Objectives

• Design and perform a set of experiments to observe the impact of limited resources on female bean beetle (Callosobruchus maculatus) egg laying behavior.

Introduction

The availability of resources is an important consideration when studying the reproductive behaviors of animals. When environmental conditions result in limited resources, juvenile growth and offspring survival may be poor due, at least in part, to maternal and/or offspring competition for those resources. One aspect of reproduction that can be altered when the availability of resources changes is the amount of parental resources (metabolic energy and/or time) invested in each offspring. This investment can be crucial for the reproductive success of the animal by enhancing or reducing the survival of the offspring. Many studies in seabirds have shown that secondary offspring are only produced and nurtured to maturity when food resources are abundant. In food limited situations, seabirds, such as Nazca booby, only produce one fledgling per breeding season (Clifford and Anderson 2001).

Numerous studies on insects have examined how resource allocation and reproductive output differ under optimal and food limited scenarios. For example, Boggs and Freeman (2005) have shown that female Speyeria mormonia butterflies have smaller body size, forewing length, and decreased survival if they are food-limited as larvae. Similarly, early work by Smith and Fretwell (1974) predicted that where juvenile growth and/or survival are poor, larger propagules (e.g. eggs or seeds) would be produced.

In this laboratory exercise, you will design and implement an experiment examining the effects of limited resources on egg production in the bean beetle, Callosobruchus maculatus.

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 beans and 60mm petri dishes will be available. Dissecting scopes can be used for identifying the sex of the beetles. You will also be provided with a Motic camera and laptop computer to photograph and measure the size of eggs laid. Your laboratory instructor will demonstrate the use of the Motic camera and program to you.

Experimental Design

Since larval bean beetles have no means of moving out of the bean they are living in, choices made and resources provided by their mothers greatly influence their survival and future success. We know that resource availability potentially impacts the amount and size of eggs laid by female bean beetles. Given this, female bean beetles may be very sensitive to the species and condition of the beans on which they are depositing eggs, including whether those beans already have an egg on them. As a class, we will discuss possible experimental approaches to examine whether female bean beetles change the number, size, or some other aspect of how they lay eggs under situations where resources are abundant or limited. We will design an experiment as a class so that enough data can be generated for statistical analysis.

In groups of 4 discuss the following:

• What resources could influence female egg laying behavior if limited?
• Describe at least TWO experimental designs for evaluating how female bean beetles' egg laying behavior differs under scenarios with abundant and scare resources.
• 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.

Be prepared to discuss your group's ideas with the class.

Set up the experiment designed by the class. Once your cultures of beans and beetles are ready, incubate the cultures at 30°C. During next week's lab we will collect and analyze the data.

Data Collection and Analysis

Observe your bean beetle cultures and record all data in your lab manual. We will pool the class data for class discussion and statistical analysis.

From each of your cultures:

1. Observe any patterns of oviposition. How many eggs are in the cultures? Are there more than one egg per bean? How do the different cultures compare?
2. Randomly select 5 eggs from each culture and measure the length and width of each egg using the Motic camera system.

Laboratory Discussion

Using the class data write a brief discussion of the data including the following:

1. How did female bean beetles respond to your experiment?
2. Conduct a t-test on the class's results. Do you see differences between your class's experimental and control groups? Are these differences statistically significant? Discuss the meaning of your statistical findings.
3. Discuss three ways to refine your experiment to get at more specific questions of bean beetle responses to population size and/or resource limited situations.

Literature Cited

Boggs, C.L. and Freeman, K.D. 2005. Larval food limitation in butterflies: effects on adult resource allocation and fitness. Oecologia 144:353-361.

Clifford, L.D. and Anderson D.J. 2001. Food limitation explains most clutch size variation in the Nazca booby. Journal of Animal Ecology 70: 539-545.

Smith, C.C. and Fretwell, S.D. 1974. The optimal balance between size and number of offspring. American Naturalist 108:499-506.

 

This study was written by M. Byrne and K. Heiman, 2010 (www.beanbeetles.org).

Copyright © by Mary Byrne and Kimberly Heiman, 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 the Bean Beetle Handbook for detailed information on bean beetle culture, handling techniques, and tips for how to identify the two sexes.

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 animals 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 healthy, well-populated cultures. When possible, we supply one culture to each group of students. Cultures should have at least 25 adults, so they provide enough male and female adults for student cultures. 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 the laboratory environment to ensure that they are not released to the natural environment. Disposal of cultures (and beans 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.

Experimental Design

In this laboratory exercise, students will consider the impact of limited resources on reproductive output and egg laying strategies. Following classroom and student group discussions the students designed a class experiment to test how the female egg laying behavior would be affected by limited resources.

Although this laboratory could be done without any introductory laboratory exercises, we used this lab as part of a three week module of labs using bean beetles. In addition to observing animal behavior, the three labs introduced students to experimental design, data collection and data analysis.

• During week one the student were introduced to the experimental process specifically focusing on the design and setup of experiments to test natal bean discrimination by bean beetles.
• In week two, the data from week one's experiments were collected, analyzed and discussed. Each group presented their findings to the class. Class discussion on egg laying strategies and nutrient resources availability followed with development of the experiments described here.
• Week three included data collection and statistical analysis of week two's data. Class discussion followed. Students were also given a writing assignment where they were asked to discuss the data from the three weeks and propose future experiments.

This laboratory exercise could easily be modified for more advanced students. In this study, female beetles were studied to see if egg laying strategies were modified when resources were limited. Experimental design could be modified by using other bean species, by including close observation of female behavior in the experimental protocol, by varying the number of females in different treatment groups to examine competition for resources or by following the development of the embryos from different sized eggs.

Data collection—Students typically designed experiments using 2 bean beetle cultures with bean numbers ranging from 5 to 200 beans. The students should use only the natal bean, in our case mung beans (Vigna radiata), for these experiments. The same number (students typically used 2-3 each) of female and male beetles were added to the bean cultures and allowed to incubate for seven days. Cultures were placed in 60mm petri dishes. The cultures were incubated at 30†C for 1 week, so that development and set-up of this experiment happened the first week and data collection and analysis happened the second week of lab. The entire class decided on the specifics of the experiment and set up the same experiment so there was sufficient replication of data for statistical analysis of the results.

During the second week of the lab, students were provided with a dissection microscope and a Motic video camera with software on an accompanying laptop that allows the student to measure egg size from captured images. The data collected by the students included total number of eggs laid and the width and diameter of the eggs in each culture. To facilitate measurement of eggs during the laboratory time, students chose 5 eggs from each culture for measurement and reported the average egg size to the class.

For viewing the eggs laid on beans, students used a dissecting scope with illumination from above only. The eggs are easily viewed at 10x total magnification.

Consult the Motic manual on the use of the camera and accompanying program. Note that you may have to install a video card on the computers to allow for image capture. The Motic camera comes with a number of adaptors that allow the camera to be installed on the ocular of most microscopes. We found that newer student microscopes with wide ocular pieces required the purchase of larger adaptors from Motic adding to the cost of equipment.

The measurement of captured images requires calibration. We made sure that all the student computers were calibrated prior to lab and reminded the students to work at the set total magnification of 10x.

We first demonstrated to the students how to capture an image of the eggs and the procedure for measuring egg width and length. Students had no difficulty using the program. It is important to point out to the students that when capturing the image that they make sure each egg is in full view so that accurate measurements of egg dimensions can be taken. This may require moving and holding the bean in position while capturing the image.

Data analysis—One of the objectives of this laboratory was to introduce the use of statistical analysis to introductory biology students. Using egg size and the number of eggs laid from each experimental treatment group, class data were pooled and the data were analyzed using ANOVA and t-tests.

 

Equipment and supplies

For a class of 30 students working in pairs:

• 15 Dissection microscopes equipped with Motic Cameras (Carolina Biological, Catalog Number NP-59-1282) and 15 computers to run the Motic software.
• 15 bean beetle cultures with newly emerged adults
• 60 Plastic 60mm petri dishes for student cultures
• 64 ounces of dried mung beans (organic)
• 30 soft forceps, BioquipTM featherweight forceps (Catalog No. 4748 or 4750)
• Permanent markers for labeling Petri plates

The bean beetle cultures provided to the students were placed in 150mm petri dishes to allow for easy access to the beetles.

Copyright © by Mary Byrne and Kimberly Heiman, 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.

When fewer beans were available (5 or 10 bean versus 100 or 200) we observed (1) an increase in the number of beans with multiple eggs, and (2) larger egg widths. However, there were no changes in the total number of eggs laid nor the length of each egg as bean numbers increased.

 

Figure 1. The total number of eggs laid by female bean beetles did not change as the a function of the number of beans available. Each replicate had three females and three males. There was no significant difference in the total number of egg laid between cultures with different numbers of bean (ANOVA: F(4, 29) = 0.3471, p = 0.844). Note that the number of multiple eggs on beans in different bean number treatments was not collected, so these data were not available for analysis.

 

Figure 2. The average length of eggs laid by bean beetles did not change as a function of the number of beans available. Each replicate had three females and three males. There was no significant differences between the cultures with different numbers of beans (ANOVA: F(3,39)=1.184, p= 0.330).

 

Figure 3. The average width of eggs laid by bean beetles decreased as the number of beans available increased. Each replicate had three females and three males. There was a significant differences between cultures with different numbers of beans (ANOVA: F(3, 39)=10.22, p=0.0001).

Student Handout [pdf] [docx]

Instructor's Notes [pdf] [docx]

Sample data [xlsx]

Sample data graphs [pptx]

Identifying the sexes [pptx]

Egg on bean [pptx]