Although female bean beetles (Callosobruchus maculatus) prefer to oviposit singly, multiple eggs will be deposited on a single bean when suitable oviposition substrates are scarce. The goal of this activity is to examine female choice when all of the beans available for oviposition already bear a single egg. Do individual female bean beetles distinguish between their own eggs deposited previously and eggs deposited by unfamiliar females when selecting an oviposition site? Since larval success is dependent on the resources of the bean and mortality increases with multiple eggs, it stands to reason that females may prefer to lay eggs on beans bearing the eggs of other females versus their own eggs so that her own larvae will not be in direct competition with each other. In this experiment, students are challenged to design and perform a set of experiments to determine whether female bean beetles discriminate between their own eggs and the eggs of other females when choosing to deposit a second egg on a bean that already contains an egg. Typical experiments include presenting a female with beans bearing her own eggs and beans bearing the eggs of other females and counting the number of subsequent eggs laid on each type of bean.
Topic: Reproductive Behavior, Animal Behavior
Level: Non-majors – Upper-level majors
Class Time: One 2-3 hour class period to design and setup the experiment, plus 2 fifteen minute sessions to collect and isolate females and beans bearing a single egg in a testing arena and then one 1-2 hour class period 5 days later to count the eggs.
Learning Objectives:
Design and perform a set of experiments to evaluate whether female bean beetles (Callosobruchus maculatus) discriminate between beans containing their own eggs and beans containing the eggs of other females when selecting oviposition sites.
Amy Reber1, Michael Black2 and William Said3
1Department of Biology, Georgia State University, Atlanta, GA
2Neuroscience Institute, Georgia State University, Atlanta, GA
3Department of Biology, University of Georgia, Athens, GA
Objectives
- Design an experiment to evaluate whether female bean beetles, Callosobruchus maculatus, distinguish between beans containing their own eggs and beans containing the eggs of other females when selecting an oviposition site.
Introduction
Bean beetles, Callosobruchus maculatus, are small beetles native to Africa and Asia whose entire life cycle revolves around a bean. During their relatively brief adult life (1-2 weeks), female bean beetles mate and then begin to lay eggs (oviposit) on the surface of beans. As the larvae hatch from the egg, they tunnel directly into the bean and remain there for the duration of their development until they pupate and emerge as adults approximately 3-4 weeks later (Beck and Blumer 2010). Because the larvae are completely dependent on the resources contained within the bean, selection of high quality oviposition sites by the female is critical to her offsprings future growth, survival and reproductive success (Mitchell 1975, Wasserman and Futuyma 1981, Brown and Downhower 1988). Although adult females prefer to deposit only a single egg on each available bean, multiple eggs will be deposited on the same bean if suitable oviposition substrates are limited. However, depositing multiple eggs on a single bean puts the larvae in direct competition with each other and increases overall larval mortality rates (Mitchell 1975). In a situation where the female is presented only with beans already containing an egg, it may be advantageous for her to deposit additional eggs on beans bearing the eggs of other females over beans bearing her own eggs so that her own larvae will not be in direct competition with each other. In this study, you will determine whether female bean beetles discriminate between eggs they themselves have laid and eggs laid by other females when choosing to deposit additional eggs on a bean.
Materials
In class, you will be provided with live bean beetle cultures raised on mung beans, a supply of organic mung beans, and plastic Petri dishes of various sizes. Female beetles are easily identified in the live cultures because they have two dark stripes on the posterior of the abdomen, whereas the posterior abdomen of males is uniformly light in color (Figure 1).
Figure 1. Identifying the sexes. Females have two dark stripes on the posterior of the abdomen, whereas the posterior abdomen of males is uniformly light in color. Image reprinted with permission from Brown and Downhower (1988).
Experimental Design
Prior to the laboratory class, each group should design a set of experiments to determine whether female bean beetles discriminate between eggs they themselves have laid and eggs laid by other females when choosing to deposit additional eggs on a bean. Each group will present their designs to the class and common experimental approaches will be discussed.
After you have read the background information and before the laboratory class meeting:
Describe an experimental design for evaluating whether female bean beetles discriminate between beans bearing their own eggs and beans bearing the eggs of unfamiliar females.
Predict the outcomes for the experiment.
Identify and list the variables you would manipulate in the experiment.
Identify and list the variables you would keep constant in the experiment.
List the data you would collect to determine if your predictions were true.
Describe the statistical analyses that you would carry out to test your predictions.
Come to class prepared to present your experimental design.
Beck, C.W. and Blumer, L.S. 2010. A Handbook on Bean Beetles, Callosobruchus maculatus. www.beanbeetles.org/handbook/
Brown, L. and Downhower, J.F. 1988. Analyses in Behavioral Ecology: A Manual of Lab and Field. Sinauer Associates.
Mitchell, R. 1975. The evolution of oviposition tactics in the bean weevil, Callosobruchus maculatus F. Ecology 56:696-702.
Wasserman, S.S. and Futuyma, D.J. 1981. Evolution of host plant utilization in laboratory populations of the southern cowpea weevil, Callosobruchus maculatus Fabrivius (Coleoptera: Bruchidae). Evolution 35:605-617.
This experiment was written by Amy Reber, Michael Black and William Said, 2014 (www.beanbeetles.org).
Copyright © by Amy Reber, Michael Black and William Said, 2014. All rights reserved. The content of this site may be freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are prohibited without prior written permission from the copyright holders.
Consult the Laboratory Methods section of the beanbeetles.org website for detailed information on growing cultures and handling techniques, as well as tips on identifying the sexes. Ideally two or more distinct cultures (from different sources or obtained at different times from the supplier) should be used in the experiment.
Experimental Questions
The questions that students generally address in their experiments are:
Will females lay more eggs on beans containing the eggs of other females than on beans containing their own eggs?
Will female beetles continue to lay eggs on beans that already have one or more eggs?
Will females distinguish between their own eggs and the eggs of closely related females (i.e. females from the same culture)?
Will females discriminate between beans bearing eggs that are different ages?
In their experimental designs, students should consider the following questions:
How can you control for the age and or size of the females being tested?
How can you control whether the female is a virgin or has mated previously and does this matter?
How can you control for variation among females in the total number of eggs laid and does this matter?
How can you control for bean size and weight?
How many beans bearing an egg from each female should be used in the experiment?
How many different females should contribute eggs to the beans in the testing arena and how should these be marked and arranged?
How long should the females be allowed to lay eggs in the testing arena before the eggs are counted?
Typical Experimental Designs
Collection of newly emerged male and female beetles
The collection of newly emerged males and females is most easily done by removing 20-30 beans with apparent pupation windows from the main culture. Each of these beans may be placed in a separate Petri dish or the well of a 12-well tissue culture plate where emergence can be monitored more easily.
Mating, oviposition, and bean collection
Following emergence, a single female and 2-3 males were placed into a small Petri dish (65 mm x 15 mm) containing a monolayer of mung beans that were marked with small symbols (using wax pencils or non-toxic marking pens) so that they could be identified as belonging to a particular female. Several of these dishes were set up simultaneously so that multiple females mated and began to lay eggs at approximately the same time. After a female deposited a single egg on several beans she was removed from the dish and placed into the testing arena.
Behavioral choice testing
The testing arena (a 100 x 15 mm Petri dish) contained a number of beans each bearing a single egg. Some of these beans had an egg belonging to the female being tested while other beans contained eggs from unfamiliar females. The females were left in the testing arena for 5 days to lay additional eggs.
Data Collection
Potential Difficulties
The most difficult part of this experiment is the timing. We found that it was best to use newly emerged females as they laid eggs more readily and more consistently than older females and were more likely to survive until the end of the trial than older females. The use of newly emerged females also helps to ensure greater consistency in the age of the females and age of the eggs used in the experiment. After the females mate and begin to lay eggs, beans with a single egg need to be removed for use in the experimental trials. This creates some challenges in scheduling because it can be difficult to know exactly when the females will emerge and when they will mate and begin to lay eggs.
Students will need one 2-3 hour lab session to design and set up the experiment. Then either the students or a teaching assistant/ instructor will need additional time when the females emerge to separate them into smaller dishes with males and beans so that mating and oviposition can begin. Once oviposition begins, beans with single eggs need to be removed (before the female can lay multiple eggs on a single bean), and placed into a testing arena. Finally the students will need an additional hour or two of class time to count the number of eggs laid on each bean for data analysis.
Data analysis
The data from the experiment should be the number of eggs laid on each of the two types of beans: beans beginning the experiment with a single egg deposited by the test female (self) and beans beginning the experiment with a single egg deposited an unfamiliar female (other). The difference in the average number of additional eggs on each bean type could be compared using a paired t-test or a Wilcoxon Signed-Ranks Test.
Equipment and supplies
For a class of 24 students working in groups of 4, the following equipment is recommended:
- 6 dissection microscopes (for counting the number of eggs on each bean)
- 6-12 plastic Petri dishes (100 mm x 15 mm) to serve as the testing arena
- 12 smaller plastic Petri dishes (65 mm x 15 mm) or (35 mm x 10 mm) to collect newly emerged females for oviposition
- 6 forceps for transferring the beetles and beans between dishes
- 2 cultures of bean beetles, preferably from different strains or obtained at different times (this reduces the degree of relatedness between females used in the experiment)
- organic beans (use the same type of bean as the parent cultures)
- nontoxic marking pens or wax pencils
This experiment was written by Amy Reber, Michael Black and William Said, 2014 (www.beanbeetles.org).
Copyright © by Amy Reber, Michael Black and William Said, 2014. All rights reserved. The content of this site may be freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are prohibited without prior written permission from the copyright holders.
Individual female bean beetles were placed into six different testing arenas containing 40 beans in a random configuration: 20 beans contained a single egg from the female being tested (Self) and 20 beans contained a single egg from an unfamiliar female (Other). The test females were allowed to lay additional eggs for 5 days after which time the beans were removed and the number of eggs on each type of bean was counted. Females in the experiment showed no clear preference for beans bearing their own eggs (mean±SE = 11.8±2.9) versus beans containing the eggs of an unfamiliar female (mean±SE = 9.3±2.1) when selecting an oviposition site. However, our sample size was small (N=6). The raw data are available in the Downloads section.
Figure 1. Mean number of eggs laid by a female beetle on beans containing either one of her own eggs (self) or an egg from an unfamiliar female (other) over the course of 5 days. The means are not significantly different (paired t-test, t=1.06, p=0.338, two-tailed), suggesting that females do not discriminate between their own eggs and the eggs of other females when choosing an oviposition site on beans already bearing an egg.
This experiment was written by Amy Reber, Michael Black and William Said, 2014 (www.beanbeetles.org).
Copyright © by Amy Reber, Michael Black and William Said, 2014. All rights reserved. The content of this site may be freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are prohibited without prior written permission from the copyright holders.