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.
• Design and conduct an experiment to evaluate the consequences of bean species choice by female bean beetles.

 

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).

 

Methods and Materials

Prior to class, design an experiment in which you could address the following question: Do female bean beetles choose prey beans randomly? Alternatively, do female bean beetles prefer some bean species and avoid others?

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 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:

• 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.
• Describe an ADDITIONAL experiment to evaluate the consequences of females laying eggs on different bean species.
• 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 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 pair of students. However, cultures should have sufficient beetles for multiple student groups.

 

Experimental Design

A similar exercise that is not inquiry-based is described in Brown and Downhower (1988), and the consequences of oviposition choice are limited to emergence success.

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?

For examining the consequences of oviposition choice, students generally propose to address the question of whether the host species affects offspring characteristics. The most challenging part of the experimental design for students is determining what offspring characteristics to measure. Below is a list of characteristics that can be measured in a reasonable time span.

• Time to emergence
• Size at emergence (either mass or body length)
• Emergence success

The quality of the data for emergence success will depend on the ability of students to identify eggs on beans. Students may suggest other offspring characteristics, such as lifespan, reproductive success, hatching rate, and sex ratio. Characters such as lifespan could be measured, but would add another two or more weeks to the experiment. Other dependent variables are appropriate, but difficult to measure (i.e., reproductive success and hatching rate). Finally, for other offspring traits like sex ratio, the predictions are not clear.

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

• What factors other than host type might affect offspring traits?
• How would you control for these factors?

 

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.

For the consequences of oviposition choice, one of the biggest confounding factors is the number of eggs laid on individual beans. If more than one egg is laid on a bean, then the larvae may compete for resources. Therefore, beans with single eggs should be used. Students may want to record the identity of the female that laid the egg to be able to consider differences among females in their analysis. However, data on female identity is not essential. Students can isolate beans of each species with single eggs into the wells of tissue culture plates or small Petri dishes. As the beetles emerge, students can record the offspring characteristics that they chose to measure. Accurate data on time to emergence and mass at emergence require that students check for emergence on a daily basis. As a result, measuring these life history traits may be feasible only in smaller, more advanced classes. Emergence success could be determined on a single day after sufficient time for emergence (approximately 40 days). Therefore, emergence success is more tractable for larger classes.

 

Data Analysis

The data from the ovipositition choice 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 may be conducted 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 with bean species as the factor and number of eggs as the dependent variable.

Most of the offspring traits that students measure will be continuous. Thus, ANOVA may be used to determine the effect of host species on offspring characteristics. For emergence success, the data would be the number of emerged and non-emerged beetles from each host species. Differences in emergence success could be determined using a chi-squared test.

 

Equipment and supplies

For a class of 30 students working in pairs:

• 30 magnifiers 2.5x, 4” diameter self-standing with folding base (Fisher #14-648-19 or VWR #62379-535) 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
• 35mm Petri dishes for holding individual beans OR flat-bottom tissue culture plates (6 or 12 well)
• 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 and beetle characteristics
• 0.1 mg analytical balance for weighing beans and newly emerged beetles

 

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.

The results reported below were collected William D. Shipman III, a Morehouse College undergraduate.

A fertilized female bean beetle was permitted to lay eggs on ten bean species, not including the natal bean species. The natal bean was mung (Phaseolus aureus) in this experiment. A total of 17 independent trials were conducted, each with one female. In each trial, 10 beans of each of the following were presented to a female: black-eyed peas (BEP, cowpea) (Vigna unguiculata), garbanzo (Cicer arietinum), kidney, pinto, navy, and black beans (Phaseolus vulgaris), soy beans (Glycine max), adzuki beans (Phaseolus angularis), lima beans (sieva bean) (Phaseolus lunatus), and green pea (Pisum sativum). Females were permitted to lay eggs for 96 hours before they were removed and the eggs were counted. The difference between bean types in the mean number of eggs laid per female was highly significant (ANOVA, F_9,160=15.1, p<0.0001, Figure 1). An alternative statistical analysis could be based on the frequencies of eggs (the total eggs counts) on each bean type (a chi-squared test). That analysis also indicated a highly significant difference between bean types in the number of eggs laid (X²=717.9, df=9, p<0.0001).

Figure 1. Number of eggs laid per female. The mean ± SE of eggs laid per female on each of 10 bean types is shown for the 17 independent trials conducted with females from mung beans. Each of the 17 replicates contained 10 beans of each of the 10 types, so these means are based on 170 beans of each type. The value shown at the top of each bar is the total number of eggs laid on each bean type.

The elapsed time from egg laying to the emergence of an adult (development time) and the mass of the newly emerged adult beetle was evaluated for each bean species on which eggs were laid. Beans with eggs were followed for a total of 40 days after egg laying to permit adequate time for adults to emerge. Adults only successfully emerged from adzuki beans and black-eyed peas (BEP) and the development time from black-eyed peas was significantly faster than from adzuki beans (ANOVA F_1,23=24.4, p<0.0001, Figure 2). There were no significant differences between the sexes in development time emerging from a given bean species (ANOVA F_1,23=0.5, p=0.49, Figure 2). The mass of newly emerged males and females was significantly greater for adults emerging from black-eyed peas compared to adzuki beans (ANOVA F_1,15=4.7, p<0.05, Figure 3).

Females had significantly greater mass at emergence than males (ANOVA F_1,15=6.1, p<0.03, Figure 3).

Figure 2. Development time of bean beetles emerging from adzuki beans and black-eyed peas (BEP). The mean ± SE development time (egg laying to emergence of adult) is shown for males and females emerging from the two bean species from which adults successfully emerged. Sample sizes are shown at the top of each bar.

Figure 3. Body mass of bean beetles emerging from adzuki beans and black-eyed peas (BEP). The mean ± SE mass at emergence is shown for males and females emerging from the two bean species from which adults successfully emerged. Sample sizes are shown at the top of each bar.

Student Handout [pdf] [doc]

Instructor's Notes [pdf] [doc]

Sample data [xls]

Sample data graphs [ppt]

Identifying the sexes [ppt]

Egg on bean [ppt]