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).