Objective

• Design and perform a set of experiments to evaluate whether female bean beetles (Callosobruchus maculatus) discriminate between two suitable species of beans.

Introduction

Bean beetles (cowpea seed beetles), Callosobruchus maculatus, are agricultural pest insects of Africa and Asia. Females lay their eggs on the surface of beans (Family Fabaceae). Eggs are deposited (=oviposition) singly and several days after oviposition, a beetle larva (maggot) burrows into the bean. At 30ƒC, pupation and emergence of an adult beetle occurs 21-30 days after an egg was deposited. Adults are mature 24 - 36 hours after emergence and they do not need to feed. Adults may live for 1-2 weeks during which time mating and oviposition occur. Since larvae cannot move from the bean on which an egg was deposited, the oviposition choice of a female determines the future food resources available to their offspring (Brown and Downhower 1988). As a result, it is the most critical choice a female makes for her offspring, because it will influence their growth, survival, and future reproduction (Mitchell, 1975; Wasserman and Futuyma, 1981). Although females can be induced to lay eggs (oviposit) on a wide range of bean species, very few bean species result in normal development and the successful emergence of adults. Some bean species are very clearly toxic to Callosobruchus maculatus larvae (Janzen 1977).

Materials

In class, you will be provided with live cultures of bean beetles containing adults that have been raised on mung beans ( Vigna radiata ) or adzuki beans ( Vigna angularis ). Supplies of organic mung beans and adzuki beans also will be available. 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.

Experimental Design

Since the oviposition choices of females influence the survival and future success of their offspring, females may be very sensitive to the species and condition of the beans on which they are depositing eggs. Prior to the laboratory class, each group should design a set of experiments to address whether female bean beetles discriminate between two suitable species of beans. 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 at least TWO experimental designs for evaluating whether female bean beetles discriminate between mung or adzuki beans.
• Predict the outcomes for each experiment.
• Identify and list the variables you would manipulate in each experiment.
• Identify and list the variables you would keep constant in each 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 designs.

Literature Cited

Brown, L. and J.F. Downhower. 1988. Analyses in Behavioral Ecology: A Manual for Lab and Field. Sinauer Associates, 194 pages.

Janzen, D.H. 1977. How southern cowpea weevil larvae (Bruchidae Callosobruchus maculatus) die on non-host seeds. Ecology 58:921-927.

Mitchell, R. 1975. The evolution of oviposition tactics in the bean weevil, Callosobruchus maculatus F. Ecology 56:696-702.

Wasserman, S.S. and D.J. Futuyma. 1981. Evolution of host plant utilization in laboratory populations of the southern cowpea weevil, Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae). Evolution 35:605-617.

This experiment was written by C. Beck and L. Blumer (www.beanbeetles.org).

Copyright © by Lawrence S. Blumer and Christopher W. Beck, 2009. 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 website for detailed information on growing cultures and handling techniques, as well as tips on identifying the sexes.

The experiment requires having dense cultures of bean beetles from which females can be isolated. If new cultures are initiated approximately 2 months before the lab period, there will be sufficient time for two generations of beetles, which will result in dense cultures. When possible, we supply one culture to each pair of students. However, cultures should have sufficient beetles for multiple student groups.

Experimental Design

The questions that students generally address in their experiments are:

• Do females prefer to visit the bean species from which they emerged (natal bean species) when given a choice between the natal bean and another bean species on which they will lay eggs?
• Do females preferentially lay eggs on their natal bean species when given a choice between the natal bean and another bean species on which they will lay eggs?
• Does the identity of the other bean species influence the strength of female preference for her natal bean species?
• Does the identity of the natal bean species influence the strength of female preference for her natal bean species?

Note that in any experiment in which location preference is evaluated, some animals may prefer to move in one direction regardless of the treatment conditions. Students should consider the following questions in their experimental designs:

• How can you control for potential location bias?
• How will you measure whether a female prefers to visit one bean species over another?

Oviposition will readily occur during a 48-hour period when adult females are provided with single layer of beans in a small covered dish. Although most adult females in an active culture will have been inseminated, there are always some female that may have only recently emerged (and be infertile) and others that are near the end of their adult life (and laid most of their eggs). Students should consider the following questions in their experimental designs:

• How can you account for variation among females in the number of eggs they lay?
• If females lay eggs preferentially on their natal bean species, how will you detect that preference?

A sample data entry sheet is provided in the Downloads section as a potential guide for data collection. However, it is most appropriate to guide your students to design their own experiments, a guided inquiry process, rather than giving them explicit directions on how to conduct their experiments.

Data Collection

Location data may be in the form of the number of times each female was in a given location (mung, adzuki, or neutral zone) in a three-section arena. These data could be collected by starting an experiment and checking the location of a female at fixed time intervals, for example, every 2 minutes during a 30-minute trial. Alternatively, continuous observations could be made during a fixed period of time and the total time a female spent in each location would be calculated.

The actual number of eggs laid on each of two bean species during a 48-hour period could be evaluated in an oviposition preference experiment in which a female is presented with an equal number of mung and adzuki beans. These egg laying data do not need to be collected immediately after 48-hours but the females should be removed from the experimental arenas, so students can evaluate the initial bean species choices. The eggs are glued to the beans and will remain intact on the beans. Therefore, students may count the eggs one (or even two) weeks after the start of the oviposition experiment. A 48-hour period for egg laying ensures that sufficient numbers of eggs are laid.

Data Analysis

In the location preference experiment, if the data consist of number of times a female was present in each section of the arena, then the appropriate test is a chi-squared analysis. In this analysis, students would be comparing the observed location counts to the expected location counts if the females behaved randomly. If the location preference experiment were conducted with continuous time in each section data, then a two-sample t-test could be performed to evaluate whether there were differences between the two bean species in female preference. Because females can spend time in a neutral zone, the time spent in one section with beans is independent of the time spent in the other section with beans. Therefore, a two-sample t-test is more appropriate than a paired t-test.

The data from the oviposition preference experiment should be the number of eggs laid on each of the two bean species. The appropriate statistical analysis for the egg count data is a binomial test to determine whether one bean species received more eggs than the other for a given female. The difference in the average number of eggs on each bean species across replicates also could be compared with a paired t-test.

Equipment and supplies

For a class of 30 students working in pairs:

• 30 magnifiers 2.5x, 4” diameter self-standing with folding base or dissection microscopes
• 15 bean beetle cultures with newly emerged adults
• 15 plastic petri dishes to hold beetle cultures and for picking adults females from cultures
• mung beans, Vigna radiata, (16 ounces) dried beans, organically grown
• adzuki beans, Vigna angularis, (16 ounces) dried beans, organically grown
• 30 small paint brushes
• 30 soft forceps, BioquipTM featherweight forceps (Catalog No. 4748 or 4750)
• 30 petri dishes (plastic) for holding isolated beetles (35mm) and for conducting oviposition choice experiment
• 30 petri dishes (three-section) for bean location preference experiment (make caulk line sections in standard plastic 100mm dishes using clear aquarium caulk)
• 15 countdown timers or stopwatches
• marking pens

Copyright © by Lawrence S. Blumer and Christopher W. Beck, 2009. 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.

A location choice experiment consisting of 26 independent trials (different females) was performed by Morehouse College Ecology students. In each trial, the location of a female was recorded every two minutes during a 60 minute observation period, so a total of 30 observations were made on each female. Within each trial, the location of the natal beans was switched after the first 30 minutes. All trials were conducted in 100mm dishes divided in three equal area sectors, one sector was empty, one contained mung beans and the third contained adzuki beans. Preferences for the natal bean species were observed for females from both mung and adzuki beans (Figure 1). The raw data are available in the Downloads section.

Figure 1. Number of observations in which a female was on mung beans, adzuki beans, or in an empty sector during 60 minute observation periods. A total of 30 observations were made on each of 26 different females, 13 females whose natal bean was mung and 13 females whose natal bean was adzuki. The preference for the natal beans by females from mung beans was not significant (chi-squared = 5.4, df=2 p<0.067) but the preference for the natal beans by females from adzuki beans was highly significant (chi-squared = 44, df=2 p<0.0001).

When an individual female from mung beans was provided with equal numbers of beans from the natal and non-natal species (mung and adzuki), and allowed to lay eggs for 24-hours, a very clear preference was observed in the bean species on which eggs are laid (Figure 2). The raw data are available in the Downloads section.

Figure 2.  Mean number of eggs laid by a single bean beetle on mung and adzuki beans during a 24-hour period.  The means are significantly different (paired t-test, t=2.72 df=37 p=0.0099).  Each dish contained 10 beans of each bean species and one female beetle.  These data were provided by Calvin Greene II, a Morehouse College student.

Student Handout [pdf] [doc]

Instructor's Notes [pdf] [doc]

Sample data sheet [pdf] [doc]

Sample data [xls]

Sample data graphs (Figure 1) [ppt] (Figure 2) [ppt]

Identifying the sexes [ppt]

Egg on bean [ppt]