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Modeling Insect Development in Earth’s Changing Climate Instructors Notes Experimental Design (Guided Inquiry flow) Below is an example of the flow from which we guided the students to the hypotheses and experimental design for this exercise: a. What is the “big picture” phenomenon that we are examining? b. Based on the current environmental data we have discussed what are a few hypotheses that you could make regarding what might happen to development rates in bean beetles if the global temperature were to increase? 1. Which one of these hypotheses can we test in a laboratory situation? d. How do we go about testing our hypothesis experimentally? 1. What are our potential variables? e. independent and dependent: temperature, humidity, substrate preference 1. What is the most appropriate way to analyze and interpret our data? f. Statistical analyses (T-test, ANOVA, bar graphs and interaction plots) g. How would we present our data? 1. To other scientists 2. To the general population There are two primary goals of this exercise, the first of which is to use a real world problem to get students to think critically about the effects of climate change on ecosystems as well as the secondary effects on society. The second primary goal of the exercise is to get the students to generate hypotheses and design sound experiments to test their hypotheses. We found it useful to spend 1-1.5 hours for the pre-lab in which the time is used to alternate between introduction material and open discussion for guided inquiry towards hypotheses and experimental design. If you want to begin the experiment the same day as the pre-lab, it is useful to set up embryo cultures just prior to the pre-lab so that there are embryos for the students to find. However, the set up (i.e. what kind of bean the adults come from, etc.) will depend on the variables being tested. Students need to keep track of when the eggs were laid to within one hour and then need to make sure they record the time accurately for each embryo as it reaches the pigmented endpoint. It is useful to make sure the students know the difference between a pigmented head capsule and a dead embryo. The dead embryos will sometimes have a large vacuole or air pocket on one end that might look like the pigmented region. Counting these embryos will cause a wider degree of error in the final results. It is useful to know the stability of the incubator temperature and humidity prior to the studentrun experiment(s) so that any variability in the incubator can be taken into account in the analysis of the results. Since the students had spent most of the semester writing up labs in various formal scientific formats we chose to have them present the results from this experiment in a format that

mimicked mass media journalism such as Time magazine. This way they were forced to take results (including statistical analyses) and package them in a way that did not assume high levels of science education in the reader. The students really enjoyed this challenge and engaged it with enthusiasm. There is the potential for some flexibility in the variables tested in the experiment in addition to temperature. For example, students may suggest that varying humidity, egg substrate, maternal substrate, etc. would cause development time to change. In this experiment, we suggest monitoring embryos for the formation of head capsule pigmentation (Figure 1 in Student Handout)—a convenient developmental marker that can be observed using dissecting microscopes. In order to achieve statistically significant differences in development time under different constant temperatures with small sample sizes (30-40 embryos) temperature choice is important. We found that 22.5ºC and 30ºC offer perhaps the best compromise between significantly different development times while maintaining a short experimental period. Cooler temperatures make for longer experiments (several weeks at 15ºC!). Testing, for example, 27.5ºC and 32.5ºC may not yield significant differences due to the shape of the development time curve. If you have a lot of time and would like to carefully examine the relationship between temperature and development time choosing several temperatures between 15ºC and 37.5ºC is recommended (we have observed 100% mortality of embryos at 40ºC). Once the students have decided upon the other variables for the experiment (e.g. adult beetles from two different substrates (mung and adzuki) with all eggs laid on the same substrate (black eyed peas)), the embryo collection and slide set-up is relatively fast. Students find beans with embryos (if they were not set up prior to the pre-lab then the students will need to set up the cultures immediately after the pre-lab and wait one hour for embryos to be oviposited on the beans). Ideally you will want 30-40 embryos per treatment (hopefully the students will settle on this number from a statistics perspective). We have had the most success getting multiple embryos quickly by working with stock cultures that have gone through at most two generations. To maximize oviposition rates, cultures should contain a large number of females and males that have emerged within the last 24-48 hours but not as recently as in the last 4-6 hours. It may also help to isolate the adults from beans prior to culturing embryos. We have also found that oviposition rates are highest if adults are kept at high temperatures (approximately 30 C) before and during oviposition. Beans with one embryo are placed on a small piece of clay with the embryo facing up for easy observation under the dissecting microscope. (Multiple embryos on the same bean can also be used but should both be in a position where the entire embryo can be observed without having to rotate the bean on the slide.) Up to six beans can be placed on one slide and five slides can be placed on one tray lid (Figure 2). This allows for easy transport of all embryos on one tray to be scanned for pigmentation at one time. Once the embryos are placed in the incubator, the time needs to be noted, and the embryos should NOT be removed except when scanning for head capsule pigmentation using dissecting microscopes at specific time intervals. Knowing it will take a few days to observe the first pigmentation it is good to start observing every eight hours and then switching to every four hours once pigmentation is first observed. Data Collection Data collection for the experiment is simple; individual trays of beans with embryos are removed from the incubators (one at a time) and scanned for head capsule pigmentation under a

dissecting microscope. It takes about 1-1.5 minutes per tray so you can get through a lot of embryos quickly at any given time point. (This is especially nice on the graveyard shift!) Students need to organize themselves into groups that are each responsible for a temperature/substrate combination and then make a schedule for students to check for pigmentation. They should record the absence or presence of pigmentation for each embryo during each check. Subtracting oviposition times from the time at which head capsule pigmentation is observed for each egg gives the development time (time to reach pigmentation) for each embryo. These are the data that should be analyzed statistically. Although our students checked embryos frequently after the first indications of hatch were recorded, it should be possible to see large differences in development time with less frequent (once or twice per day) checks. The standard deviation of development time tends to increase roughly linearly with the mean development time, so less frequent checks may mean that all of the hatches occur within one or two checks at high temperature while being spread out over multiple checks at low temperatures. With less frequent checks it is unlikely that a significant result will be obtained when examining the effects of variables such as maternal substrate and humidity that tend to have smaller effects on development time than temperature. We had our students make a simple template on which they list each embryo with the tray, slide and bean number and then note whether there is pigmentation or not at each time-point. Once an embryo has achieved head capsule pigmentation, it does not have to be observed at subsequent time points. The experiment is terminated when the last embryo reaches pigmentation or the remaining embryos have gone more than two time points since the last embryo has reached pigmentation. Data Analysis 1.

What is the most appropriate way to analyze and interpret our data? a. Statistical analyses: independent samples T-test and univariate analysis of variance (two-way anova) 2. What would the curve (development time vs. temperature) look like if we used a large range of temperatures?

This study was written by A. Putzke and B. Yurk, 2012 (www.beanbeetles.org).