Download EIA Buffer Concentrate (10X) 10ml $11.00 1

Temperature Dependent Changes in Hormone Levels of Developing Bean
Beetle Larvae (Callosobruchus maculatus)
Instructor’s Notes
The physiology of many organisms is linked to seasonal changes. The biochemical and
morphological changes observed during development are a product of the interactions between
genetics and the environment. Temperature is one of the most reliable indicators of seasonal
change that can induce hormonal changes in many organisms including insects. Alterations in the
concentration of hormones could affect the timing mechanisms of different growth and
developmental processes. One such hormone, ecdysone, is released in its inactive form and is
converted into 20-hydroxyecdysone (20E) which directs insect molting and metamorphosis.
Alterations in the levels of 20E could therefore affect the timing of molts in beetle larvae during
development. Temperature increases have been reported to shorten the generation time of bean
beetles, but the effect of temperature changes on 20E titers during beetle development is not
clear. In this activity, students will evaluate the effects of environmental temperature on 20E
titers in developing bean beetles at different instars.
Experimental Design
The student handout is written in such a way as to guide students through the process of
designing the experiment. However, the following questions can be used to direct students as
they devise their hypotheses and develop their experimental design.
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What is the objective of this experiment?
Given that temperature increases shorten generation times and 20E controls insect
molting and metamorphosis, what do you think will happen to the levels of 20E at a
specific developmental stage if temperature was to decrease or increase?
What hypothesis could you develop to evaluate the effects of temperature on 20E titers?
How would you test your hypothesis?
What variable (s) would you manipulate?
What are the dependent and independent variables?
o Incubation temperature?
o Incubation period?
How would you analyze the data collected after performing enzyme immunoassay (EIA)?
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The goal of this activity is to allow students to develop a biological question and then devise a
series of experiments that would enable them to gather evidence to support or refute their
hypothesis. This experiment requires that cultures containing dense populations of recently
emerged bean beetles be isolated prior to the lab. The time allotted for the introduction to this
study, and for open discussion to guide students in selecting variables, planning procedures,
controlling variables, planning measures, and finding flaws through questioning may require 1 to
1.5 hours. You may find it useful to allow students to spend additional time setting up beetle
cultures, learn to use the incubator (assess temperature stability), and discuss the principles and
procedures associated with EIA (also known as ELISAs, enzyme-linked immunosorbent assays).
Additionally, the availability of EIA practice kits (item # 10009658, Cayman Chemicals) may
allow students who are not familiar with this technique to practice the EIA assay prior to the
actual analysis of specimens. The basics of EIAs are described in a Cayman Chemical article
(Cayman 2014).
As a part of the experimental protocol students will need to track when the eggs are laid. This
may require each group to make daily observations to determine when oviposition has occurred.
Depending on how many mating pairs are placed in each dish, bean collection after oviposition
may take from 1to 5 days. Each group must maintain a record of when each bean with a newly
laid egg was removed from the petri dish and placed in the incubator, record the incubation
temperature, and the incubation period. The range of incubation temperatures used is limited by
the number of incubators available; however, keep in mind that changes in temperature can only
be within defined limits. It may be useful for students to perform the experiments using the
incubation temperatures listed in the “Handbook on Bean Beetles” (Beck and Blumer 2014)
which list the generation times associated with specific incubation temperatures. Of course
temperatures other than those listed in the Handbook may be used.
My students chose to collect samples every 7 days at each incubation temperature. Alternatively,
students could vary the sampling interval since the generation time of bean beetles decreases
with increasing temperatures. At temperatures of 30°C, 25°C, and 22°C generation time is 3-4,
5-6, and 7 weeks, respectively. Data from each group were compiled to undergo statistical
analysis.
Equipment and Supplies
For a class of twenty students working in pairs:
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10 bean beetle cultures with newly emerged adults
Plastic petri dishes (100 x 15mm) to serve as mating chambers, and for housing
developing beetle larvae in incubation chambers.
Marking pens
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Insect culture incubators (item # 173150), Carolina Biological.
Benchmark Plus Microplate Spectrophotometer (BIO RAD) with absorbance set at
415nm.
Frozen larvae (15 beetles/tube) were homogenized (glass tube mortar and Teflon pestle)
in 500 μl of ice-cold 75% aqueous methanol.
Table top centrifuge (homogenate centrifuged at 13,000g for 10 min and supernatant
extracted)
EIA was used to estimate 20E titers in extract of beetle larvae.
EIA Supplies* (20 students)
Cayman Chemical Company
(800) 364-9897
www.caymanchem.com
Item
Quantity
Price
Unit
20-Hydroxyecdysone EIA Antiserum
Item # 482202
100dtn
$42.00
1
20-Hydroxyecdysone AChE Tracer
Item # 482200
100dtn
$124.00
1
Precoated (Mouse Anti-Rabbit IgG)
EIA 96-Well Strip Plate Item # 400004
each
$21.00
2
EIA Buffer Concentrate (10X)
Item # 400060
10ml
$11.00
1
Ellman’s Reagent
Item # 400050
100dtn
$10.00
1
UltraPure Water
Item # 400000
1L
$14.00
1
Wash Buffer Concentrate (400X)
Item # 400062
5ml
$8.00
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*Note: Procedures for making EIA solutions and performing the EIA assay are listed in the
inserts that accompany the reagents. Prices are in US dollars as of February 2014.
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Sample Collection and Analysis
Sample collection for the experiment involves the removal bean beetles from insect incubators
set at specified intervals post-oviposition. Approximately 20-25 beans should be collected for
each sampling interval. Samples collected at various sampling intervals were stored at -20°C.
Beans were dissected and beetle larvae were removed and homogenized in a 75% aqueous
methanol solution (15 beetles / 500 μl). The homogenate was centrifuged and the supernatant
was stored at -20°C. Titers of 20E were assayed using EIA. The solutions needed to perform
EIA, which are listed above, can be obtained from Cayman Chemicals. Protocols for the
preparation of assay specific reagents, and procedures for performing the EIA assay are
contained in inserts provided by Cayman.
Data Analysis
Within temperature and between different temperature analyses of multiple 20E titers (at
different larval stages) can be performed with an analysis of variance or a non-parametric
alternative such as a Kruskal-Wallace test. Comparisons between two 20E titers, such as an early
larval stage and a late larval stage evaluated at the same temperature, or between two different
temperatures at the same larval stage may be performed using a two sample test such as a t-test
or the non-parametric Mann-Whitney test.
Literature Cited
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.
Cayman Chemical. 2014. Enzyme Immunoassays: The Details.
https://www.caymanchem.com/app/template/Article.vm/article/2155.
This experiment was written by Sherman Ward and Regina Knight-Mason, 2014
(www.beanbeetles.org).
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