2013 Reduced Gravity Education Flight Program Drosophila Ethanol Sensitivity and Metabolism in Altered Gravity
Flight Date
April 9, 2013
PRINCIPAL INVESTIGATORS
Debora Wines, Ph.D.; Life Science Instructor, Billings Central Catholic High School, Billings, MT
CO-INVESTIGATORS
Luke Muskett; Billings Central Catholic High School
Sammy Elliott; Billings Central Catholic High School
Austin Van Delinder; Billings Central Catholic High School
Lauren Lee; Billings Central Catholic High School
Ian Byorth; Billings Central Catholic High School
Riley Nichols; Billings Central Catholic High School
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Abstract
Enzyme activity is an essential part of all biological processes. Enzyme function is dependent on
complex three-dimensional shapes that are held together through weak intramolecular
interactions. Microgravity could potentially disrupt these weak bonds, resulting in deleterious
changes in enzyme function, which could affect normal physiological functions. The question
addressed in our research was “Is sensitivity to and metabolism of ethanol affected in altered
gravity?” The enzyme that we focused on was alcohol dehydrogenase, which metabolizes
ethanol. Fruit flies were utilized as a model system because of extensive ethanol-related research,
as well as a similar alcohol dehydrogenase enzyme and intoxication signs to those of humans.
Both wild type (normal) and the adh mutants (lacking alcohol dehydrogenase) were utilized for
behavioral and biochemical assays. Sensitivity to ethanol was quantified by determining ST50s,
the time taken for 50% of exposed flies to become immobilized. On the ground, the ST50 for
both strains of flies averaged about 20 minutes. On the zero gravity flight, technical difficulties
as well as the complex interaction of intoxication behavior and effects of micro- and
hypergravity prevented the determination of clear ST50 values. Measurements of ethanol levels
in flies showed that flies in altered gravity metabolize nearly twice as much ethanol as flies on
the ground. Interestingly, adh flies show marked metabolism of ethanol. Comparisons between
wild type and adh strains indicate that the activity of the alcohol dehydrogenase enzyme is
impacted by altered gravity, but that other enzymes are likely to be involved as well. Our results
show clear impacts of altered gravity on ethanol metabolism utilizing an in vivo system.
Enzyme activity is essential to all life processes, and changes in enzyme function underlie many
diseases, perhaps including those that affect astronauts, such as bone loss. Studying enzymes in
microgravity could provide new insights into disease and offer new insights into potential
pharmaceuticals.
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Introduction
Enzyme function is essential for all biological processes. Enzymes depend on complex threedimensional shapes stabilized by weak intramolecular interactions for their activity. Our
experiments question the possibility that microgravity could potentially disrupt or alter these
weak bonds, affecting enzyme activity, which could affect physiological functions. The fruit fly,
Drosophila melanogaster was selected as a model system for this study, with the focus on
ethanol sensitivity and metabolism. Drosophila is one of the most thoroughly studied model
organisms in biology, with a high degree of molecular similarity to mammals, including genes
and enzymes involved in the actions of ethanol. Fruit flies exhibit behavioral changes upon
ethanol exposure similar to those exhibited by intoxicated humans, and the enzyme alcohol
dehydrogenase (adh) is a principle enzyme involved in ethanol metabolism in both humans and
flies. The experiment's focus is the effect of altered gravity on the activity of alcohol
dehydrogenase (adh), utilizing biochemical assays to measure the metabolism of ethanol or
behavioral assays to measure flies' physiological responses to ethanol. The experimental design
provides an indirect measurement of the function of alcohol dehydrogenase in altered gravity,
and hence addresses the general question of whether in vivo enzyme activity is influenced by
alterations in gravity.
Statement of the Research Objectives
1. Conduct a biochemical assay to compare metabolism of ethanol in fruit flies on the ground to
that of flies exposed to gravitational oscillation in parabolic flight. Flies will be exposed to
ethanol vapors on the ground immediately before flight, allowed to recover for approximately 2
hours on the ground or on the Zero G flight, then frozen for subsequent determination of body
ethanol levels. Recovery ethanol levels will be compared to initial body ethanol levels to
determine how effectively ethanol was metabolized.
2. Conduct a behavioral assay to examine sensitivity of fruit flies to ethanol. To quantify
intoxication, the standard time taken for 50% of a population of 20 flies to become immobilized
(the ST50) will be determined under standard conditions on the ground, and also during
gravitational oscillations in parabolic flight. ST50's will be determined from video captured
during the flight.
3. Utilize analyses of Drosophila lacking a functional alcohol dehydrogenase gene (adh mutant)
as a control for biochemical and behavioral assays to help understand effects due to alterations in
this enzyme's activity.
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Methods and Materials
Fruit Flies
Drosophila melanogaster strains were obtained from Carolina Scientific and maintained on
Nutri- Fly™ fly food (Genesee Scientific). Wild type strains and the adh mutant lacking a
functional alcohol dehydrogenase enzyme were utilized. Flies were anesthetized with carbon
dioxide for handling. Cultures were maintained at room temperature, and males were isolated
within two days of eclosing. Experiments were conducted with male flies within one week of
eclosion.
Figure 1. Exposure vials are housed in a lexan rack. Flugs with
ethanol are enclosed in lexan lids with syringes affixed. Slidable
tabs allow ethanol vapors to reach the vials housing the flies.
Syringes allow placement of the flugs adjacent to the screen lids
of the vials. Fly behavior was filmed for subsequent analysis.
Ethanol Exposure
Everclear (95% ethanol) was purchased from local liquor stores by our teacher and dyed blue for
visibility. Flies were exposed to ethanol vapors in plastic fly vial with ethanol pipetted onto
Flugs (cellulose acetate stoppers; Genesee Scientific). For the Zero-gravity flight, special vial
lids were designed to contain the ethanol vapors and to allow a simple method to begin ethanol
exposure (shown in Figure 1). An MQ-3 ethanol sensor (Spark Fun Electronics) operated by an
Arduino microcontroller was utilized to test all exposure lids for the zero gravity flight for
leakage of ethanol vapor.
Measurement of Fly Ethanol Levels
Microfuge tubes containing 20 flies were homogenized in 500µl Tris (7.5) and centrifuged for 20
min. 10µl samples were utilized in duplicate tests utilizing the Ethanol L3K® assay (Sekisui
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Diagnostics) with modifications (Heberlein Lab, personal communication). Measurements were
conducted with a Tecan Infinite M200 with a Quad4 monochromator. Data was analyzed with
the Magellan v.6.4 software.
Determination of ST50s
ST50 is the time it takes for 50% of fruit flies being exposed to ethanol to become sedated, or
unable to right themselves (Maples and Rothenfluh, 2011). Flies were exposed to either 4 ml or
500µl of ethanol pipetted onto a Flug. ST50s were determined from analysis of behavior filmed
utilizing video cameras mounted on tripods on the ground, or cameras mounted on ball head
camera stands on the zero gravity flight (shown in Figure 1).
Results
Table 1 and Figure 2 summarize our experimental data regarding metabolism of ethanol by fruit
flies in altered gravity. Initial ethanol levels were similar in adh and wild type flies, and were 460
and 475 mg/dl, respectively. After two hours of recovery on the ground, levels dropped to 206 or
263 mg/dl, and after two hours of recovery on the zero gravity flight, levels dropped to 156 or
123 mg/dl for the adh and wild type flies, respectively.
Table 1. Drosophila Ethanol Levels1
Sample2
Strain3
Exposure4
mg/dl average5
(# of samples)
Initial Time Point
2 hrs Ground
2 hrs Flight
Initial Time Point
2 hrs Ground
2 hrs Flight
Control
Control
adh
adh
adh
Wild Type
Wild Type
Wild Type
adh
Wild Type
exposed
exposed
exposed
exposed
exposed
exposed
none
none
460.3 (8)
206.3 (8)
155.9 (10)
474.8 (8)
262.6 (8)
123.3 (10)
115.9 (26)
155.6 (26)
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Ethanol levels were measured utilizing the Ethanol L3K® Assay on pooled samples of 20 flies
Initial time Point, Ground, and Flight flies were exposed to ethanol for 40 minutes on the ground. Initial
time Point flies were frozen immediately after exposure; Ground flies recovered in normal gravity for 2
hours in new vials before being frozen; Flight flies recovered on the zero gravity flight.
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Wild Type flies are normal; adh strain has an inactive alcohol dehydrogenase gene
4
Ethanol exposure was to 4ml of ethanol for 40 minutes. Control flies were exposed to no
ethanol and were frozen alongside the initial time point, ground, and flight exposed flies.
5
Numbers are averages expressed in milligrams per deciliter. Sample size is number of
vials analyzed, each containing 20 flies.
2
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Figure 2. Ethanol levels in Flies
Data is from Table 1. The vales of ethanol are expressed in mg/dl and represent the average value for all
of the samples used in that category. The control samples were not exposed to ethanol. The initial time
point represents the ethanol levels of the flies that were exposed to ethanol and then frozen. The recovery
flies were exposed to ethanol for 40 minutes and then allowed to recover for 2 hours on the ground or
aboard the altered gravity flight. Bars shown indicate 5% error both below and above the average
(Microsoft Excel).
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Table 2 displays our preliminary data regarding the sensitivity of fruit flies to ethanol as measured with
ST50 determinations. Values are shown for both wild type and adh flies, with exposures utilizing flugs
with 4.0 ml of ethanol, or 0.5 ml of ethanol. The average ST50, or time taken for 50% of the flies in a
vial of 20 flies to become immobilized, ranged from approximately 21 to 23 minutes. The trials with 0.5
ml of ethanol were conducted utilizing the exposure chambers designed for the zero gravity flight. This
volume was chosen to minimize any chance of leakage of ethanol fumes during the flight. No ST50
values were obtained for the exposures on the zero gravity flight, however, due to technical problems and
the complexity of the interactions between effects of ethanol and altered gravity on fly behaviors (see
Table 3).
Table 2. Determination of ST50's1
Ground2
ST50 (min)
Exposure3
Wild Type4
adh5
4 ml/flug
23.3
20.7
0.5 ml/chamber
22.6
21.5
Sample Size6
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4
1
Intoxication was quantified by determining the ST50, the time taken for
50% of the flies to pass out from ethanol exposure. On the flight ST50 were
unable to be determined.
2
Preliminary tests were done on the ground prior to the flight to get a
baseline quantitative data point to be compared to the ST50’s on the flight.
3
Initial exposures were 4 ml of ethanol but were reduced to 500 microliters
due to safety considerations on the flight
4
Wild type flies are normal flies.
5
adh flies are mutants that lack the alcohol dehydrogenase enzyme
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Vials of 20 flies were tested to find an ST50; sample size is the number of
vials averaged to determine the ST50
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Table 3 summarizes fly behaviors observed on the zero gravity flights. The behavior of flies that
were not intoxicated was not affected in altered gravity, and was indistinguishable from behavior
on the ground. As flies became intoxicated, their behavior was altered in ways that had been seen
on the ground, such as less negative geotaxis (spending more time lower in the vial and on the
bottom of the vial). However, as the flies became more intoxicated, changes in gravity affected
behaviors dramatically. When partially intoxicated in microgravity, the flies spun around
because of their wing vibrations and twitching (spinners). Flies that were completely passed out
floated around in microgravity (floaters) because there was no movement of their wings, and also
slammed to the bottoms of the vials in hypergravity. Quantifiable data was not obtained from
these experiments
Table 3. Observation of Fly Behavior in Altered Gravity7
Ethanol
Ground 1
Flight 2
Exposure 3
Zero Time
50% ST50 5
ST50
2 times ST50
Crawl on the sides of vials,
negative geotaxis4
More time on bottom of vial,
some incapacitated, random
twitching, difficulty climbing
above the base
½ intoxicated, nearly all
crawling on or near bottom,
rapid twitching and wing
flutters
All on bottom, little crawling,
and twitching
Crawl on sides of vials,
negative geotaxis
Some flies begin to drop off
sides of vial
Floaters and spinners in
microgravity, free fall in
hypergravity, some hooked to
bottom6
All floaters, spinners, or
clinging to bottom
1
Flies exposed to Ethanol on the Ground
Flies exposed to Ethanol on the Zero-G Flight
3
Exposed to 500μl Ethanol; times are approximations
4
Crawling up to the top of the vial
5
ST50 is the standard time taken for 50% of a vial of flies to pass out; 50% ST50 is an estimate
of approximately half that time
6
Floaters are totally passed out, spinners have vibrating wings which cause them to wildly spin
in microgravity; floaters free fall to the bottom of the vial in hypergravity; some flies stay
hooked to the bottom of the vial with their claws
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Behaviors were similar for wild type and adh flies
2
Discussion
Our results indicate that enzyme activity is affected in altered gravity. Both the wild type and
adh strain metabolized significantly more ethanol on the flight than on the ground. Comparisons
of adh and wild type patterns of metabolism clearly show differences which indicates effects on
the alcohol dehydrogenase enzyme. However, the fact that metabolism in the adh strain was
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greatly impacted by altered gravity indicates that other genes are involved, and suggests that
altered gravity may affect many or all enzymes. This experiment had a large sample size with
minimal variation; however, we would like to repeat this experiment to see if the results are
consistent. We would also like to test other Drosophila mutants that affect alcohol metabolism to
see if the affect varies.
The purpose of the behavior experiments was to see if flies exposed to ethanol in altered gravity
would have altered sensitivity to ethanol. The ST50 test was utilized as it is a simple assay on the
ground to show differences in ethanol sensitivity in different strains of fruit flies. Unexposed,
normal flies show no unusual behavior in Micro or Hyper gravity but when the flies become
intoxicated, their behavior becomes complex. Effects of alterations in gravity interacting with the
effects of intoxication cause the flies to exhibit behavioral traits such as spinning uncontrollably
when the fly’s wings twitch and floating when passed out due to intoxication. This made the
identification of an ST50 indeterminable. A different assay would be required to conduct this
experiment in the future.
Conclusion
Our research indicates that enzyme activity is affected by altered gravity. This could have major
implications for future NASA research because enzyme activity controls all biological functions.
Future research of enzyme structure in microgravity could provide insight as to why the
astronauts experience health problems, such as bone loss, on the ISS. It could also provide
information about how to design pharmaceuticals to be more effective.
Outreach Items
The outreach items used were a slinky, an hour glass, and a wind-up chicken. The slinky was
most affected by altered gravity. In hyper gravity the slinky stretched to the floor without any
added force. In microgravity, when placed on top of the glovebox, the slinky floated away
without stretching instead of stretching and falling off the glovebox. It only stretched with added
force.
The hour glass was also affected by the changes in gravity. The sand flowed faster in hyper
gravity but in microgravity the sand did not flow at all. It floated inside the glass and did not
separate.
The chicken was least affected by the change in gravity. The time it took to unwind was the same
with normal gravity and microgravity. In hyper gravity, the chicken was only able to walk in
place. With normal gravity the chicken walked on the ground. During microgravity the chicken
floated and did flips while the legs moved.
Bibliography
"Flies In Space - Drosophila: Life Cycle." Flies In Space - Drosophila: Life Cycle. Web.
http://quest.nasa.gov/projects/flies/lifeCycle.html. 05 Sept. 2012.
"Life Cycle of the Fruit Fly." Life Cycle of the Fruit Fly. Web.
http://www.woodrow.org/teachers/bi/1994/life_cycle.html. 07 Sept. 2012.
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Maples, T., Rothenfluh, A. A Simple Way to Measure Ethanol Sensitivity in Flies. J. Vis.
Exp.(48), (2011). http://www.jove.com/video/2541/a-simple-way-to-measure-ethanol-sensitivityin-flies. Sept. 2012.
Maroni, G., and C.C Laurie-Ahlberg. "Genetic Variation in the Expression of ADH in
DROSOPHILA MELANOGASTER." NCBI. Web.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1201870/. 15 Oct. 2012
Sekisui Diagnostics. Ethanol Assay. Assay. 28 January, 2010. September-April, 2012-13.
Shohat-Ophir, Galit. Ethanol Absorption Assay. Assay. 2004. September-April, 2012-13.
Shohat-Ophir, Galit. "Sexual Deprivation Increases Ethanol Intake in Drosophila."
Sciencemag.org. Web. http://www.sciencemag.org/content/335/6074/1351. 12 Sept. 2012.
Acknowledgements
Dr. Adrian Rothenfluh answered questions about vials, lids, and explained his video about
exposing fruit flies to ethanol and the idea of finding a ST50 to use in the experiment.
Dr. Galit Shohat-Ophir, Ulrike Heberlein and Dr. Sharmila Bhattacharya answered technical
questions and provided protocols regarding Drosophila ethanol exposure.
Dr. Florence Gold gave constant guidance in what needed to be done to complete a project for
NASA
Genesee Scientific provided advice on our project and donated valuable equipment.
Dr Brian Stephens of the University of Houston at Clear Lake for assistance with the ethanol
assay and access to a university lab and equipment.
Dave DeBats and Exxon Mobil, and the Widdicombe family gave generous financial support for
this project.
Our principal Mr Sheldon Hanser and school board of Billings Central, created the STEM class
and provided many of the resources necessary for completing our experiment. Dr. Craig Pierson,
Dr. Mark Elison, and many members of the Billings Central Faculty provided valuable advice
and assistance. Don Larson and Warren Schaff provided technical support throughout the
project.
CONTACT INFORMATION
Debora Wines, Ph.D., Instructor of Life Sciences
Billings Central Catholic High School
3 Broadwater Ave
Billings, 59101
406.861.0728
dwines@billingscatholicschools.org
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