Bio 3B
Fall 2015
Kara Kirkpatrick, Richard Niederecker, Samyar Attarian
The Effect of Caffeine on the Resistance to Heat in the fruit flies Drosophila melanogaster
Abstract
Drosophila melanogaster is the species being tested. The production of heat shock
proteins (HSPs) is known to increase under stressful situations, and in other conditions as well,
such as development. An increase in heat shock proteins allows for proteins to, essentially, be
renatured. The renaturation of proteins allows for an increased lifespan. It is hypothesized that
caffeine, a stressor, will induce heat shock factors (HSFs) to produce more HSPs, and extend the
lifespans of Drosophila melanogaster in elevated temperature conditions, compared to the
lifespans of those without caffeine. Drosophila melanogaster were split into control groups and
test groups. Test groups were introduced to a caffeinated medium and control groups were given
uncaffeinated medium. Both groups were kept in an incubator at 32°C. Two tests were
performed: the first measured lifespans of flies beginning at the pupal stage, and the second
measured the lifespans starting from the larval stage. Lifespans were measured and analyzed
using unpaired, one-tailed t-tests. The average lifespan of the caffeinated flies introduced at the
pupal stage was 5.05±0.28 days, and uncaffeinated fruit flies’ average lifespans were 4.27±0.32
days. The average lifespan of the caffeinated flies introduced at the larval stage was 2.49±0.12
days, and the average lifespan of uncaffeinated fruit flies was 4.85±0.27 days. The caffeinated
flies that were introduced to heat at the pupal stage had a significantly longer lifespan than the
flies introduced without caffeine (p=0.0347, N=60). However, the caffeinated flies that were
introduced to heat at the larval stage had a significantly shorter lifespans than the flies introduced
to heat without caffeine (p=1.45x10-13, N=120).
Introduction
Drosophila melanogaster is a species of fruit fly which produces heat shock proteins
(HSPs) as a response to stressful situations, as well as unstressed situations. HSPs are
specifically chaperone proteins that correct the denaturation of proteins under strain. The
expression of heat shock proteins is regulated by the heat shock transcription factors (HSFs) in
response to various stressful inducers, such as elevated temperatures, oxidants, heavy metals, and
bacterial and viral infections. HSFs can also be stimulated for non-stress conditions such as
growth and development proliferation of cells (Prikkala, 2001).
The purpose of this experiment is to determine if drugs, specifically caffeine, are inducers
of HSFs to help with fruit flies’ resistance to heat. Caffeine was found to help the development
of Drosophila melanogaster in a heated environment compared to heat alone (Shiffman, 2014).
Caffeine supplementation has been shown in a previous study to increase the production of
HSP72 (heat shock protein 72) in humans exercising greater than that of humans exercising
alone (Whitham, 2006). In another experiment, determining if heat shock proteins were
expressed in response to elevated temperatures in humans, participants refrained from
consumption of caffeine for 24 hours before tests (Iguchi 185). This omission of caffeine allows
one to assume that caffeine does have an effect on the release of heat shock proteins. In the
amoeba Dictyostelium Discoideum, caffeine introduction was found to increase concentrations
of HSP102, and HSP69, but not found to increase concentrations of HSP90, HSP85, HSP72,
HSP50 and HSP29; thus showing that caffeine induces specific HSPs (Hagmann, 1986). An
increase in heat shock proteins decreases the total amount of denatured proteins, therefore
allowing the flies to endure higher temperatures. It is predicted that the Drosophila melanogaster
ability to overcome environmental stresses can be enhanced through the consumption of
caffeine, while under heated conditions, which induces HSFs to produce more HSPs, ultimately
extending the lives of the fruit flies.
Methods
The objective of this research experiment is to determine if caffeine has an effect on flies’
resistance to heat. In this experiment, the researchers Richard, Kara, and Samyar cultivated three
colonies, each containing 120 Drosophila melanogaster. Each colony was divided amongst 12
fruit fly culture tubes, while remaining at room temperature with a controlled medium in each
tube. These flies were separated into two groups: the control and the test group. The control
group, consisting of 60 flies, was placed in an incubator at 32°C, to simulate a heated
environment. The test group, consisting of 60 flies, was also kept at a ratio of ten flies per tube,
each with a 1% caffeinated medium. The test group was placed in the same incubator at 32°C.
The flies were inspected as frequently as possible (every one-to-three days) to determine the
length of their lifespans. The average lifespan between caffeinated and uncaffeinated flies was
analyzed using a one-tailed, unpaired t-test.
To prepare the medium for the first colony, five mL of dried medium and and five mL of
water were measured using a 25 mL graduated cylinder. This amount of medium dried quickly
in the incubator, therefore, the amount of medium was doubled for the second and third colonies.
To prepare the medium for the second and third colonies, 10 mL of water was measured for each
fly culture tube using a 10 mL beaker. To keep an equal ratio of water to medium, the same 10
mL beaker was used to add 10 mL, or about 3.24 grams, of dried medium. For all of the
colonies, the 1% caffeine solution was prepared by mixing one gram of concentrated caffeine in
100 mL of water. Undissolved caffeine was removed from solution using gravity filtration with
a #41 ashless filter paper cone and a small funnel. To prevent dehydration, ten drops of water
per visit were added to the incubated medium.
Lifespans of the wingless flies were measured from different development stages
between the first and the last two colonies. All the larvae in the first colony were developed into
pupae at room temperature with uncaffeinated medium before they were tested for lifespan in
heated conditions, with and without caffeine. The second and third colonies were tested in heat,
starting from the larval stage, so that the larvae could be immersed in the caffeinated medium.
Results
Duration of the lifespans were measured differently for the first colony and for the second and
third colonies. Lifespans for the first colony were measured from placement in heat as pupae
until death, and from the larval stage until maturation into the pupal stage for the second and
third colonies, as shown in Figures 1 and 2.
Figure 1. Colony one data collected over ten days.
Figure 2. Colonies 2 and 3 data collected over fourteen days and eleven days.
The pupae and larvae that did not develope were assumed to have deceased prior to the second
observation. Based on this information, a one-tailed, unpaired t-test was performed to analyze
the heat resistance of the flies starting from the pupa stage. This test revealed that the first
colony of caffeinated flies had an average lifespan of 5.05±0.28 days and the uncaffeinated flies
had an average lifespan of 4.27±0.32 days (Figure 3.).
Figure 3. Mean life spans of caffeinated and uncaffeinated Drosophila melanogaster in colony
one. The average lifespan of caffeinated fruit flies was 5.05±0.28 days and the average lifespan
of uncaffeinated fruit flies was 4.27±0.32 days. Error bars are mean ± SEM. A one-tailed,
unpaired t-test revealed that caffeinated fruit flies had a significantly longer lifespan than
uncaffeinated fruit flies (p=0.0347, N=60).
A second one tailed unpaired t-test was performed on the second and third colony data to test the
heat resistance of the flies starting from the larval stage. This test revealed that the second and
third colony caffeinated flies had an average lifespan of 2.49±0.12 days and the uncaffeinated
flies had a lifespan of 4.85±0.27 days (Figure 4.).
Figure 4. Mean life spans of caffeinated and uncaffeinated Drosophila melanogaster in colony
two. The average lifespan of caffeinated fruit flies was 2.49±0.12 days and the average lifespan
of uncaffeinated fruit flies was 4.85±0.27 days. Error bars are mean ± SEM. A one-tailed,
unpaired t-test revealed that caffeinated fruit flies had significantly shorter lifespans than
uncaffeinated fruit flies (p=1.45x10-13, N=120).
Starting from the pupal stage, the average lifespan of caffeinated flies was found to be
significantly longer in flies that were given caffeine under heated conditions, based on the one
tailed unpaired t-test (p=0.0347, N=60). However, starting from the larval stage, the average
lifespan of uncaffeinated flies was found to be significantly longer than the lifespans of those
flies given caffeine (p=1.45x10^-13, N=120).
Discussion
Different techniques used in this experiment showed different results. Colony one
supported the hypothesis that caffeine and heat, combined, increases the lifespan of adult fruit
flies compared to those that were not given caffeine. The following conclusion can be
tentatively drawn according to the data collected on colony one: each stress, alone, is not able to
stimulate enough HSP production to increase survivability. The combined stresses of heat and
caffeine is hypothesized to increase heat shock proteins to an amount that is capable of
prolonging the lives of caffeinated Drosophila melanogaster. However, upon further analysis of
this data, it was found that the flies that developed in the caffeinated medium did not actually
live longer than those that developed in the uncaffeinated medium. The average lifespan of the
developed caffeinated flies was found to be 6.72±0.26 days, whereas the average uncaffeinated
lifespan was found to be 8.43±0.53 days. The results are due to the fact that 33 caffeinated flies
developed and 14 uncaffeinated flies developed; the amount of caffeinated flies that developed
was more than double the amount of uncaffeinated flies that developed. This shows that the
caffeinated flies did not necessarily live longer, but that more of them survived to adulthood,
possibly due to the caffeine. To further test the hypothesis, the flies must be developed in the
caffeine under heat to determine life span.
The testing on colonies two and three, on the other hand, gave contradicting results.
Colonies two and three accepted the null hypothesis that caffeine and heat, combined, decreased
the lifespan of adult fruit flies compared to those that were not given caffeine. This data is
supportive of the theory that caffeine and heat are independent stresses to one another, so when
these stresses are combined, the lifespan of Drosophila melanogaster is decreased. This could
be due to various factors. In colonies two and three, larvae were introduced to the medium, as
opposed to the technique used in colony one, where pupae were isolated outside of the medium,
and could not consume the 1% caffeine solution until adulthood. This technique used in colony
one allowed the the Drosophila melanogaster to mature into adulthood, but they weren’t under
caffeinated conditions until adulthood. Looking back to colonies two and three, the larvae were
able to consume the caffeinated medium, introducing it into their systems before reaching
adulthood. This technique used in the later colonies was expected to show more accurate results,
but more extensive testing is needed to allow the larva and pupa to reach adulthood.
Sources of uncertainty in this experiment include the development of the pupa in
caffeine, and the stability of the medium under heat. In the first colony, less than half of the
uncaffeinated flies developed in comparison to the caffeinated flies. It is uncertain if this result
is due to the medium, for the pupa were not consuming the medium. In the second and third
colonies, the medium became brown and rotten smelling after a couple of days. The effect of
these adverse conditions on the lifespan of the flies is unknown, but it may have unavoidably
shortened the lifespan of the flies, independent of the effects of caffein or heat.
Overall, it was found that caffeinated adult Drosophila melanogaster have seemingly
longer lifespans than uncaffeinated adult Drosophila melanogaster when introduced at the pupal
stage, but significantly shorter lifespans when introduced at the larval stage. According to the
data pertaining to introduction at the pupal stage, flies that developed did not actually live longer,
but more flies lived. Reasons for the difference in response to combined caffeine and heat,
between developing Drosophila melanogaster and adult Drosophila melanogaster, are not
definitely known, however, some hypotheses can be deduced from these differences. In the first
colony, the flies were found to develop into adults more when caffeine was introduced, however,
their lifespans were not longer than those of the uncaffeinated flies. This shows that the caffeine
is an added stress on the flies in addition to the heat they are exposed to. This was further
confirmed with the second and third colonies. One approach is that the larvae could be more
sensitive to stressful situations than adult fruit flies are. This could allow an increased number of
caffeinated flies from colony one to develop into adulthood. In contrast, in colonies two and
three, this would cause a decrease in the development of caffeinated larvae into pupa. Stating
this, it can be deduced that caffeine increases flies’ likelihood to develop.
References
Hagmann, J. (1986) “Caffeine and Heat Shock Induce Adenylate Cyclase in
Dictyostelium Discoideum.” The EMBO Journal 5.13: 3437–3440. Print.
Iguchi, Masaki (2012) “Heat Stress and Cardiovascular, Hormonal, and Heat Shock
Proteins in Humans.” Journal of Athletic Training 47.2: 184–190. Print.
Prikkala Lila, Sistonen Lia, Nykanen Paivi (2001). Role of heat shock transcription
factors in regulation of the heat shock response and beyond. The Journal of the
Federation of American Societies for Experimental Biology. Vol.15 no. 7, pp
1118-1131
Shiffman Benjamin, Soliman Kareem (2014) “The effect of heat and caffeine on the
development of fruit flies (Drosophila melanogaster). Department of Biological
Sciences Saddleback College.
Whitham Martin, Walker Gary J., Bishop Nicolette C. (2006). Effect of caffeine
supplementation on the extracellular heat shock protein 72 response to exercise.
American Physiological Society Journal of Applied Physiology. Vol. 101 no. 4, pp
1222-1227.
Budget
Wingless Fruit Fly Starter Culture (Drosophila melanogaster)- $7.99
CVS Caffeine Tablets-$11.99
Drosophila melanogaster fruit fly media 1.5lbs- $11.99
36 Drosophila culture vials, plugs, caps- $50.45
Mini Incubator-$580
Total Budget- $662.33