Female bean beetles discriminate among different beans during oviposition. The choice that they make influences the growth and survivability of their offspring. Typically they show a preference for natal beans, however even among a large selection of different beans they favor a particular subset. The question is how do beetles determine an appropriate bean on which they could lay their eggs. This experiment ideally follows an experiment where students determine that female bean beetles are somewhat choosy in regards to their prey bean. In this experiment, students are encouraged to design an experiment where they determine what sensory structures would be important in discriminating among bean species. By prompting them to think about how they discriminate the qualities of items in their environment, they can explore the role that beetle sensory structures play in discriminating bean qualities. Experiments that examine sensory detection include ablation of antenna, distal or whole antenna, ablation of the palps, and/or painting of the eyes. Evaluation of the role of sensory structures includes examining on which beans eggs are laid and changes in the placement of the eggs.
Topic: Sensory Systems
Level: Introductory – Intermediate majors
Class time: One class period of 1.5 hours to set up experiment and another class period of 2 hours to collect and analyze data. The experiment runs 48 hours and for a once a week lab, students can remove the beetles at that time and collect their data on their assigned lab day.
Barbara Musolf
Department of Natural Sciences, Clayton State University, Morrow, GA 30260
Objectives
- Design and perform a set of experiments that examine the role of sensory structures on female bean choice.
- Design and perform a set of experiments that determine which sensory structures have the greatest affect on bean choice or egg distribution.
Introduction
Bean beetles, Callosobruchus maculatus, are agricultural pest of beans native to Africa and Asia.The common name for this beetle is the cowpea weevil and indeed this beetle thrives on the cowpea or black-eyed pea (Vigna unguiculata). Beetles typically specialize in a host bean or family of beans, which has led to speciation and development of mechanisms to overcome plant defenses. For the beetle to be successful, they need to choose the appropriate host bean. Beans have a number of sensory qualities that could be detected by the beetle. The question is which sensory quality is important to the beetle and how does the beetle detect that quality.
Materials
In class you will be provided with live cultures of bean beetles that have been raised on mung beans (Vigna radita) or black-eyed peas (Vigna unguiculata). Supplies of other bean species that are of different sizes, textures and colors will be provided so that you can test the beetlesí ability to discriminate among different beans. Such bean species will include garbanzo (Cicer arietinum), black beans (Phaseolus vulgaris), adzuki beans (Vigna angularis), urad beans (black gram) (Vigna mungo), lima beans (sieva bean) (Phaseolus lunatus), and green pea (Pisum sativum). Spring scissors will be provided to perform ablations and paint and a brush with a single hair will be provided to paint the eyes to block vision. A freezer will be used to anesthetize the beetles and ice chambers constructed from petri dishes will be used to keep them anesthetized while you are performing your sensory ablations.
Experimental Design
In a previous experiment that you did with bean beetles, you found that female bean beetles preferred particular host beans for their offspring. Prior to class, you should design a set of experiments that could determine how females discriminate among different host beans. The designs will be discussed in class and a common experimental approach will be determined.
Use results from your prior experiment and the Bean Beetle Handbook to provide you with background information and bring with you to class responses to the list of items below.
- What are the different characteristics of a bean species that could be detectable by a beetle?
- How would a female bean beetle detect some of these characteristics?
- Describe an experimental design for determining how you could test the way a female bean beetle can discriminate a host bean.
- Predict the outcome for the experiment.
- Identify and list the variables you would manipulate in the experiment.
- Identify and list the variables you would keep constant.
- List the data you would collect to determine whether your prediction was correct.
- Describe the statistical analyses that you would carry out to test the strength of your prediction.
Literature Cited
Brown, L. and J.F. Downhower.1988.Analyses in Behavioral Ecology:A Manual for Lab and Field.Sinauer Associates Publishers.
Mitchell, R.1975.The evolution of oviposition tactics in the bean weevil, Callosobruchus maculatus F.Ecology 56:696-702.
Szentesi, ¡. 1976. The effect of the amputation of head appendages on the oviposition of the bean weevil, Acanthoscelides obtectus say (Coleoptera: Bruchidae). Symp. Biol. Hung. 16: 275-281.
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 study was written by B. Musolf, 2010 (www.beanbeetles.org).
Copyright © by Barbara Musolf, 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 experiment requires having dense cultures of bean beetles from which females can be isolated. Cultures on black-eyed peas will generally have produced two generations of beetles in a period of two months. Cultures on mung beans may take a little longer. To provide some uniformity in egg-laying behavior, we generally provide virgin bean beetles and allow the males and females to mate during the time of the experiment. To isolate virgin beetles, remove from the cultures those beans that are beginning to show an emergence window and isolate them in a culture dish. The beetles generally will emerge within the week.
Experimental Design
Questions that students generally address in their experiments include:
- Do females use vision to identify a particular color of bean?
- Do females use their antennae to detect a particular odor of the bean?
- Is there a particular region of the antenna that is important in detecting odor of the bean?
- Do females use their palps to taste the bean or determine its texture?
Because this lab builds on the lab where the students discover that bean beetles are somewhat choosy in regards to where they lay their eggs, many of the same protocols apply. In the prior experiment they learned to control for the different states of the female by choosing to work with virgin females and males. They also provided a variety of beans (10 varieties at 10 each) that numbered approximately 100 to make sure that there continued to be a choice. In this lab, they could reduce the number of varieties and use the information from previous labs to choose beans that clearly were ignored and those that were favored. Choosing beans that clearly were not favored will provide a good test of whether the sensory system that they are investigating is important in detecting bean characteristics.
In generating the experimental design, students should first consider the following questions:
- How can we determine whether a particular sensory structure is important?
- How do we choose which beans to use in the experiment?
- Will putting a beetle in the dark produce the same results as blinding the beetle?
Once you have determined that sensory ablation may be the only feasible way to test the importance of sensory structures then you need to address the following questions?
- How can we immobilize the beetles while doing the sensory ablations?
- Which sensory ablations can we do that will not impair the beetlesí ability to lay eggs?
- Can we do more than one sensory ablation?
- How can we control for the stress of ablating a sensory structure?
- If we anesthetize the beetles, how can we control for the effects of anesthetization?
Notes on beetle anesthetization: We constructed an ice chamber for the beetles from two petri dishes (see description in equipment supplies). We then placed both experimental and control beetles in the chamber and placed the ice chamber in a -4°C freezer for 75 s. The time required for a beetle to become anesthetized can vary around that time, with larger beetles requiring more time. We found that the beetles remained immobile for about 10 minutes, which was enough time for the students to perform their ablations or paint the eyes.
Details on ablation techniques:
Antennal ablations: Student spring scissors are used to cut the antenna at the base for a full antennal ablation or at a point where there is a difference in color (light and dark brown segments) that differentiate between the distal and proximal antenna. The rationale for distinguishing the different parts of the antenna is that they may be involved in detecting different sensory information.
Eye ablation: A dark colored acrylic paint can be used to cover the eyes. A color is preferable to black to determine that the eyes are completely covered and that they have remained covered for the duration of the experiment. The paint is applied using a single hair attached to a stick. The single hair needs to be repeatedly dipped into the paint and allowed to dry to form a small bead. The small dried bead of paint should then pick up sufficient wet paint to apply over the eye. Students may need to practice this technique before becoming adept or an experienced intern may be able to help with this ablation. The paint can be dislodged by the beetle over a period of time. Paint generally stays on for 48 hours, however, students need to check for presence of the paint before collecting data.
Palp ablations: Student spring scissors can be used to cut both pairs of palps. Start at the midline of the mouth and cut away from the midline. This will help extend the palps so that they may be cut more easily.
Data collection—The easiest parameters for the students to measure are the number of eggs laid on each bean and the total number of eggs on each type of bean. As in other labs, the beetles are left in the petri dish with the selected beans for a 48-hour period to provide sufficient time for eggs to be laid. The beetles can be removed after that period of time and the eggs counted in the next lab period (within two weeks) to evaluate bean choice and distribution.
Data analysis—Two types of data could be collected. One could be the number of eggs on particular varieties of beans and the other is the number of beans that have multiple eggs as opposed to a single egg. Ablation of the distal part of the antenna can disrupt the pattern of egg laying. A Chi-square test can be used to test the null hypothesis and whether the distribution of the eggs on the different bean species is random. A student t-test could be used to examine variation in egg distribution by comparing the number of eggs found on each bean for control and experimental treatments. An analysis of variance test (ANOVA test, the non-parametric Kruskall-Wallis test) could be used to compare the average number of eggs found on each bean species across replicates.
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 for each replicate of the sensory ablation experiment
- 30 plastic 35mm Petri dishes for holding isolated beetles
- 15 chilling plates each made with a 35mm plastic Petri dish inside a 100mm plastic Petri dish. The 35mm dish is held in place in the center of the larger dish with Vaseline. The larger plate is then half-filled with water, allowing some of the water to seep under the smaller dish. The two dishes are placed in the freezer and used when the water is frozen. Make sure that there is no water or ice in the smaller petri dish. The beetles can drown in a small amount of water.
- 16 ounces of four bean species, dried and organically grown, if possible. Two species should be favored beans and the other two should be less favored by the beetles. Beans typically favored for oviposition include mung beans, adzuki beans, black-eyed peas, and black beans. Those less favored include garbanzo beans (chick peas), urad beans, lima beans, and green pea
- 15 Student spring scissors for antennal and palp ablations (Fine Science Tools, Inc., Straight 91500-09)
- 30 wooden applicator sticks with a single paint brush hair glued on to the tip. Let a small drop of paint dry on the brush to pick up enough wet paint to cover the eye.
- Dark acrylic paint (DecoArt, Halloween paints dry quickly, adhere permanently to different surfaces)
- 30 soft forceps, BioquipTM featherweight forceps (Catalog No. 4748 or 4750) or 30 small paint brushes for moving beetles from one dish to another
- A freezer at -4°C
Copyright © by Barbara Musolf, 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.
Preliminary experiments on the ablation of sensory structures were performed by a Rocio Salomon, student intern and I in Summer 2009 at Clayton State University. We used 10 different kinds of beans and included their natal bean, mung. The other beans were adzuki, garbanzo, black, navy, urad, pinto, kidney, green pea, and lima. We used 10 of each bean and placed them in a 150 mL petri dish with a virgin female and male for 48 hrs. We then counted the number of eggs on each bean to see if the distribution changed.
Figure 1. The beetles that lacked a distal antenna did not appear to have their egg laying diminished by the sensory ablation, since they laid more eggs than control beetles. The bean beetles that had the palps ablated laid many fewer eggs. The visual and complete antennal ablations did not have the same number of beetles sampled, however it is likely that they were not greatly affected by the ablations.
Figure 2. Sensory ablation of the different sensory structures did appear to alter the bean choice of the bean beetles. Painting the eyes did seem to reduce the inhibiting affects of garbanzo beans, which were avoided by all the other beetles.
Figure 3. Sensory ablations alter the distribution of eggs on the beans. The distal antenna appears to be important for detecting the egg laying pheromone and if the experimental numbers of the complete antennal ablations were greater, then the lack of an antenna would show a similar result. The lack of palps does not appear to be as important for the egg laying pheromone. Vision may play a role but we would have to increase the number of experiments to see if a difference exists.