis that galls occur independently of chewing damage
I have known a number of people who conducted research on gall-forming insects, and indirectly inspired the idea of this lab, including Tom Whitham, Peter Price, and their numerous graduate students and postdocs at Northern Arizona University. Karen Clancy kindly answered questions about Pontania gall life history and provided several photographs for this experiment. Daniel Beck has used this activity several times in general ecology and provided critical feedback; the written and oral report evaluation sheets are modified from his ideas. This contribution to TIEE benefited greatly from detailed comments by Bruce Grant and an anonymous reviewer.
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