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. Drosophilamelanogaster 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 Drosophilamelanogaster 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 DictyosteliumDiscoideum, 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 uncaffeinatedDrosophila 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 uncaffeinatedDrosophila 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 theDrosophila 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 DictyosteliumDiscoideum.” 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, NykanenPaivi (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