Objectives

• Evaluate characteristics of the prey (beans) of bean beetles, Callosobruchus maculatus, that could influence prey choice.
• Design and perform an experiment to determine whether female bean beetles distinguish among prey species.

Introduction

Bean 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. Larval growth and pupation occur inside the bean and are consequently difficult to observe. 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 7-10 days 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. The choice of prey bean is the most critical choice a female makes for her offspring, as it will influence their growth, survival, and future reproduction (Mitchell, 1975; Wasserman and Futuyma, 1981).

Materials

In class, you will be provided with live bean beetle cultures and supplies of dried beans (seeds) from a variety of species. The seeds available for this experiment will include: mung beans (Phaseolus aureus), black-eyed peas (cowpea) (Vigna unguiculata), garbanzo (Cicer arietinum), kidney, pinto, and black beans (Phaseolus vulgaris), soy beans (Glycine max), adzuki beans (Phaseolus angularis), urad beans (black gram) (Vigna mungo), fava beans (horsebean) (Vicia faba), lima beans (sieva bean) (Phaseolus lunatus ), and green pea (Pisum sativum). 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 determine whether female bean beetles discriminate among bean species. 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:

• List characteristics of bean species that might be important to a female bean beetle, and how you would measure these characteristics.
• Describe an experimental design for evaluating whether female bean beetles discriminate among different bean species.
• 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.

Literature Cited

Brown, L. and J.F. Downhower. 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 D.J. Futuyma. 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 L. Blumer and C. Beck (www.beanbeetles.org) and is based on an experimental protocol originally published by Luther Brown and Jerry F. Downhower (Brown and Downhower, 1988).  

Copyright © by Lawrence S. Blumer and Christopher W. Beck, 2008. 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 group of students. However, cultures should have sufficient beetles for multiple groups.

Experimental Design

Questions that students generally address in their experiments include:

• Do females prefer to lay eggs on a particular species of bean?
• Do females actively avoid laying eggs on a particular species of bean?
• Is oviposition substrate choice by females determined by the size of a species of bean?

In their experimental designs, students should consider the following questions:

• How would you control for female preference for the bean species from which she emerged?
• How would you control for the possibility that females will lay their eggs on the first species of bean they encounter?

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 females 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 a particular bean species, how will you detect that preference?

Data Collection

The actual number of eggs laid on each of ten 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 each bean species. In this experiment, we do not use the natal bean species to control for a bias toward that species. Generally, about 10 beans of each species in a 150mm Petri dish are appropriate. If the beans are randomly arranged throughout the dish, females will be equally likely to encounter each bean species. 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

The data from the experiment should be the number of eggs laid on each bean species. The appropriate statistical analysis for the egg count data is a chi-squared test to determine whether the distribution of eggs on the bean species differed from random. The null hypothesis is that females will lay an equal number of eggs on each bean species. The chi-squared test can be carried out for individual replicates or for all replicates pooled. The difference in the average number of eggs on each bean species across replicates also could be compared with a one-way ANOVA (or the non-parametric Kruskal-Wallis test) with bean species as the factor and number of eggs as the dependent variable.

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 150mm Petri dishes for picking adults females from cultures
• 30 plastic 150mm Petri dishes for each replicate of the oviposition substrate choice experiment
• 30 plastic 35mm Petri dishes for holding isolated beetles
• 16 ounces of each the following bean species, dried and organically grown, if possible: mung beans, adzuki beans, black-eyed peas, garbanzo, kidney, pinto, black beans, soy beans, urad beans, fava beans, lima beans, and green pea
• 30 small paint brushes
• 30 soft forceps, BioquipTM featherweight forceps (Catalog No. 4748 or 4750)
• 30 vernier calipers for measuring bean characteristics
• 0.1 mg analytical balance for weighing beans

This experiment is based on an experimental protocol originally published by Luther Brown and Jerry F. Downhower (Brown and Downhower, 1988).

Copyright © by Lawrence S. Blumer and Christopher W. Beck, 2008. 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.

Oviposition preference experiments were performed by Morehouse College Ecology students in 2000 and 2001. In each trial, a female was placed in a petri dish with 10 beans from each of a variety of species (10 species in 2000 and 12 species in 2001) not including the bean species from which that female had emerged (the natal bean species). Females had been reared on either black-eye pea or mung beans. Fifteen independent trials (different females) were carried out in 2000 and 21 trials in 2001. All trials were conducted in 150mm petri dishes.

In 2000, the total number of eggs laid differed significantly among bean species for females reared on black-eye pea (chi-square = 181.7, df=9, P<0.0001) and for females from mung beans (chi-square = 412.2, df=9, P<0.0001) (Figure 1a). In addition, the total number of beans with eggs varied among different bean species for females from black-eye pea (chi-square =145.6, df=9, P<0.0001) and for females from mung beans (chi-square =161.3, df=9, P<0.0001). However, the pattern of oviposition did not differ based on maternal host (chi-square =2.7, df=7, P=0.91) (Figure 1a). Both maternal hosts were removed from this last analysis and lentils also were removed, because no female ever laid eggs on lentils. The average number of eggs varied among bean species for females from black-eye pea (Kruskal-Wallis chi-square=39.0, df=9, p<0.0001) and for females from mung beans (Kruskal-Wallis chi-square=35.0, df=9, p<0.0001) (Figure 1b). The raw data are available in the Downloads section.

 

Figure 1a. Total number of eggs laid by individual females on 10 species of beans other than the natal bean species. A total of 15 independent trials were performed (different females) in 2000. Each replicate evaluated the oviposition choices of a given females on ten beans of each bean species.

Figure 1b. The average number of eggs (per replicate) laid by individual females on 10 species of beans other than the natal bean species. A total of 15 independent trials were performed (different females) in 2000. Each replicate evaluated the oviposition choices of a given females on ten beans of each bean species.

In 2001, similar results were observed. The total number of eggs laid differed significantly among bean species for females from black-eye pea (chi-square = 301.3, df=11, P<0.0001) and for females from mung beans (chi-square = 149.8, df=11, P<0.0001) (Figure 2a). The total number of beans with eggs also varied among different bean species for females from black-eye pea (chi-square =193.9, df=11, P<0.0001) and for females from mung beans (chi-square =103.7, df=11, P<0.0001). Unlike in 2000, the pattern of oviposition did differ based on maternal host (chi-square =44.3, df=10, P<0.0001) (Figure 2a). Both maternal hosts were removed from this last analysis. The average number of eggs varied among bean species for females from black-eye pea (Kruskal-Wallis chi-square=57.1, df=11, p<0.0001) and for females from mung beans (Kruskal-Wallis chi-square=40.0, df=11, p<0.0001) (Figure 2b). The raw data are available in the Downloads section.

Figure 2a. Total number of eggs laid by individual females on 12 species of beans other than the natal bean species. A total of 21 independent trials were performed (different females) in 2001. Each replicate evaluated the oviposition choices of a given females on ten beans of each bean species.

Figure 2b. The average number of eggs (per replicate) laid by individual females on 12 species of beans other than the natal bean species. A total of 15 independent trials were performed (different females) in 2001. Each replicate evaluated the oviposition choices of a given females on ten beans of each bean species.

Student Handout [pdf] [doc]

Instructor's Notes [pdf] [doc]

Sample data [xls]

Sample data graphs [ppt]

Identifying the sexes [ppt]

Egg on bean [ppt]