One of the most important decisions a female makes is how much energy to invest in reproductive parameters such as egg size and clutch size. A factor often constraining maternal investment in offspring is the nutritional status of the parent. This laboratory activity investigates how a female bean beetle's nutritional history (that is, the type of bean in which she was raised) influences the investment she makes in her offspring. Before the lab, students look up the nutritional content of two different bean types (mung beans and blackeye peas). With the help of the instructor, students develop a hypothesis regarding the effect of maternal nutritional history on the amount of energy invested in her offspring, develop an experimental procedure for testing their hypothesis, perform the experiment, and analyze the data using appropriate statistical tests. A typical student experiment involves (1) rearing virgin female and male bean beetles in mung beans and blackeye peas, (2) allowing mung bean-raised females to mate with mung bean-raised males and blackeye pea-raised females to mate with blackeye pea-raised males, (3) placing the resulting inseminated females on mung beans for a specified period of time, (4) measuring the sizes and numbers of eggs produced by the females, and (5) comparing the data from mung bean-raised females with the data from blackeye pea-raised females. In a postlab discussion, conclusions are drawn from the data, implications of the conclusions are discussed, and directions for future research are proposed.
Topic: Reproductive Physiology
Level: Intermediate or upper-level majors
Class time:
- Discussion (either in lecture or in a lab period prior to the one in which the experiment is designed) of the natural history of bean beetles and of parental reproductive investment strategies (50 minutes)
- Laboratory period in which beetles are mated and placed on new beans (75 minutes)
- Second laboratory period in which egg number data (75 minutes) and/or egg size data (90 minutes) are gathered
- Follow up discussion (either in lecture or in a subsequent lab period) (20 minutes)
Karin Gastreich and Gregory Fitch
Department of Biology, Avila University, Kansas City, MO 64145
Objectives
- Design and complete an experiment testing the relationship between nutritional status and parental investment.
- Based on natural history observations, develop original hypotheses and testable predictions regarding mate choice and assessment in bean beetles.
Introduction
One of the most important decisions an animal makes is how much energy to invest in its offspring (see Alcock, chapter 12). Parental investment can take many forms, including egg size and clutch size (in females), provision of spermatophores or other sources of nutrition to a mate (in males), and care of the offspring after they have hatched (for both males and females). In general, parental investment in offspring is constrained by many factors, including nutritional status of the parent. Parental investment is also subject to trade-offs, in that resources invested in offspring cannot be used for other activities, such as finding more food or another mate.
In this lab, we will investigate parental investment by the female bean beetle Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae). Because their larvae feed exclusively on beans, these beetles are agricultural pests that affect staple foods in tropical and subtropical environments (Beck and Blumer 2009). Adults do not feed, and spend their short lifespan mating and laying eggs. Females have two potential sources of energy to invest in egg laying. One, they have the resources they captured while feeding as larvae on their host bean. Two, they receive a 'spermatophore' from the male that may provide additional nutrition.
There is no extended parental care in bean beetles, so the sum total of a female's investment in her offspring can be assessed via her oviposition (egg laying) behavior: Where does she lay her eggs? How many does she lay? How big are they? How long does she spend looking for a place to lay them? These are all questions we might look at when assessing maternal investment in female C. maculatus. In this experiment, we will look at how a female's nutritional history affects her maternal investment.
Before coming to class
- Review the handout on Callosobruchus maculatus (Beck and Blumer 2009) for basic natural history information about the beetle.
- We can readily raise bean beetles on mung beans, blackeye peas, and adzuki beans. Come to class with a written summary of the nutritional content of these beans.
- Come to class ready to discuss how we could vary the nutritional history of female bean beetles, and how we might measure maternal investment.
Experimental Methods
We will develop our experimental protocol as a group. However, here are some basics on handling bean beetles with which you should be familiar before coming to class.
Preparing Virgin Adults
Prior to the lab period, virgin females and males will be raised on two kinds of beans using the following protocol:
a) Place 150 mung beans in a large Petri dish. Place 150 blackeye peas in a different large Petri dish. Place 15 female bean beetles from an existing culture (maintained on mung beans) into each of the two Petri dishes. Allow the beetles to remain on the beans and lay eggs for one week.
b) At the end of one week, remove the beetles. Discard any beans bearing no eggs.
c) Place each egg-bearing bean into a separate well of a tissue culture plate. Use one tissue culture plate (or one set of plates) for mung beans, and a different tissue culture plate for blackeye peas. Allow adults, all of which will be virgins, to emerge from these beans. Both females and males will be used in the experiment.
Virgin adults used in this experiment will be 3 to 5 days old.
Observations of Mating Behavior
Regardless of our experimental protocol for assessing maternal investment, as part of this activity we will observe mating behavior in the beetles, using the following protocol:
a) Place one virgin female in a small Petri dish. Be sure to keep track of whether the female developed in a mung bean or in a blackeye pea by labeling the small Petri dish.
b) Place one male into each small Petri dish containing a virgin female. Observe and/or film each pair of beetles until they mate. Make detailed notes regarding your qualitative assessment of the mating behavior.
Measuring Maternal Investment
We will determine the details of our experimental protocol in class, but as you think about the experimental design you will want to keep in mind the following:
a) Should we use just one kind of bean? If yes, which one: mung or blackeye peas?
b) How many beans should we provide for each female?
c) How should we measure 'egg size'?
d) What kind of statistical comparison do you think we can use to compare the two groups?
Literature Cited
Alcock, John. 2005. Animal Behavior, Eighth Edition. Sinauer Associates, Inc. Sunderland, MA. pp405-435.
Beck, Christopher W. and Lawrence S. Blumer. 2009. A Handbook on Bean Beetles, Callosobruchus maculatus. www.beanbeetle.org
This study was written by K. Gastreich and G. Fitch, 2010 (www.beanbeetles.org).
Copyright © by Karen Gastreich and Greg Fitch, 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.
Introduction
This lab was designed as part of an upper level Animal Behavior class for biology majors. It complements a general unit on parental care and parental investment. By the time the lab is introduced, students have learned basic tenets of parental investment theory, and they have reviewed some examples of parental investment trade offs in animals other than bean beetles. The purpose of the lab is to apply student understanding of parental investment to the specific case of maternal investment in bean beetles.
Pre Lab Preparation by the Instructor
This lab involves some care and time in its preparation, since sufficient females of egg laying age that have been raised on two kinds of beans must be available by the time the students begin the lab.
Raise virgin females on two kinds of beans using the following protocol:
(a) Place 150 mung beans in a large Petri dish. Place 150 blackeye peas in a different large Petri dish. Place 15 female bean beetles from an existing culture (maintained on mung beans) into each of the two Petri dishes. Allow the beetles to remain on the beans and lay eggs for one week.
(b) At the end of one week, remove the beetles. Discard any beans bearing no eggs.
(c) Place each egg-bearing bean into a separate well of a tissue culture plate. Use one tissue culture plate (or one set of plates) for mung beans, and a different tissue culture plate for blackeyed peas. Allow adults, all of which will be virgins, to emerge from these beans.
Introducing Students to the Lab
Prior to the lab, review with students general factors that can affect parental investment for both males and females. Have student consider the question of how parental investment - specifically, maternal investment - can be measured.
Present the natural history of bean beetles in class and use this as a launching pad for a general discussion of the specifics of parental investment in bean beetles. Have students work in groups to identify factors that might affect bean beetle female investment in egg or clutch size. Develop a class list of these factors, focusing on nutrition and nutritional history as the focus of this lab.
Pre Lab Assignment for the Students
In addition to reading the introduction in the lab handout, students must complete a brief literature report on the nutritional/caloric content of mung beans versus blackeye peas. The instructor may also request research on other topics relevant to the lab, including bean beetles, egg production and parental investment.
In Lab Preparation for Students
Work with the students to develop a clear statement of the hypothesis, such as the following:
If nutritional state influences the number and size of eggs in bean beetles, then females with different nutritional history will differ in the size and number of eggs they lay.
Based on the background research performed by the students prior to the lab, develop a testable prediction. For example:
Females raised on mung beans will lay more/larger eggs than females raised on blackeye peas.
Develop experimental materials/methods and experimental procedures with students. Key questions that need to be addressed as part of setting up the experimental design include:
- Should we use just one kind of bean as the substrate for new eggs laid by females with different nutritional histories? If yes, which one: mung or blackeye peas?
- How many beans should we provide for each female?
- How should we measure 'egg size'?
- What kind of statistical comparison do you think we can use to compare the two groups?
In our classroom, we used only mung beans as the new egg substrate, placing 50 beans with each female. Egg size was estimated by measuring egg length and width using a Moticam 352. After mating, females were allowed to lay eggs for 2 days before eggs were counted and measured. In addition, students decided to mate virgin females with males raised on the same kind of bean (e.g., mung females with mung males; blackeye pea females with blackeye pea males).
Sample Experimental Protocol Designed by the Class:
Two to five days after the emergence of adult females:
(a) Place each virgin female obtained from the prior step into a small Petri dish containing 50 mung beans. (Be sure to keep track of whether the female developed in a mung bean or in a blackeyed pea by labeling the Petri dish.)
(b) Place one male raised on the same type of bean as the female into each small Petri dish containing a virgin female. Observe and/or film each pair of beetles until they mate. Make notes regarding your qualitative assessment of the mating behavior.
(c) Allow females two days for egg laying. Count the total number of eggs laid by each female by observing the entire surface area of each bean.
(d) To gather data regarding egg size, measure the length and width of ten eggs (chosen randomly) for each female. These measurements will require the use of a camera system (such as a Moticam 352 camera) mounted on a microscope together with software such as Motic. Capture and save images of each of ten randomly chosen eggs from each female. Determine the size of each egg by measuring the width and length of the egg. These measurements can be used to estimate the area of the egg by assuming the egg is an ellipse and using this formula: area = pi(0.5*length)(0.5*width). For each female, determine mean egg size by taking the average of the ten individual egg sizes.
Data Analysis for the Sample Experimental Protocol:
- For the group of females that developed in mung beans, find the mean number of eggs produced per female. Also, use the data from each individual female about mean egg size to determine the mean egg size produced by this group of females.
- Repeat using data from the females that developed in blackeyed peas.
- Use a t-test to determine if the difference (if any) between the two means for egg number per female is statistically significant.
Alternatives to the Sample Experimental Protocol
(a) Obtain and weigh a virgin female, then place this female into a small Petri dish containing 50 mung beans. (Be sure to keep track of whether the female developed in a mung bean or in a blackeye pea by labeling the Petri dish.)
(b) Place one male raised on the same type of bean as the female into each small Petri dish containing a virgin female. Observe and/or film each pair of beetles until they mate. Make notes regarding your qualitative assessment of the mating behavior.
(c) Although females will be allowed ten days for egg laying, egg size will be determined after two days of egg laying, by following steps d, e, and f below.
(d) After two days of egg laying (that is, during the next class meeting), randomly choose ten eggs from each female to gather data about egg size. This will require the use of a camera system (such as a Moticam 352 camera) mounted on a microscope together with software such as Motic or ImageJ. Capture and save images of each of ten randomly chosen eggs from each female. Determine the size of each egg using one of the two following procedures: (i) Measure the length and width of the egg, and use these measurements to estimate the area of the egg assuming the egg is an ellipse using this formula: ellipse area = (pi) (0.5 x length) (0.5 x width). Alternatively, (ii) use Motic software to draw over the egg using the Irregular shape tool, and allow the software to automatically calculate the area of the egg, or use ImageJ software to outline the egg with the Freehand selection tool and then select Analyze, Measure from the menu bar to get the area enclosed. Note: for either the Motic or the ImageJ software, it is necessary to calibrate the system before making measurements.
(e) Return the beans containing the eggs that were measured in the prior step to the dish from which they came.
(f) For each female, determine mean egg size by taking the average of the ten individual egg sizes.
(g) After five days of egg laying, move each female to a new dish of 50 mung beans. (This step prevents the number of available beans without eggs from becoming limited.)
(h) Aften ten total days of egg laying (five days in each dish), count the total number of eggs laid by each female. For a given female, this will involve observing the entire surface area of each bean from both dishes in which the female spent time.
Data Analysis for the Alternative Protocol:
- For the group of females that developed in mung beans, find the mean number of eggs produced per female. Also, use the data from each individual female about mean egg size to determine the mean egg size produced by this group of females.
- Repeat using data from the females that developed in blackeye peas.
- Female body size may influence egg size and/or number of eggs laid. To compare females developing in different bean types without the confound of female body size, perform an ANOVA (regression analysis) of egg number with females' body size as a covariate. Repeat, replacing egg number with egg size.
- (Optional): Using the data from females that developed in mung beans, use regression analysis to examine the relationship between egg size and number of eggs laid. Do females that lay fewer eggs tend to lay larger eggs?
Alternative Bean Substrates
Other bean species could be used to obtain female beetles with different nutritional histories. Adzuki beans will foster normal development but at a much slower rate than either mung or black-eye peas. Forcing females to lay eggs on beans that they would not normally use would permit instructors to obtain adult females with a broader range of nutritional histories.
Follow-Up Discussion
Follow-up discussion was completed in two parts. Part one involved using natural history observations on beetle mating behavior to develop hypotheses and testable predictions regarding the criteria used by males and females to assess mates. Part two focused on the data from the egg laying experiment, determining whether the data supported the hypothesis, and discussing the broader implications of the results. Key questions for part two included:
- How should the data be interpreted? What can you conclude about the relationship between nutritional history and egg laying in females?
- What are the implications of egg number and/or egg size for fitness?
- Given these results, what are some possible directions for further research?
Materials and Equipment
- Culture of bean beetles (Callosobruchus maculatus)(Carolina Biological 144180)
- Mung beans (Phaseolus aureus, or Vigna radiata)
- Blackeye peas (Vigna unguiculata)
- Plastic Petri dishes, 150 mm (Fisher Scientific 08-757-145) (at least 2 for a class of 20 students)
- Plastic Petri dishes, 35 mm (Fisher Scientific 08-757-100A) (at least 2 for each lab group of 4 students)
- 12-well tissue culture plates (at least 4 for a class of 20 students) (Falcon, available from Fisher Scientific)
- Soft forceps (Ward's Natural Science 14 V 0520) or small paint brushes (1 for each lab group of 4 students)
- Milligram balance capable of weighing bean beetles
- Permanent ink marker
- Microscope with camera system (Moticam 352 or similar)
- Motic or ImageJ software
In an experiment performed by undergraduate students during the fall 2009 semester, no significant difference (p = 0.44; n = 10 females raised in mung beans; n = 11 females raised in blackeye peas) was found between the mean number of eggs laid by female bean beetles raised in mung beans (16.9 +/- 8.4 eggs per female) and the mean number of eggs laid by female bean beetles raised in blackeye peas (13.5 +/- 11.0 eggs per female). In this experiment, virgin female beetles raised in mung beans were mated with virgin male beetles raised in mung beans, and the resulting mated females were placed in a small Petri dish containing 50 mung beans and allowed to lay eggs for 48 hours. Also, virgin female beetles raised in blackeye peas were mated with virgin male beetles raised in blackeye peas, and the resulting mated females were placed in a small Petri dish containing 50 mung beans and allowed to lay eggs for 48 hours. Data (means +/- standard errors) were analyzed using a student's t-test.
Figure 1. Number of eggs laid per female based on maternal bean type
In the same experiment, no significant difference (p = 0.09; n = 83 eggs on mung beans; n = 62 eggs on blackeye peas) was found between the size of eggs laid by female bean beetles raised in mung beans (0.0082 +/- 0.0006 mm2 per egg) and the size of eggs laid by female bean beetles raised in blackeye peas (0.0079 +/- 0.0009 mm2 per egg). In this experiment, virgin female beetles raised in mung beans were mated with virgin male beetles raised in mung beans, and the resulting mated females were placed in a small Petri dish containing 50 mung beans and allowed to lay eggs for 48 hours. Also, virgin female beetles raised in blackeye peas were mated with virgin male beetles raised in blackeye peas, and the resulting mated females were placed in a small Petri dish containing 50 mung beans and allowed to lay eggs for 48 hours. Data (means +/- standard errors) were analyzed using a student's t-test.
Figure 2. Mean egg size based on maternal bean type