Objective

• Design and perform a set of experiments to evaluate whether female bean beetles (Callosobruchus maculatus) discriminate between different size beans of the same species.

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 30°C, pupation and emergence of an adult beetle occurs 25-30 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 occurs.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.Supplies of organic mung of various sizes 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 size 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 different bean sizes within one bean species and the consequences of those choices.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 different bean sizes with one bean species.
• Describe an ADDITIONAL experiment to evaluate the consequences of females laying eggs on beans of different sizes.
• 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 Publishers.

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 study was written by C. Beck, S. Migabo and L. Blumer, 2010 (www.beanbeetles.org).

Copyright © by Christopher W. Beck, Saphida Migabo, and Lawrence S. Blumer, 2011. 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 “A Handbook on Bean Beetles, Callosobruchus maculatus” for detailed information on growing cultures, handling techniques, and methods of safe disposal (available for downloading at: http://www.beanbeetles.org/handbook). In addition, tips on identifying the sexes including pictures of a male and female are available at: http://www.beanbeetles.org/handbook/#IS.

The student handout is written as a guided inquiry that allows students to design their own experiments, rather than instructors giving students explicit directions on how to conduct their experiments. No matter the exact experiment that students design, the experiments will require 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 group of students. However, cultures should have sufficient beetles for multiple groups. Newly emerged cultures work better for this experiment than older cultures.

Instructors should caution students to prevent the accidental release of bean beetles from the laboratory environment. Callosobruchus maculatus is a potential agricultural pest insect that is not distributed throughout the United States and Canada. It is essential that you keep your cultures secured in a laboratory environment to ensure that they are not released to the natural environment. Disposal of cultures (and beans (seeds) exposed to live beetles of any life cycle stage) requires freezing (0°C) for a minimum of 72 hours prior to disposal as food waste. If you have any questions about the handling or disposal of bean beetles, please contact Larry Blumer at lblumer@morehouse.edu or 404 658-1142 (voice or FAX). Information also is available at: www.beanbeetles.org in the Handbook section.

Experimental Design

Our students have successfully conducted this experiment using both very short-exposure and long-exposure protocols. Students typically design experiments in which female beetles choose between two categories of beans and are permitted to lay eggs on those beans. The species of bean is the same but the quantity of the bean resource is different. For example, we have had students conduct experiments in which each replicate contained 15 whole mung beans and 15 split mung beans in a 60mm dish (or 10 of each category in a 35mm dish). A female and a male bean beetle were introduced to each dish and the female was permitted to lay eggs for a minimum of 24 hrs or as long as 7 days. Alternatively, students could sort whole mung beans (visually judging size) to create sets of large beans and small beans. A similar experiment with 15 large and 15 small beans per replicate could be conducted, and run for 24 hrs to 7 days. If similar numbers of beans of each size category were presented to a female, the experiment could be permitted to run until each female laid all her eggs and died (7-10 days from adult emergence). The difference in the quantity of the bean resource may be documented at the start of an experiment by weighing the beans in each size category or measuring beans with vernier calipers. In all experiments, the data collected are the number of eggs laid on beans in each category of size.

This experiment may be readily modified for more advanced students by using it as an alternative means of studying intra-specific competition. Beck and Blumer (Intraspecific Competition in Bean Beetles) previously developed a competition protocol in which female beetles chose between beans with or without an egg from another female, so females were choosing based on the number of competitors already present. In the current study, female beetles would choose between beans based on differences in the resources actually available, not the number of potential competitors. The experiment could be elaborated by using other bean species (such as black-eye peas or adzuki beans) and by evaluating differences in successful development and adult body size for eggs laid on different size beans.

Data analysis—The total number of eggs laid on each of the two size categories may be analyzed using a binomial test. This is more appropriate than a chi-squared test since there are only two categories in a given experiment. Alternatively, the mean number of eggs laid on each bean size category per replicate may be evaluated using a paired-t test or a non-parametric pairwise test (Wilcoxon sign-rank test). These statistical tests are easily performed using the on-line VassarStats website: http://vassarstats.net.

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
• 60 plastic 60mm Petri dishes for new cultures
• Plastic 35mm Petri dishes for isolating adults (minimum 60, but reused)
• 32 ounces each of dried mung beans and split mung beans
• 30 small paint brushes
• 30 soft forceps, BioquipTM featherweight forceps (Catalog No. 4748 or 4750)
• 0.1mg analytical balance for weighing beans (more than one balance would permit more efficient data collection by students)
• 30 vernier calipers for measuring bean size
• permanent markers for labeling Petri plates

Copyright © by Lawrence S. Blumer and Christopher W. Beck, 2011. 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.

The following data were collected by students in the non-majors Science and Society course at Morehouse College in Fall 2009. A total of 26 replicates of 15 whole and 15 split mung beans were conducted. Each replicate contained one female and one male bean beetle and egg laying was permitted for 48 hours. The average per bean mass of 15 whole beans was 0.060g and that of the split beans was 0.028g.There were significantly more eggs laid on whole beans (260) compared to split beans (26) (binomial test, p<0.000001) with an average per replicate of 10 eggs on whole beans and only 1 egg on split beans (paired t-test, t=4.22 df=25 p=0.0003) (Figure 1). A total of 16 replicates of 15 large and 15 small (all whole beans) also were conducted. The average per bean mass of 15 large beans was 0.071g and that of the small beans was 0.054g.Again, there were significantly more eggs laid on large beans (169) compared to small beans (130) (binomial test, p=0.027) with an average per replicate of 10.6 eggs on large beans and only 8.1 eggs on small beans (paired t-test, t=3.48 df=15 p=0.003) (Figure 1). Note that split beans differ from whole beans in both size and quality, which likely accounts for the much greater discrimination by female bean beetles.

Figure 1. Female discrimination of bean size in short exposure experiments.The mean+SE of the number of eggs laid by female bean beetles per replicate are shown for 16 replicates containing 15 large and 15 small mung beans, and 26 replicates of 15 whole and 15 split mung beans.The differences in eggs laid on large versus small and whole versus split were significant (paired t-tests, p<0.05 for both comparisons).

A long-exposure protocol experiment was conducted by students at the University of Northern British Columbia in Spring 2010. A total of 76 replicates were prepared that consisted of 10 whole and 10 split mung beans on which a single inseminated female was permitted to lay eggs until she died.The average per bean mass of the whole beans was 0.059g and that of split beans was 0.036g in this experiment.As in the short-exposure experiment, there was a significant bias toward whole beans.The bias was highly significant in this long-exposure experiment with more eggs laid on whole beans (2020) than on split beans (151) (binomial test, p<0.000001).The average number of eggs per replicate laid on whole beans was 27 while on split beans it was 2 (paired-t test, t=23.8 df=75 p<0.0001) (Figure 2). A total of 76 replicates of 10 large and 10 small (all whole beans) also were conducted.The average per bean mass of 10 large beans was 0.089g and that of the small beans was 0.037g.There was a highly significant bias by females toward larger beans as there were significantly more eggs laid on large beans (1786) compared to small beans (860) (binomial test, p<0.000001) with an average per replicate of 23.5 eggs on large beans and only 11.3 eggs on small beans (paired t-test, t=12.67 df=75 p<0.0001) (Figure 2).

Figure 2. Female discrimination of bean size in long-exposure experiments.The mean+SE of the number of eggs laid by female bean beetles per replicate are shown for 76 replicates containing 10 large and 10 small mung beans, and 76 replicates of 10 whole and 10 split mung beans. The differences in eggs laid on large versus small and whole versus split were highly significant (paired t-tests, p<0.000001 for both comparisons).

Student Handout [pdf] [doc]

Instructor's Notes [pdf] [doc]

Sample data [xls]

Sample data graphs [ppt]

Identifying the sexes [ppt]

Egg on bean [ppt]