Bean beetles (Callosobruchus maculatus) are phytophagous insects that lay their eggs on the surface of several species of beans in the family Fabaceae. Larval development is completed within the bean. Therefore, we would expect strong selection when the beetle switches to a new host. Students are provided with live cultures of beetles containing adults that have been raised on mung beans (Vigna radiata) for many generations; they also are given cultures that were originally grown on mung beans, but were recently switched to another bean species. In this exercise, students design and conduct an experiment to determine whether beetles have adapted to the new host. In the first class period, students design and set up the experiment. In subsequent sessions, they isolate beans with eggs and record data on life history traits including time to emergence and mass at emergence to evaluate the success of beetles on their historic and novel hosts.
Topic: Evolution
Level: Upper-level majors
Class time: one 2-3 hour class period, plus 1 hour class period 48 hours later, 15 minutes daily for 2 weeks about 4 weeks later
Christopher W. Beck1 and Lawrence S. Blumer2
1Department of Biology, Emory University, Atlanta, GA 30322
2Department of Biology, Morehouse College, Atlanta, GA 30314
Introduction
If individuals of a species are adapted to a particular environment, any change in the environment may lead to reduced fitness. As a result, a rapid evolutionary response to environmental changes can be advantageous. Environmental changes that might lead to an evolutionary response include changes in the local environment, changes in the global environment (e.g., global climate change), or changes in the natural range of environments that a species inhabits due to range expansion. In phytophagous (phyto=plant, phagous=eating) insects, different species or different populations of the same species are often specific to a particular host plant species (i.e., are specialists). Therefore, a change in the availability of a particular host plant or the introduction of a new host plant may lead to a shift in the host plant used, which in turn could lead to strong natural selection for adaptation to the new host plant. Adaptation after host shift in herbivorous insects has been documented in a wide range of species (Via 1990). In some species, the evolutionary response of insects to a new host can be very rapid. For example, soapberry bugs (Jadera haematoloma) historically used balloon vine (Cardiospermum corindurn) and the soapberry tree (Sapindus saponaria) as their host (Carroll et al. 1997). However, in the 1950s, the goldenrain tree (Koelreuteria elegans) was introduced into Florida. By 1990, soapberry bugs that had switched to using goldenrain trees as a host had evolved shorter beaks. In addition, when soapberry bugs from both balloon vine and goldenrain tree were reared on goldenrain tree, those that had switched to using goldenrain trees were larger and developed more rapidly on goldenrain (Carroll et al. 1997). Similarly, in the checkerspot butterfly Euphydryas editha, females evolved a preference for a novel host and rejected their native host in just seven years (Singer et al. 1993).
Bean beetles (cowpea seed beetles), Callosobruchus maculatus, are agricultural pest insects of Africa and Asia. Females lay their eggs on the surface of beans of several species in the family Fabaceae. Although bean beetles are generalists, females prefer to lay eggs on their natal host (Messina 2004). Eggs are deposited (=oviposition) singly. Several days after oviposition, a beetle larva (maggot) burrows into the bean and cannot move from the bean on which an egg was deposited. As a result, the quality of the food resources available in a bean will influence the developing individual’s growth, survival, and future reproduction (Mitchell 1975, Wasserman and Futuyma 1981). At 30°C, pupation and emergence of an adult beetle occurs 25-30 days after an egg is deposited, completing one generation of the life cycle. Adults are mature 24 - 36 hours after emergence and they do not need to feed. Adults may live for 1-2 weeks during which time mating and oviposition occurs. Because the ability to use the resources of the host bean efficiently is important in determining larval growth, survival, and future reproduction, we would expect populations to adapt rapidly to the host plant species that are available.
Experimental Design
In class, you will be provided with live cultures of bean beetles containing adults that have been raised on mung beans (Phaseolus aureus), for a large number of generations and other bean beetle cultures that were originally from mung beans but were switched to adzuki beans, (Phaseolus angularis), or black-eye peas (Vigna unguiculata) about 18 generations ago. Supplies of each bean type also will be available. Female beetles are easily identified in the live cultures because they have two dark stripes on the posterior of the abdomen, whereas the posterior abdomen of males is uniformly light in color.
Prior to the laboratory class, each student should design an experiment or set of experiments to address whether rapid adaptation to a novel host has occurred in the bean beetle cultures that were recently switched to a new host bean. Each individual will discuss his or her experimental design with others in a small group, and each group will present a consensus design to the class. Based on the experimental designs presented by the groups, we will discuss common experimental approaches for the entire class.
After you have read the background information and before the laboratory class meeting:
- Describe at least one experimental design for evaluating whether adaptation to a novel host species has occurred.
- Predict the outcomes for the experiment.
- List the dependent variables you would measure to determine if your predictions were true.
- Identify and list the variables you would manipulate in each experiment.
- Identify and list the variables you would keep constant in each experiment.
- Describe what comparisons between treatments you would need to make to test your predictions.
Come to class prepared to present your experimental designs. Each individual will share his or her experimental design with their group and then the group will present their consensus experimental design to rest of the class. Together, we will develop a class-consensus experimental design.
Literature Cited
Brown, L. and J.F. Downhower. 1988. Analyses in Behavioral Ecology: A Manual for Lab and Field. Sinauer Associates Publishers, Sunderland, MA.
Carroll, S. P., H. Dingle, and S. P. Klassen. 1997. Genetic differentiation of fitness-associated traits among rapidly evolving populations of the soapberry bug. Evolution 51:1182-1188.
Messina, F. J. 2004. How labile are the egg-laying preferences of seed beetles? Ecological Entomology 29:318-326.
Mitchell, R. 1975. The evolution of oviposition tactics in the bean weevil, Callosobruchus maculatus F. Ecology 56:696-702.
Singer, M. C., C. D. Thomas, and C. Parmesan. 1993. Rapid human-induced evolution of insect host associations. Nature 366:681-683.
Via, S. 1990. Ecological genetics and host adaptation in herbivorous insects - the experimental study of evolution in natural and agricultural systems. Annual Review of Entomology 35:421-446.
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 C. Beck and L. Blumer, 2008 (www.beanbeetles.org).
Copyright © by Lawrence S. Blumer and Christopher W. Beck, 2009. 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 Laboratory Methods section for detailed information on growing cultures and handling techniques, as well as tips on identifying the sexes.
The experiment requires having dense cultures of bean beetles from which females can be isolated. Beetles should be from cultures reared on a natal host and from cultures switched to a new host several generations prior to the experiment. 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 dense cultures. When possible, we supply one culture of each type (natal and new host) to each group of students working in pairs. However, each culture should have sufficient beetles for use by multiple student groups. Sufficient cultures for one class section can be established in less than an hour. Once cultures are established, they do not need to be monitored or recultured until 2 months later.
Experimental Design
Students often design reciprocal transplant experiments in which females from each host lay eggs on their natal host or an alternative host. However, students may have difficulty determining what dependent variables to measure. Time to emergence, body size at emergence, and emergence success can be measured in a reasonable time span. Students may suggest other offspring characteristics, such as lifespan, reproductive success, hatching rate, and sex ratio. Characters such as lifespan could be measured, but would add another two or more weeks to the experiment. Other dependent variables are appropriate, but difficult to measure (i.e., reproductive success and hatching rate). Finally, for other offspring traits like sex ratio, the predictions are not clear.
Each student should set up a single replicate of each treatment combination of the reciprocal transplant experiment. To set up a replicate, a single female from a stock culture (either mung or adzuki/BEP) is added to a 35mm Petri dish with a monolayer of beans (either mung or adzuki/BEP). Oviposition will readily occur during a 48-hour period. Although most adult females in an active culture will have been inseminated, some females may have only recently emerged (and be infertile) and others are near the end of their adult life (and laid most of their eggs). Replication in the class will allow for failures in egg laying.
Data Collection
For offspring life history traits, one of the biggest confounding factors is the number of eggs laid on beans. If more than one egg is laid on a bean, then the larvae may compete for resources. As a result, only beans with single eggs should be used in tabulating data. Students may want to record the identity of the female that laid the egg to be able to consider differences among females in their analysis. However, data on female identity is not essential. Students can isolate beans of each species with single eggs into the wells of tissue culture plates or small Petri dishes. As the beetles emerge, students can record the offspring characteristics that they chose to measure. Accurate data on time to emergence and mass at emergence require that students check for emergence on a daily basis. As a result, measuring these life history traits may be feasible only in smaller, more advanced classes. The daily checks take between 15 – 30 minutes depending on the number of beetles that have emerged on that day. Students carry out these checks outside of class time. Emergence success could be determined on a single day (potentially as a 1 hour part of a longer lab period) after sufficient time for emergence (approximately 40 days). Therefore, emergence success is more tractable for larger classes.
Data Analysis
Because the experiment is a 2 x 2 factorial design, the data are most appropriately analyzed with a multifactor ANOVA, assuming a normal distribution, which is commonly the case. If adaptation has occurred, we would expect a significant interaction effect between maternal host and offspring host, with offspring having higher fitness on the same host as their mother. In bean beetles, some life history traits differ between the sexes. Therefore, the analysis should be done for males and female separately. Students also could look at correlations between life history traits.
Copyright © by Lawrence S. Blumer and Christopher W. Beck, 2009. 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.
Students in the Fall 2006 ecology course at Emory University carried out a reciprocal transplant experiment using bean beetles from mung bean and black-eye pea stock cultures. Females from the two host types (maternal hosts) were allowed to lay eggs on either mung beans or black-eye peas (offspring hosts). Beans with single eggs were isolated into wells of tissue culture plates and maintained at 30C until emergence. Students recorded sex, time to emergence, and mass at emergence.
Offspring host did not have a significant effect on percent adult emergence for either maternal host (BEP: X2 = 0.44, P = 0.51; mung: X2 = 0.38, P = 0.54).
Because females are significantly larger than males in bean beetles, the data for the sexes were analyzed separately. For both males and females, mass at emergence was not significantly affected by maternal host, offspring host, or the interaction between the two (2 x 2 ANOVA: P>0.07 for main effects and interaction for both sexes).
In contrast, the interaction between maternal host and offspring host significantly influenced time to emergence for both males (F1, 99 = 9.4, P < 0.01) and females (F1, 118 = 6.3, P = 0.01). Interestingly, however, time to emergence was longer when offspring were reared on the host from which their mother emerged.
