Objectives

• Design and perform a set of experiments to evaluate whether female bean beetles (Callosobruchus maculatus) discriminate between two suitable species of beans.
• Design and conduct an experiment to evaluate the consequences of bean species choice by female bean beetles.

 

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 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.
• Describe an additional experiment to evaluate the consequences of females laying eggs on 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, 2009-2010. 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?

This experiment may be used as a follow-up to an experiment on oviposition substrate choice (Oviposition substrate choice by bean beetles), in which students determine on which species of beans, other than their natal host, females will lay eggs. The results from that experiment can be used to determine the alternate bean species for this experiment. Different groups of students in the class may want to carry out the same experiment with different alternate bean species and then compare their results. Different groups of students also could use beetles that are from different 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.

For examining the consequences of natal choice, students generally propose to address the question of whether the host species (natal or non-natal) 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

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.

For the consequences of oviposition choice, one of the biggest confounding factors is the number of eggs laid on beans. If more than one egg is laid on a bean, then the larvae will 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. Consequently, measuring these life history traits may be feasible only in smaller, more advanced classes. Linear measures of body size (such as elyta length) or emergence success could be determined on a single day after sufficient time for emergence (approximately 40 days). Therefore, body size and emergence success variables are more tractable for data collection in larger classes.

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.

Most of the offspring traits that students measure will be continuous. As a result, t-tests can be used to determine the effect of natal versus non-natal host species on offspring characteristics. For emergence success, the data would be the number of emerged and non-emerged beetles from each host species type. Differences in emergence success could be determined using a chi-squared test or Fisher’s exact 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 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) (Falcon 351008) 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
• 35mm Petri dishes for holding individual beans OR flat-bottom tissue culture plates (6 or 12 well)
• 0.1mg analytical balance for weighing beetles
• 30 vernier calipers for measuring beetle size or inexpensive microscope eyepiece video cameras (Moticam 352, Carolina Biological 591282) that permit image capture and measurement on a computer via USB connection

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

A fertilized female was permitted to choose between laying eggs on mung beans or adzuki beans in this experiment. A total of 14 independent trials were conducted in which 10 beans of each species were presented to a different female that had emerged from mung beans. Females were allowed 96 hours to lay eggs before being removed. The mean number of eggs laid per female on adzuki beans was significantly greater than on mung beans (paired t-test, t=2.602, df=13, p=0.02, Figure 1). An alternative statistical analysis could be based on the frequencies of eggs (the total eggs counts) on each bean species. That analysis also indicated a highly significant difference between bean species in the number of eggs laid (binomial test, p<0.001).

Figure 1. Number of eggs laid per female. The mean ± SE of eggs per female laid on either adzuki or mung beans is shown for the 14 independent trials conducted with females from mung beans. Each of the 14 replicates contained 10 beans each of these two species, so these means are based on 140 beans of each species. The value shown at the top of each bar is the total number of eggs laid on each bean species.

The elapsed time from egg laying to the emergence of an adult (development time) and the mass of the newly emerged adult beetle also was evaluated for the two bean species in this experiment. Beans with eggs were followed for a total of 40 days after egg laying to permit adequate time for adults to emerge. The development time from mung beans was significantly faster than from adzuki beans (ANOVA F[1,73]=116.9, p<0.0001, Figure 2). There were no significant differences between the sexes emerging from a given bean species. The mass of newly emerged males and females was significantly greater for those adults emerging from mung beans compared to adzuki beans (ANOVA F1,73=9.9, p=0.002, Figure 3). Females had significantly greater mass than did males (ANOVA F[1,73]=22.3, p<0.0001) and there was a significant interaction between bean and sex on emergence mass (ANOVA F[1,73]=5.7, p=0.02, Figure 3). Although the number of eggs on, and therefore larvae in, a given bean can influence development time and adult mass at emergence, there were no consistent significant relationships between either development time or mass and the number of eggs laid on a given bean for either males or females. This suggests that these results are not confounded by competition between larvae in beans that received more than one egg.

Figure 2. Development time of bean beetles emerging from adzuki and mung beans. The mean ± SE development time (egg laying to emergence of adult) is shown for males and females emerging from these two bean species. Sample sizes are shown at the top of each bar.

Figure 3. Body mass of bean beetles emerging from adzuki and mung beans. The mean ± SE mass at emergence is shown for males and females emerging from these two bean species. Sample sizes are shown at the top of each bar.

Student Handout [pdf] [doc]

Instructor's Notes [pdf] [doc]

Sample data sheet [pdf] [doc]

Sample data [xls]

Sample data graphs (Figures 1-3) [ppt]

Identifying the sexes [ppt]

Egg on bean [ppt]