Reproduction though required for survival of a species is an energetically costly endeavor for an individual. Energy gained as the larval bean beetle feeds on its bean host is used for survival, growth and ultimately reproduction after pupation is complete. Since adult bean beetles do not require food or water following pupation, energy consumption can be directly correlated with longevity in the adult. In this lab, students are tasked with designing an experiment to determine which components of reproduction are physiologically costly for the beetle. Basic experiments include examining the cost of behaviors such as mating and egg laying, but extensions of this experiment include looking to see if there are correlations between factors such as the number of eggs laid, the number of available mates and the natal bean, with lifespan of the adult beetle.
Topic: Reproductive physiology
Level: Non-major and Introductory majors
Class Time: This lab is designed as a multi-week experiment with the initial introduction to bean beetles and set-up taking place during the students' weekly lab period (2-3 hours). Group members are then responsible for checking on their experiment (approx. 5-10 minutes) every day (30-40 days) until all beetles have died
Learning Objectives:
Design and conduct an experiment to determine which components of reproduction are physiologically costly for bean beetles.
Emily Boone
Department of Biology, University of Richmond, Richmond, VA
Objectives
- To identify factors in the reproductive cycle of a bean beetle that are potentially energy costly
- To design and execute an experiment to test which factors are most energetically costly for the bean beetle
Introduction
The principle of allocation states that life histories of organisms are based on a series of trade-offs designed to maximize the overall fitness of an individual. In other words, if organisms use energy for one function such as reproduction than the amount of energy available for other functions is reduced (Cody 1966). In juveniles of a species, energy is divided between survival (for example, obtaining nutrients, escaping from predators, dealing with environmental changes) and growth. Once the organism has reached sexual maturity however, those same energy resources are now divided among survival, growth and reproduction. For many organisms, the cost of reproduction comes in the form of reduced longevity (Yanagi and Miyatake 2003, Paukku and Kotiaho 2005). In this lab, you will examine how different reproductive costs influence the life span of the bean beetle, Callosobruchus maculatus. Bean beetles are ideal organisms for examining reproductive costs because they only feed during the larval stage. This lack of feeding as an adult means that the beetle has a finite amount of energy resources to draw on as it completes its lifecycle.
Materials
In class you will be provided with live bean beetle cultures that have been raised on mung beans. These cultures will be used to help you practice identify the sexes. You also will be provided with cultures on mung beans and black eyed peas that contain beetles which have not yet emerged. Your instructor will show you how to recognize beans that have started to form windows indicating that a beetle will emerge within the next few days. These beans can then be placed into individual wells in 6 or 12 well plates in order to isolated virgin individuals to use in your experiment. Petri dishes will also be provided to use as housing during the actual experiment.
Experimental Design
Prior to lab review the bean beetle handbook (www.beanbeetles.org/handbook) to become familiar with your organism.
In groups of 4, discuss the following:
1. What costs do you think a beetle might incur in the reproduction process?
2. Do these costs differ by sex?
3. How can you measure the cost of each of these processes (i.e. what data will you collect? hint: review your bean beetle lifecycle)?
4. Are there other environmental factors that might influence the amount of resources available for reproduction that need to be considered?
As a group decide on a question that you would like to ask regarding the costs of reproduction in bean beetles, formulate a hypothesis and design an experiment to test your hypothesis.
Cody, M.L. 1966. A general theory of clutch size. Evolution 20:174-84.
Paukku, S. and J. Kotiaho. 2005. Cost of reproduction in Callosobruchus maculatus: effects of mating on male longevity and the effect of male mating status on female longevity. Journal of Insect Physiology 51:1220-6
Yanagi, S. and T. Miyatake. 2003. Costs of mating and egg production in female Callosobruchus chinensis. Journal of Insect Physiology 49:823-27.
This experiment was written by Emily Boone, 2014 (www.beanbeetles.org).
Copyright © by Emily Boone, 2014. 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.
This laboratory activity was designed as part of an introductory level integrative physiology class for biology majors. It corresponds with an introduction to general reproductive physiology in their lecture class. By the time this lab is introduced, students have already had practice designing their own experiments within a guided inquiry type environment and have been introduced to basic statistical analysis techniques.
Introduction of Laboratory
Students are given the bean beetle handbook (www.beanbeetles.org/handbook) prior to the first lab to familiarize themselves with their new study organism. At the beginning of lab, we review the bean beetle lifecycle as a class before breaking into groups to answer the questions posed in the student handout. We then reconvene as a class to discuss the questions and review the materials available to them before individual groups are asked to design their own experiments. This lab can be successfully performed in small groups (3-4) or can be set up as a class depending on the type of question asked, the number of beetles available and the number of replicates you wish to conduct.
Sample Experimental Question
The initial question asked in this lab was simply what are the costs of reproduction in bean beetles? Students identified multiple components of reproduction that could be easily studied including 1) the cost of producing gametes 2) the cost of mating and 3) the cost of egg laying.
Alternative/follow-up questions asked by students:
Does the number of eggs laid by the female affect longevity?
Does the natal bean influence the amount of energy stores available to (and therefore longevity of) the beetle?
Does the species of bean available to lay eggs on affect beetle longevity?
Does the time at which a mate is introduced affect longevity?
Does the number of mates/number of mating events affect longevity?
Sample Experimental Protocol
As the beetles do not feed as adults, the cost for each of these acts was measured by simply calculating longevity for each beetle. Students recorded the adult emergence and death days for each individual in order to determine lifespan. After identifying the reproductive components that they wanted to study, students were asked to consider how they would isolate each component (gamete production, mating and egg laying). The following treatment groups were then selected:
1) Unmated, where newly emerged virgins were placed in individual petri dishes for the duration of the experiment in order to determine the cost of simply being female versus male (gamete production).
2) Mated, where a male and female pair were placed together in a petri dish.
3) Mated with beans, where a mated pair was placed in a petri dish with a layer of beans on which to lay eggs.
Students were provided with bean beetle cultures that were within a few days of having adults emerge and asked to isolate beans that had an identifiable pre-emergence window present on the bean. Note: you may have the students place beans into individual wells in 6 or 12 well plates to isolate emerging virgins however it is not advisable to use these plates for the entire experiment as we have found that given time beetles will manage to climb out of the wells into the spaces between the lid and the curvature of the well. For this reason we used petri dishes to house individuals after emergence. Although sex ratios tend to be 1:1 in beetle cultures, isolation of extra beans was encouraged in order to insure an adequate number of beetles would emerge.
For the first few days, students came in daily to separate beetles into treatment groups as they emerged. After that daily checks were made to determine the death date for each individual. The experiment continued until all beetles had died.
Data Collection
We originally piloted this lab as a class (15 students) in order to have a large number of replicates (50 for each treatment group) and still insure that the outside time commitment was reasonable for the students (as this lab requires students to check on the beetles every day including weekends, student groups signed up on a calendar to check on the beetles so that the individual time commitment outside of lab was not as great). Students were given a 2 hour time frame in which they needed to collect the data each day. As error rates tend to increase as the number of individuals involved increase, I have found that if you are doing this as a class experiment, the easiest way to collect the data is to have the students record the emergence and death days directly onto the individual petri dishes as they check them rather than relying on the use of a separate data sheet. Petri dishes whose beetle(s) are deceased can then be placed on a designated benchtop for the data entry into a central spreadsheet. If working in smaller groups, the number of replicates can be cut back in order to make daily data collection reasonable for your students. I find it helps to reiterate what dead means to the class prior to starting the data collection otherwise less active beetles tend to be classified as such before they have actually died.
Data analysis
Two types of data could potentially be collected. One type would be the lifespan of the beetles, which could be analyzed using an ANOVA. A second possible type could be the number of eggs laid. This could be compared to the lifespan of the female using a correlation analysis.
Equipment and supplies
For a class of 30 students working in groups of 3:
- 10 bean beetle (Callosobruchus maculatus) colonies (on mung beans) with beetles ready to emerge within a few days (additional colonies on black eyed peas can be provided for students wishing to examine effects of natal bean type)
- 16 ounces of mung beans, Vigna radiata, dried beans
- 16 ounces of one or more of the following bean species, for students wishing to examine the effect of bean type: black-eyed peas (Vigna unguiculata), garbanzo (Cicer arietinum), kidney, pinto, black beans (Phaseolus vulgaris), soy beans (Glycine max), lima beans (Phaseolus lunatus), and green pea (Pisum sativum)
- 30 soft forceps, BioquipTM featherweight forceps (Catalog No. 4748 or 4750) or 30 small paint brushes for moving beetles from one dish to another
- 120 plastic 150mm Petri dishes for each replicate of the oviposition substrate choice experiment (only 4 dishes per replicate for each group as the female and male mated beetles can be housed together)
- 30 Sharpie marking pens to record dates on petri dishes
This experiment was written by Emily Boone, 2014 (www.beanbeetles.org).
Copyright © by Emily Boone, 2014. 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.
Preliminary data were collected by University of Richmond Integrated Physiology students in the spring of 2013. Fifty replicates of female unmated, male unmated, female mated, male mated and female mated with beans were initially set up in 150mm petri dishes. Due to time constraints at the end of the semester we had to end the experiment after 30 days even though not all of the beetles had died so some treatment groups had fewer than 50 data points collected. We found reproduction had a significant affect on lifespan (ANOVA, F(4,218) = 28.42, p < 0.0001) (Figure 1). There was no difference in lifespan between unmated males (n = 46) and either unmated (n = 52) (p = 0.8560, Tukey's) or mated females without beans (n = 25) (p = 0.9763, Tukey's). Similarly, there was no significant difference between mated females without beans (n = 25) and unmated females (n = 52) (p = 0.9999, Tukey's). Mated males (n = 50) however had shorter lifespans than either unmated sex (n = 52 female, 50 male) or mated females without beans (n = 25) (p < 0.01, Tukey's,). Mated females housed in dishes with a layer of mung beans (n = 50) had lifespans considerably shorter than all other treatments (P < 0.01, Tukey's). However, no correlation was found between the lifespan of females and the number of eggs that they laid (Linear regression, t = -1.55, r2 = 0.048, p = 0.064, one-tailed, df = 47).
Figure 1. Average (mean±SE) lifespan of virgin bean beetles compared to those allowed to mate in the presence or absence of mung beans. Unless noted otherwise, all females were housed in dishes without access to beans on which to lay eggs. Mating by males and egg laying by females had significant negative affects on lifespan (ANOVA: F(4,218) = 28.42, p < 0.0001).
This experiment was written by Emily Boone, 2014 (www.beanbeetles.org).
Copyright © by Emily Boone, 2014. 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.