Environmental cues influence insect physiology causing fluctuations in the levels of several hormones that regulate development. These hormonal changes occur at different developmental periods, and influence a number of physiological processes. Temperature is a primary mediator of changes in insect development. Changes in thermoperiod induce alterations in the levels of the hormone ecdysone which mediates insect molting and metamorphosis. Ecdysone therefore produces stage-specific changes that determine the outcome of each developmental transition. In this experiment, students will be challenged to design and perform a set of experiments to correlate changes in the levels of the active form of ecdysone, 20-hydroxyecdysone (20E), with changes in temperature during the development of bean beetle larvae. Typical experiments will involve incubating bean beetle larvae at various temperatures, and measuring the levels of circulating 20E at different intervals using an enzyme immunoassay (EIA). Data obtained from these experiments will provide information that could be used to clarify our understanding of how environmental cues influence the development of bean beetle larvae, and by extension, development in other insect species.
Topic: Developmental Biology
Level: Upper-level majors
Class Time: one 2 hour class period to discuss and set-up the experiment, 10 minutes daily for up to 5 days to collect beans after oviposition and place in incubator, 10 minutes to collect beans at different intervals after incubation at designated temperatures, two- 2 hour class periods to perform EIA analysis.
Learning Objectives:
Design and perform a set of experiments to investigate alterations in the levels of the hormone 20-hydroxyecdysone with changes in temperature during the development of bean beetle larvae.
Sherman A. Ward and Regina Knight-Mason
Department of Biology, Virginia State University, Petersburg, VA 23806
Objectives
- Design and perform a set of experiments to correlate alterations in the levels of the hormone 20-hydroxyecdysone with changes in temperature during the development of bean beetle larvae.
Introduction
After fertilization, adult female bean beetles (Callosobruchus maculatus) deposit their eggs (oviposition) on the surface of a bean seed, and a few days later beetle larvae burrow through the seed coat into the bean. Larval growth and development continues as they feed on the endosperm of the seed completing four instars inside of the bean upon which the egg was laid. The time from oviposition to the emergence of an adult bean beetle is determined both by its genotype and the environment. Temperature has a significant impact on the size of intervals of offspring development. Within defined limits, generation time is shortened when temperature is increased. The generation time of beetle larvae maintained at 22°C is 7 weeks, whereas cultures of beetle larvae incubated at 25°C emerged 5-6 weeks after oviposition. When temperature is increased to 30°C, generation time is decreased to 3-4 weeks (Beck and Blumer, 2014). The development of insect larvae is also mediated by endogenous regulatory mechanisms including chemical signaling. The steroid hormone ecdysone is an example of one such signal that is associated with bean beetle development. The active form of ecdysone, 20-hydroxyecdysone (20E), coordinates the differentiation of adult structures during insect molting and metamorphosis (Dubrovsky, 2005). In insects, the level of 20E has been reported to fluctuate during molting, raising the possibility that alterations in the levels of this hormone could affect the timing of molts at different developmental stages (Gelman, Blackburn, and Hu 2002; Noriega, Ramberg, and Hagedorn 2002). 20E interacts with other regulatory hormones to produce stage-specific changes that determine the outcome of each developmental transition. Therefore, since 20E has such a significant role in insect development, it is important to determine if this hormone plays a role during development in this organism, and if so, the effect of temperature on the levels of 20E in bean beetle larvae.
Materials
The following materials will be available in class:
- Live cultures containing adult bean beetles.
- A supply of black-eyed peas or mung beans.
- Insect incubators (temperature controlled)
- Plastic petri dishes.
- Dissecting microscopes.
- Reagents needed to perform enzyme immunoassay for 20E analysis (EIA).
- Sandwich bags, tape, and markers for labeling and storing beetles.
Experimental Design
Temperature has been shown to influence the generation time of bean beetles, and 20E has been reported to direct metamorphic changes in insects. Changes in environmental temperature may therefore alter the level of 20E in developing bean beetles. Prior to the laboratory class each group will design a set of experiments to examine the influence of temperature on the level of 20E in bean beetle larvae at different intervals post-oviposition. Each group will then present their experimental design and methods for discussion by the class.
After you have read the background information and before the laboratory class meeting:
- Describe at least one experimental design for evaluating whether fluctuations in temperature induces alterations in the levels of 20E in developing bean beetles.
- Predict the outcomes for the experiment that support your hypothesis.
- Identify and list the variables you would manipulate in the experiment.
- Identify and list the factors that would remain constant in the experiment.
- Identify the data you would collect to test your predictions.
Each student should come to class prepared to present their experimental design. Each student will then share their design with their group, and the group will agree upon a design and present it to the class. The class will then agree upon a common experimental design.
Beck, C.W. and Blumer, L.S. 2014. A Handbook on Bean Beetles, Callosobruchus maculatus. 12 pages. Privately published booklet posted at: http://www.beanbeetles.org/handbook.
Dubrovsky, E.B. 2005. Hormonal cross talk in insect development. Trends in Endrocrinology and Metabolism. 16(1):6-11.
Gelman, D.B., Blackburn, M.B., and Hu, J.S. 2002. Timing and ecdysteroid regulation of the molt in last instar greenhouse whiteflies (Trialeurodes vaporariorum). Journal of Insect Physiology. 48:63-73.
Noriega, R., Ramberg, F.B., and Hagedorn, H.H. 2002. Ecdysteroids and oocyte development in the black fly Simulium vittatum. BMC Developmental Biology 2002 2:6.
This experiment was written by Sherman Ward and Regina Knight-Mason, 2014
Copyright © by Sherman A. Ward and Regina Knight-Mason, 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.
Objectives
- Design and perform a set of experiments to correlate alterations in the levels of the hormone 20-hydroxyecdysone with changes in temperature during the development of bean beetle larvae.
Introduction
After fertilization, adult female bean beetles (Callosobruchus maculatus) deposit their eggs (oviposition) on the surface of a bean seed, and a few days later beetle larvae burrow through the seed coat into the bean. Larval growth and development continues as they feed on the endosperm of the seed completing four instars inside of the bean upon which the egg was laid. The time from oviposition to the emergence of an adult bean beetle is determined both by its genotype and the environment. Temperature has a significant impact on the size of intervals of offspring development. Within defined limits, generation time is shortened when temperature is increased. The generation time of beetle larvae maintained at 22°C is 7 weeks, whereas cultures of beetle larvae incubated at 25°C emerged 5-6 weeks after oviposition. When temperature is increased to 30°C, generation time is decreased to 3-4 weeks (Beck and Blumer, 2014). The development of insect larvae is also mediated by endogenous regulatory mechanisms including chemical signaling. The steroid hormone ecdysone is an example of one such signal that is associated with bean beetle development. The active form of ecdysone, 20-hydroxyecdysone (20E), coordinates the differentiation of adult structures during insect molting and metamorphosis (Dubrovsky, 2005). In insects, the level of 20E has been reported to fluctuate during molting, raising the possibility that alterations in the levels of this hormone could affect the timing of molts at different developmental stages (Gelman, Blackburn, and Hu 2002; Noriega, Ramberg, and Hagedorn 2002). 20E interacts with other regulatory hormones to produce stage-specific changes that determine the outcome of each developmental transition. Therefore, since 20E has such a significant role in insect development, it is important to determine if this hormone plays a role during development in this organism, and if so, the effect of temperature on the levels of 20E in bean beetle larvae.
Materials
The following materials will be available in class:
- Live cultures containing adult bean beetles.
- A supply of black-eyed peas or mung beans.
- Insect incubators (temperature controlled)
- Plastic petri dishes.
- Dissecting microscopes.
- Reagents needed to perform enzyme immunoassay for 20E analysis (EIA).
- Sandwich bags, tape, and markers for labeling and storing beetles.
Experimental Design
Temperature has been shown to influence the generation time of bean beetles, and 20E has been reported to direct metamorphic changes in insects. Changes in environmental temperature may therefore alter the level of 20E in developing bean beetles. Prior to the laboratory class each group will design a set of experiments to examine the influence of temperature on the level of 20E in bean beetle larvae at different intervals post-oviposition. Each group will then present their experimental design and methods for discussion by the class.
After you have read the background information and before the laboratory class meeting:
- Describe at least one experimental design for evaluating whether fluctuations in temperature induces alterations in the levels of 20E in developing bean beetles.
- Predict the outcomes for the experiment that support your hypothesis.
- Identify and list the variables you would manipulate in the experiment.
- Identify and list the factors that would remain constant in the experiment.
- Identify the data you would collect to test your predictions.
Each student should come to class prepared to present their experimental design. Each student will then share their design with their group, and the group will agree upon a design and present it to the class. The class will then agree upon a common experimental design.
Beck, C.W. and Blumer, L.S. 2014. A Handbook on Bean Beetles, Callosobruchus maculatus. 12 pages. Privately published booklet posted at: http://www.beanbeetles.org/handbook.
Dubrovsky, E.B. 2005. Hormonal cross talk in insect development. Trends in Endrocrinology and Metabolism. 16(1):6-11.
Gelman, D.B., Blackburn, M.B., and Hu, J.S. 2002. Timing and ecdysteroid regulation of the molt in last instar greenhouse whiteflies (Trialeurodes vaporariorum). Journal of Insect Physiology. 48:63-73.
Noriega, R., Ramberg, F.B., and Hagedorn, H.H. 2002. Ecdysteroids and oocyte development in the black fly Simulium vittatum. BMC Developmental Biology 2002 2:6.
This experiment was written by Sherman Ward and Regina Knight-Mason, 2014
Copyright © by Sherman A. Ward and Regina Knight-Mason, 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.
These sample results were collected by students at Virginia State University who conducted an experiment with beetles raised at three temperatures, 22°C, 25°C and 30°C. The concentrations of 20-hydroxyecdysone (20E) were measured in pg/ml using EIA methods described in the Instructor's Notes. The concentration of 20E increased with temperature (Table 1) and this increase was significant (ANOVA F=5.93 p=0.023). The concentration of 20E has a decreasing trend with the age of larvae and pupae (Figure 1), but this trend was not statistically significant with our small sample size (Table 2). The raw data from this experiment are available in the Downloads data spreadsheet.
Table 1. Concentrations of 20-hydroxyecdysone (20E) in beetle larvae and pupae incubated at different temperatures. One sample was collected every seven days (7, 14, 21 and 28 days after egg laying) at each temperature in this experiment. There was a significant increase in concentration of 20E with increasing temperature (ANOVA F=5.935 p=0.023).
| Temperature (°C) | 20E Concentration (pg/ml Mean±SE) | N |
|---|---|---|
| 22° | 89±35 | 4 |
| 25° | 310±44 | 4 |
| 30° | 381±92 | 4 |
Table 2. Concentration of 20-hydroxyecdysone (20E) in bean beetle larvae and pupae at different developmental time points. One sample was collected from each of three different temperature conditions (22°C, 25°C and 30°C) at each time point. There was no significant affect of age on 20E concentration (ANOVA F=0.791 p=0.698).
| Age (days) | 20E Concentration (pg/ml Mean±SE) | N |
|---|---|---|
| 7 | 358±105 | 3 |
| 14 | 236±99 | 3 |
| 21 | 268±115 | 3 |
| 28 | 179±110 | 3 |
Figure 1. Concentration of 20-hydroxyecdysone (pg/ml) as a function of larvae and pupae age. One sample of 20-25 beetles was collected at each temperature (22°C, 25°C and 30°C), at each time point (7, 14, 21 and 28 days after egg laying). Although 20E has a decreasing trend with age at both 22°C and 30°C, that trend was not observed at 25°C.
This experiment was written by Sherman Ward and Regina Knight-Mason, 2014 (www.beanbeetles.org).
Copyright © by Sherman A. Ward and Regina Knight-Mason, 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.