Objectives

• Brainstorm characteristics (female, male, and environmental) that may influence how female bean beetles respond to approaching males.
• Design and perform an experiment that determines how one of these characteristics may affect how females respond to approaching males.

Introduction

Polyandry is a common mating system in which females mate with more than one male during the breeding season. Types of animals exhibiting polyandry are incredibly diverse and include some birds (e.g., red-winged blackbirds), insects (e.g., honeybees), mammals (e.g., chimpanzees) and fish. Benefits associated with polyandry include receiving good genes from a mate and increased assurance that eggs will be fertilized (Alcock 2010). Females may also receive gifts (e.g., food) when they mate with males (Alcock 2010). Costs associated with polyandry include time and energy spent searching for additional mates, risk of losing parenting help from a male that detects female cheating, and health risks (e.g., STDs and injury during mating; Alcock 2010). Given the costs that may accompany multiple matings, examining female choice associated with mating is important in understanding polyandrous mating systems.

Female bean beetles (Callosobruchus maculatus) are polyandrous (Arnqvist et al. 2005) and are injured when the spines on the male’s intromittent organ penetrate the female genital tract (Crudgington and Siva-Joth 2000). Female bean beetles have limited ways to deter approaching males. Essentially, they can kick or move away (Savalli and Fox 1999). Female bean beetles exhibit premating activity that includes a fixed standing posture accompanied by contraction and relaxation of the abdomen (Rup 1986). Rup (1986) stated that female bean beetles always terminate the mount by dragging and kicking the male, indicating that female choice plays a role in mating.

Which male a female “chooses” may be driven by characteristics of the male, characteristics of the female herself, or characteristics of the environment. The main question to be addressed in this experiment is: What characteristics influence female responses to male courtship attempts?

Materials

• Bean beetles raised on mung beans and bean beetles raised on black eyed peas
• Petri dishes (35 mm and 90 mm)
• Unused mung beans and black eyed peas
• Rulers
• Markers
• Dissecting scopes
• Forceps or small paintbrushes
• Stopwatches
• Other assorted items that your experiment requires

Experimental Design

The research question is: What characteristics influence female responses to male courtship attempts? Your specific hypothesis and prediction will be determined by your group, as this is an open-inquiry lab.

Week 1: You may use online resources to assist you in the following activity.

• Brainstorm characteristics of males that might influence a female’s choice to mate with them.
• Brainstorm characteristics of females that might influence their choice to mate with males.
• Brainstorm characteristics of the environment that might influence a female bean beetle’s choice to mate with males.

Week 2: Complete the following in lab.

As a group, choose one characteristic from one list above on which to focus for your experiment.

Research question:

Research hypothesis:

Research prediction:

Null hypothesis:

Prediction associated with null hypothesis:

Describe your methods:

• list the variables you will manipulate in your experiment
• list the variables that you will keep constant in your experiment
• describe the data you will collect (to determine if your prediction supports your hypothesis) and how frequently you will collect data
• list each possible outcome for your experiment
• describe your experimental set-up (pictures may help), focusing on how variables will be manipulated, characteristics of beetles you’ll use, and how many replicates you’ll have of each condition
• state the statistical test that you will use to analyze your data and justify use of this test.

** You will write a report of your study. Details are provided in a separate document.

Literature Cited

Alcock J. 2010 Animal Behavior. 9th ed. Sunderland, MA: Sinauer Associates.

Arnqvist G, Nilsson T, Katvala M. 2005. Mating rate and fitness in female bean weevils. Behav Ecol 16:123-127.

Crudgington HS and Siva-Jothy MT. 2000. Genital damage, kicking and early death – the battle of the sexes takes a sinister turn in the bean weevil. Nature 407:855-856.

Rup RJ. 1986. Mating and its attendant behavior in Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J Stored Prod Res 222:77-79.

Savalli UM, Fox CW. 1999. The effect of male mating history on paternal investment, fecundity and female remating in the seed beetle Callosobruchus maculatus. Funct Ecol 13:169-177.

This experiment was written by K. Buckholz and S. Larimer.  

Copyright © by Kimran Buckholz and Samantha Larimer, 2012. 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.

Experimental Design

Two important caveats:

1. Our student groups did not observe a lot of beetle mating, as can be seen in the examples of student data. The students observed the beetles for 10-30 minutes. Typically, mating is observed in approximately 15 minutes. However, extending observation time may result in more matings. Additionally, isolating beans and collecting males and females upon emergence would better ensure that virgins/young animals are used. Since mating seems to occur at greater frequencies when the animals are young, this method might improve the chances of observing mating. We have also separated males from females for up to 18 hours prior to experiments. Although this did not dramatically increase mating frequency observed by one student group, this method could be played around with.
2. Although we claim that we are testing female choice in these experiments, you should engage in a discussion with your students about why these experiments don’t rule out the influence of the male on mating behavior. What happens prior to mating and during mating is likely a complicated set of male-female interactions and male-female choice. You could focus on the percent of mating attempts that are successful because this may imply that females have decided to allow a mating. However, rape occurs among animals, so even these data could be influenced by the male. Larger males may have more of a coercive influence on females than smaller males. Likewise, other factors such as prior mating experience, age, etc. could influence the degree to which males “have their way” with the females, regardless of female choice. You can discuss this confounding factor with the students and ask them to brainstorm ways around it.

Given that this study is open-inquiry, experimental design among student groups varies. Many possible characteristics associated with the female, male and environment can be tested. We have generated a list of possible characteristics associated with each non-mutually exclusive hypothesis. Some characteristics will be easier to test than others, depending on the level of the course and time devoted to the activity.

H1: Female characteristics influence female reactions to approaching males.

P1: Female reproductive status (virgin or experienced) influences her reaction to males.

P2: Length of time since female has mated influences her reaction to males.

P3: Female body size (mass, length, length of elytra) influences her reaction to males.

P4: Female ability to detect pheromones influences her reaction to males.

P5: Female age influences her reaction to males.

H2: Male characteristics influence female reactions to approaching males.

P1: Male reproductive status (virgin or experienced) influences female reaction.

P2: Male body size (mass, length, length of elytra) influences female reaction.

P3: Deficiency in male physical characteristics (altered wings, antennae, legs) influences female reaction.

P4: Male age influences female reaction.

P5: Persistence of male influences female reaction.

P6: Relatedness to female influences female reaction.

P7: Relatedness to males with which the female has previously mated influences female reaction.

P8: Female familiarity with the male influences female reaction.

P9: Various genetic characteristics of the male influence female reaction.

H3: Environmental characteristics influence female reactions to approaching males.

P1: Temperature of environment during mating influences female reaction.

P2: Female access to water/nutrients in water influences female reaction.

P3: Number of eggs already on beans in environment influences female reaction.

P4: Type (natal species vs. other species, size, etc.) of beans available influences female reaction.

P5: Number of beans available influences female reaction.

P6: Density of males and/or females in the environment influences female reaction.

Students have a tendency to want to put multiple males in with a female to test her reaction to the males. They should be asked to consider how one male might influence another male’s interaction with a female, since this might be a confounding factor. If male body size is being tested, the order in which a female is exposed to a small male vs. large male should be randomized. Students should also consider the value of allowing the females to rest/recover for a short time, between exposures to different males. Students should certainly be encouraged to set up replicates in their experimental design.

Data collection

Prior to determining the specific group project, students need to complete the brainstorming activity. Depending on student knowledge, you can ask them to conduct online searches for characteristics that influence mate choice among animals (either in lab or as homework). Keywords include: female mate choice, male mating, environment mating, polyandry, etc. If the students already understand mate choice, simply brainstorming this information (either in lab or as homework) may be sufficient.

In non-major or introductory courses, you can provide articles for them to read either while they are brainstorming characteristics that influence mating or as they are figuring out their own experiment. In upper-level classes, you can require that they find articles on mate choice among bean beetles. These will be helpful when figuring out their group experiments.

A list of helpful resources follows:

Alcock J. 2010 Animal Behavior. 9th ed. Sunderland, MA: Sinauer Associates.

Arnqvist G, Nilsson T, Katvala M. 2005. Mating rate and fitness in female bean weevils. Behav Ecol 16:123-127.

Crudgington HS and Siva-Jothy MT. 2000. Genital damage, kicking and early death – the battle of the sexes takes a sinister turn in the bean weevil. Nature 407:855-856. ** particularly helpful, as images of genital damage and intromittent organ are included

Fox CW, McLennan LA, Mousseau TA. 1995. Male body size affects female lifetime reproductive success in a seed beetle. Anim Behav 50:281-284.

Kishi S, Nishida T, Tsubaki Y. 2009. Reproductive interference determines persistence and exclusion in species interactions. J Anim Ecol 78, 1043-1049.

Maklakov, A.A.; Arnqvist, G. 2009. Testing for direct and indirect effects of mate choice by manipulating female choosiness. Current Biology 19 1903-1906

Miyatake T, Matsumura F. 2004. Intra-specific variation in female remating in Callosobruchus chinensis and C. maculatus. J Insect Physiol 50:403-408.

Moya-Larano J, Fox CW. 2006. Ejaculate size, second male size, and moderate polyandry increase female fecundity in a seed beetle. Behav Ecol 17(6):940-946.

Rova, E.; Björklund, M.; Bilde, T. Can Preference for Oviposition Sites Initiate Reproductive Isolation in Callosobruchus maculatus? 2011 PLoS ONE 6 e14628

Rup RJ. 1986. Mating and its attendant behavior in Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J Stored Prod Res 222:77-79. ** particularly helpful, as a mating ethogram is included

Savalli UM, Fox CW. 1999. The effect of male mating history on paternal investment, fecundity and female remating in the seed beetle Callosobruchus maculatus. Funct Ecol 13:169-177.

Ursprung C, den Hollander M, Gwynne DT. 2009. Female seed beetles (Callosobruchus maculatus), remate for male-supplied water rather than ejaculate nutrition. Behav Ecol Sociobiol 63:781-788.

Characteristics (independent variables) that have been investigated by students in our courses include:

• effects of male body size (students measured either body length or width, with a small and larger size able to be measured)
• putting the beetles in the freezer for approximately 75 seconds allows body measurements to be taken upon retrieval from the freezer
• water availability (students allowed females to drink water from a moistened cotton swab for 10 minutes, observed using a dissecting scope; gel ("water gel from water absorbing crystal polymers") used for feeding insects could also be used as a water source)
• temperature (students used lamps with incandescent bulbs and compact fluorescent bulbs to test effects of different environmental temperatures; another group of students used ice or a heating pad under the Petri dish)
• size of environment (students used two different sizes of Petri dishes)
• quality of environment (students had different numbers of beans in the Petri dish)
• time since female last mated (students used females from mixed sex colonies or females that had been removed from their mixed sex colony to reside in a female-only dish, 18 hours prior to the experiment)

Students have collected behavioral data involving male-female interactions (dependent variables) that include: chase, contact of any sort, antennate (touch with antenna), attempt mount, mate. Examples of variables that were controlled include: temperature, light/dark cycles, humidity, dimensions of the environment, number of females in the dish, number of males in the dish, number of beans in the dish, bean type, etc. Likewise, these could be variables that are manipulated depending on experimental design. Replicate number has varied from 3-10, depending on methodology. Students have observed 6-12 females in their experiments. Initial data collection has occurred on the same day of experimental design; however, that also could vary with methodology. Students have successfully collected data throughout the week (approximately 3 times) following initial data collection, with data analysis conducted during the next lab period. Duration of data collection can vary, though.

Data Analysis

Non-majors were not expected to statistically analyze their data. Qualitative comparisons of graphed data were required. However, in upper-level classes, the experiment lends itself well to statistical analysis. Type of statistics varies depending on the question asked. Students who conduct experiments that present the same females with 2 different choices and collect quantitative data on these choices could use a paired t-test (or the non-parametric equivalent: the Wilcoxon Signed Ranks test). Students who carry out experiments that have females exposed to only one testing variable could use the unpaired t-test (or the non-parametric equivalent: the Mann-Whitney U). Students who record presence or absence of an activity (such as whether or not mating occurred) can use a Chi square test.

Equipment and Supplies

Typical supplies that will be needed for a class of 30 students include:

• Bean beetles raised on mung beans and bean beetles raised on black eyed peas: you can choose to have dishes for the entire class to use or 1-2 dishes per lab table. You can also decide whether or not to create special colonies that would be available to students (such as virgins or animals that have been separated for a given amount of time).
• Petri dishes (35 mm and 90 mm): small Petri dishes are easy to work with, but if students would like to vary environment, having different sizes of Petri dishes allows them to manipulate environmental space. Depending on the number of beetles put in each dish, each student can observe a dish or multiple students can observe a dish. As an estimate, having 1-2 dishes available for each student to use during their observations seems sufficient.
• Unused mung beans and black eyed peas
• Rulers: 1 per student
• Stopwatches: 1 per student or several per table
• Markers: 1 per student
• Dissecting scopes: 2 per table
• Forceps: 1 pair per student (Paintbrushes also work well for transferring beetles.)
• Other assorted items that group experiments require

Additional specific supplies will vary depending on the experiments designed by students. Supplies that have been used in previous semesters include:

• 4 lamps: 2 with incandescent bulb (heat given off) and 2 with compact fluorescent bulb (less heat given off) (to test effects of temperature)
• Ice
• Heating pads
• Moistened cotton swabs to test effect of female exposure to moisture; gel ("water gel from water absorbing crystal polymers") used for feeding insects could also be used as a water source

 

This experiment was written by K. Buckholz and S. Larimer.

Copyright © by Kimran Buckholz and Samantha Larimer, 2012. 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.

Non-majors class:

I. Question: How does a male's body size affect a female bean beetle's choice to mate?

Hypothesis: A female will be selective in her mate choice relative to a male's body size.

Prediction: Females will choose males that have the largest body size.

Methods: Our experiment focused on the male's body size and how females would respond to it. We collected data by procuring seven sets of male beetles and seven female beetles. During each procedure, we tested a large male (4mm wide) and a small male (3mm wide), each in separate Petri dishes, with one female to see what male she would choose. After leaving the female in with the large male for 15 minutes, we placed the female in her own dish for five minutes to recover from possible mating exercises. We then put her in the other dish with the small male. We watched the pre-mating behaviors, which include, but are not limited to, the male pursuing the female, the male feeling her with his antennae, and the male preparing to mount (Rup 1986). When the male was starting to mount the female, we separated them. In this way, the female would still be seeking a male for the remainder of the experiment. Each female was exposed to a large male and to a small male per trial, with two trials being conducted (Fig. 1). The purpose of the two trials was to alter the order of large male and small male exposure for each female. We gave her rest-time in between the first and second trials.

Results and Conclusions: An attempt at mating occurred between three females and small males, while an attempt at mating occurred between two females and large males (Fig. 1, Table 1). Qualitatively, male size does not seem to influence attempted matings. (This group's data did not adequately differentiate between the order of large and small males presented to females in trial 1 vs. 2.) Potentially, a longer male-female interaction time and/or using virgin or young females may affect the mating interactions. Additionally, attempted mating only somewhat addresses female choice, since male choice is probably an important factor in the occurrence of mating.

Figure 1. The number of attempted matings in two trials in which females were exposed to large and small males.

Table 1. Data from the two trials in which females were exposed to large and small males.

Note: "Mated" refers to an attempted mating.

II: Same body size question, hypothesis and prediction as in part I. Similar methods were used except that eight replicates were set up, in which each female was exposed to a small male and a large male in a randomized order. Females and males were allowed to interact for 10 minutes. Female acceptance (see Fig. 3-4) was defined as the female "allowing" the male to antennate her, mount her, or mate with her.

Results and Conclusions: Responses by eight females to large males and to small males were measured (Fig. 2). One female accepted the mating attempt by a large male. One female rejected the mating attempt by a large male and two females rejected the mating attempts by small males.

Fig. 2. Female responses to advancing males.

Chasing did not differ between small and large males (Fig. 3). Two large males and one small male felt a female with their antennae and attempted to mount a female. One large male mated with a female, while no small male mated with a female. In conclusion, only four of the sixteen males attempted to mate with a female. Therefore, results are inconclusive. Ages of males and females could have influenced the results negatively.

Fig. 3. Males' behavioral interactions with females.

III. Question: Does exposure to water influence female bean beetle response to approaching males?

Hypothesis: Drinking water influences a female's response to males.

Prediction: When the female bean beetle consumes water, she will be less likely to mate with the male than the female bean beetle that doesn't consume water.

Methods: We chose to investigate the effect that water in the female bean beetles' environment has on female response to males. We used six female subjects per day, three of which were exposed to water prior to introducing the males into the environment and three of which were not exposed to water prior to male introduction. We first put the females into a Petri dish with the end of a Q-tip that had been placed in water for ten seconds. After we exposed the females to the water source for at least ten minutes, we then removed the source and placed a male and five mung beans that had not had any eggs laid on them into the dish with the female and observed closely for 15 minutes. Beans were placed in the dish with each female to encourage the mating/egg laying process. We then recorded our results. We performed the same experiment on two additional days. We tested 18 female subjects total, 6 per day.

Results and Conclusions: On the first day of observations, only one of six females mated with a male, and she was not exposed to water prior to mating. On the second day of observations, only one female out of six mated with a male, and she was also not exposed to water prior to mating. On the third day of observations, no female mated with a male.

Two of the nine females (22%, Fig. 4) not exposed to water mated with males. No female exposed to water mated with a male. Exposure to water may influence mating with males, but the small number of females not exposed to water that actually mated with males makes the results inconclusive. This group did not adequately observe drinking by the females (using dissecting scopes), so they did not quantify the extent to which females exposed to water actually drank water. Also, using younger or virgin females may have altered the results.

Figure 4. Mating tendencies of females exposed to water or not, prior to introduction of males.

IV. Question: Does environmental temperature influence female response to approaching males?

Hypothesis: Environmental temperature affects female response to males that approach.

Prediction: Females will be more likely to mate in warm vs. cooler environments.

Methods: Incandescent lighting was used to create a warmer environment, while compact fluorescent lighting was used to create an environment in which light was still emitted, but less heat was generated. Thermometers registered the incandescent environment at 30°C and the compact fluorescent environment at 25.3°C. Four Petri dishes with a male, female and 4 mung beans were set under each lamp for 5 minutes to acclimate to the lamps (although male and female interactions could also occur during this time). The beetles were observed for 30 minutes and mating was noted. Three trials (of 4 dishes) were run.

Results and Conclusions: Across the three trials, results were inconsistent (Fig. 5). In trial one, more matings occurred under incandescent lighting (heat) than compact fluorescent lighting (less heat). The opposite occurred in trial three, while in trial two, mating occurred only under incandescent lighting. Across the three trials, eleven matings occurred at higher temperatures, while seven occurred in cooler temperatures. In conclusion, temperature could have an influence on mating behaviors.

Figure 5. Comparison of number of matings between male and female bean beetles
when exposed to incandescent lighting (heat) vs. compact fluorescent lighting (less heat).

Upper-level class:

I. Question: Does environment size affect female mate choice? (This question was designed by the students. Admittedly, their experimental design doesn't differentiate between male choice and female choice.)

Hypothesis: How much time females spend with a male will be affected by environment size.

Prediction: If placed in a smaller dish, males and females will spend more time in physical contact than in a larger dish.

Relevant methods: Fifteen mung beans were added to each dish, 8 small dishes (3.5 cm diameter) and 8 large dishes (9 cm diameter). (Males and females had both come from colonies with a long history of living on mung beans.) One male and one female were given 15 minutes to interact in each dish. Interaction between individuals was defined as any time the two individuals were physically in contact. Duration of contact was measured using a stopwatch. Males and females had been housed in separate sex colonies for 4 hours prior to testing.

Results and Conclusions: Most bean beetles didn't interact with one another at all (11 out of 16). Those that did interact in the small dish maintained a longer duration of contact than those that interacted in the large dish (Fig. 7; mean ± SE 19.25 ± 10.2 sec and 0.63 ± 0.5 sec, respectively), but, not surprisingly given the variation, this difference was not significant (Mann Whitney U: p = 0.371).

Figure 6. Duration of contact time between males and females.

I. Question: Does the quality of the environment for egg laying influence desire to mate?

Hypothesis: The more beans there are in a Petri dish, the less time the bean beetles will interact.

Prediction: If 2 bean beetles of the opposite sex are in a Petri dish of 22 beans, then they will spend less time interacting than those bean beetles in a one bean Petri dish.

Relevant methods: Beans were mung beans. Petri dishes were 3.5 cm in diameter. Beetles were given 15 minutes to interact. Interaction was defined as any physical contact between beetles.

Results and Conclusions: There was large variation in time spent in contact between males and females (Fig. 7), but this variation did not appear to be related to number of beans in the dish (Mann Whitney U: p = 0.527).

Figure 7. Duration of time males and females remained in contact in Petri dishes
with 1 mung bean and Petri dishes with 22 mung beans.

III. Question: Do periods of abstinence influence females' willingness to mate?

Hypothesis: The longer a female has gone without mating the more they are willing to be mated with.

Prediction: The females who have gone about 18 hours without mating will choose to mate more than the females who were immediately previously in a colony with males.

Relevant methods: Dishes were 6 well plates with circular wells 2 cm in diameter, 3 cm in height. Three black eyed peas were placed in each dish, as were 2 males and 1 female. Individuals were allowed 30 minutes to interact, and it was recorded whether or not the animals mated during that time, where mating was described as 1 minute or more of mounting of the female by either male. Ten females were tested that had been separated from males for 18 hours prior; 10 females were tested directly from the colony.

Results and Conclusions: Very few animals mated (2 out of 20). Both of those matings occurred in the wells with females that had been unable to mate for the previous 18 hours, but the numbers are too low to draw any conclusions. The difference between these two groups is not significant (Chi square: X² = 2.5, df = 1, p = 0.1138).

Powerpoint slides with the figures are available in the Downloads section.

Student Handout [pdf] [docx]

Instructor's Notes [pdf] [docx]

Lab report instructions [pdf][docx]

Sample data graphs and tables [pptx]

Identifying the sexes [ppt]