A Comparison of Caloric and Protein Restriction in Drosophila melanogaster Sebastien Deveau STEM Research Project Massachusetts Academy of Math and Science December 3, 2012
Caloric and Protein Restriction 1 Abstract...........................................................................................................................................................2 Introduction..................................................................................................................................................3 Literature Review.......................................................................................................................................4 Methodology..............................................................................................................................................14 Results..........................................................................................................................................................16 Data Analysis and Discussion.............................................................................................................19 Conclusions.................................................................................................................................................20 Limitations and Assumptions.............................................................................................................21 Applications and Future Experiments............................................................................................22 Literature Cited.........................................................................................................................................23 Acknowledgements.................................................................................................................................24
Caloric and Protein Restriction 2 Abstract The relationship between caloric restriction and the longevity of invertebrates has been shown to be inversely proportional. However, the relationship between protein restriction and longevity has not yet been extensively studied. It has been hypothesized that Drosophila melanogaster cultured with restricted protein will have a longer lifespan than those cultured with restricted calories. The fruit flies were separated into vials, in which half were kept at a constant caloric content through the addition of sugar. The other half did maintain a constant caloric content but did have analogous dietary nutrition otherwise. The flies were then allowed to reproduce in their specified media. After the eggs were laid, the parental generation was removed and the F1 developed into the imago stage. At this point, the flies were separated by gender and allowed to complete their life. Their longevity was measured by the number of days the flies survived from the day of separation to the day of death. Because the diets were shown to produce a higher longevity, and because Drosophila melanogaster are purported to be good models of human physiology, speculations can be made about future areas of research in humans.
Caloric and Protein Restriction 3 Introduction
Scientists have used Drosophila melanogaster, or the common fruit fly, in
research experiments for many years. Due to their ease of use, similarity to humans, simplicity, and invertebrate nature, they pose a large role in the evolution of scientific discovery. An older area of research deals with the diets of the Drosophila melanogaster and the best way to culture them. Scientists conducted experiments of this nature to show that results from Drosophila melanogaster can be extrapolated to mice and other vertebrates.
Calorie restriction has been an area of interest over the years regarding
Drosophila and the increase of their lifespan. This phenomenon also relates to humans because more and more people are struggling with dietary issues. The main concern that people face when dealing with diets is whether or not it is more beneficial to consume fewer calories or consume more or less of the right nutrients.
In Drosophila melanogaster, this experiment is extremely easy to perform
because one of the main food ingredients that they consume and need is yeast. By comparing environments with varied amounts of yeast but a constant caloric content against environments with varied amounts of yeast but not a constant caloric content, it is possible to end this confusion and finally say which diet is best for longevity. Because of their purported similarity to humans, an experiment regarding the benefits of caloric restriction versus the benefits of nutrient restriction performed on Drosophila melanogaster may suggest some areas for human research.
Caloric and Protein Restriction 4 Biology of Drosophila melanogaster For years, scientists have studied in Drosophila melanogaster because of their ease of use, basic genetic structure, and similarity to humans. Drosophila, or fruit flies, are very useful models for studying human genetics. However, their use is not just limited to genetic research. The flies are also good models for many other humanlike qualities. This allows them to help to extrapolate possible human reactions, which allow for experiments to be performed on humans safely. Some of these experimental areas include a shared pathway of intercellular signaling, tumor formation, developmental patterning, learning and behavior, metastasis, neuronal degeneration, and the effect of drugs on behavior and neurotransmitters (Wixon et al., 2000). There are four main stages to the development of Drosophila: the egg, the larvae, the pupa, and the adult. The larvae also undergo three different stages, or instars, that start during the early phase of the larvae stage and are signified by a molting process. The length that the Drosophila remain in each of the primary stages is temperature reliant. At a temperature of twenty degrees Celsius, the Drosophila maturate from the egg to larval stage in approximately eight days while the pupal stage is nearly 6.3 days long. During the larval stage, the Drosophila melanogaster is the most active until the third instar, or final molting period. During this period, the fruit fly removes itself from the food and climbs the side of the test tube in preparation for the pupal stage. Due to the high activity of the insects during this stage, the food media that they are raised on becomes littered with many channels and ruts. From this stage, it is easiest to discern whether or not the developing
Caloric and Protein Restriction 5 generation will be successful based on their inclination to consume the food media (Demerec, 1967). A crucial focus point when working with Drosophila melanogaster is food restriction, which can lead to greater longevity and an overall decrease in morality in many different types of life forms. Food restriction extends the lifespan of many different organisms, and mammals are not an exception. Experiments regarding dietary restriction (DR), performed by scientists, have also been shown to reduce age-‐related damage, which allows the organism to be in a younger state for a longer amount of time (Chapman and Partridge, 1996, Chippindale et al., 1993, and Partridge et al., 1987). This has led to the theory that DR could reduce the rate at which life forms age. Studies regarding mortality have helped to support this idea because typically when any being ages, their mortality rate increases almost exponentially; therefore, lifespan can either be extended because of a lowered starting rate of mortality or a reduction to the level of increase in mortality. In Drosophila melanogaster, DR has a severe affect and the rate of mortality changes quickly between the control and DR values with a simple change in their nutritional regime. Therefore, DR does not affect the rate at which the fly ages (Partridge et al., 2005).
Table 1. Lifespan increase, recorded over the years, due to a 37% dietary restriction in Drosophila melanogaster (Partridge et al., 2005).
Caloric and Protein Restriction 6 An important feature about the adult Drosophila melanogaster is the ease of distinguishing the males and females. There are several external features to look for in their shape and overall appearance that allow for determining gender, including the pointed abdomen of the female, the dark markings in the abdomens that vary for sex, and the number of segments in the abdomen. This is extremely important because the male and female Drosophila vary greatly in their responses to experimental behavior. It is also exceedingly important to have both male and female fruit flies together in an environment; otherwise, all of the eggs that the females lay will be sterile, and there will be no progeny (A. Consilvio, personal communication, October 2012). Culturing Drosophila melanogaster Drosophila rely on fermenting fruits as their main food source as well as their primary breeding grounds, and as such they can survive on almost any aging fruit or fermenting medium. The main components that are necessary in any culture medium, however, are sugar, yeast, and agar. Without the sugar, the yeast will not grow; without the yeast, the larvae will not survive; and without a solid consistency, the medium will simply fall out of the test tube during the removal process. Temperature plays a key role in the longevity of the Drosophila melanogaster because they are exothermic organisms. As noted above, in a temperature of twenty degrees Celsius, it takes the flies eight days to move from the egg-‐larval stage to the pupa stage, and it takes 6.3 days to move from the pupa to imago stage. In a temperature of 25 degrees, on the other hand, it takes the flies approximately five days to move from the egg-‐larval stage to the pupa stage, and 4.2 days to move from
Caloric and Protein Restriction 7 the pupa stage to the imago stage. In total, the difference between the growth periods is nearly five days where the cycle can be completed in ten days at 25 degrees and in fifteen days at twenty degrees. Moreover, the flies should not be kept at temperatures far below twenty degrees or far higher than 25 degrees. When kept at low temperatures the life cycle takes too long to be of use, the larval stage takes 57 days to complete at ten degrees, and at higher temperatures, such as 30 degrees, the flies are susceptible to becoming sterile (Demerec, 1967). Previous Studies on Yeast Consumption and Longevity Multiple studies in this area have focused on the yeast content of the media of the Drosophila melanogaster; however, the way in which the experiments were performed produced vague results. Countless experiments have shown that Drosophila grow better if provided a food source that has yeast; however, the larval stage of Drosophila, which has been largely ignored in past experiments, benefits from the presence of yeast the most out of all the other stages of the life cycle. Numerous experiments were done to understand this relationship between the Drosophila melanogaster and yeast (Northrop, 1917).
Table 2. Number of flies produced with varying diets of yeast, banana, casein, and sugar (Northrop, 1917).
Caloric and Protein Restriction 8 A summary of the data in table 2 demonstrates that without the addition of yeast the flies were unable to produce a second generation. Additional experiments were also undergone to learn the appropriate amount of yeast needed to allow the flies to hatch from egg to pupa in the least amount of time. Additionally, the experiment measured the resulting time taken for them to continue on the cycle from the pupa stage and on to the imago stage. The results concluded that the appropriate amount of yeast is about one half the amount of banana, or the main food source, by mass measured in grams. To determine why the yeast was so important to longevity the scientists also developed experiments to test what exactly it was that the Drosophila needed to survive. Using the liver, kidney, and pancreas of a dog and the liver of a mouse the biologists made a food medium that could actually sustain the flies. Although this media did not sustain the flies as well as the traditional yeast enriched one, the diet did show that the substance in yeast, which is needed for the flies’ development, is also found in this mixture of tissues (Northrop, 1917). Previous Studies on Caloric Content and Longevity Multiple studies in this area have focused on the caloric content of the media of the Drosophila melanogaster; however, the experiments performed do not cover all of the aspects of caloric content. It was discovered that not only does food restriction prolong life, but caloric restriction, an aspect of food restriction, also results in a greater life span. The commonly recorded effects of calorie restriction, or CR, are a decrease in body temperature, insulin levels, blood glucose, body fat, and overall weight (Guarente & Picard, 2005).
Caloric and Protein Restriction 9
The idea that animals under the influence of CR undergo a decrease in
metabolic rate was the originally accepted belief about CRs; however, further research upon the subject matter found that this was a myth. In C. elegans and budding yeast (Saccharomyces cerevisiae), it was shown that the metabolisms of the organisms actually sped up while placed on a CR diet, contrary to the previous findings. This diet has also exhibited an ability to stall diseases or prevent their overall appearance in the future (Guarente & Picard, 2005). In Drosophila specifically, the dilution of yeast has been shown to prolong the average lifespan. The introduction of the diet to Drosophila demonstrates that CR will only affect the short-‐term uncertainty of life, not their long-‐term wellbeing. This shows that either the CR sets the amount of damage needed to cause death to a higher amount, or that it causes a damage reversal in the body of the fly (Guarente & Picard, 2005). In another experiment performed by Mair and his colleagues, it was shown that calorie restriction has an unusual effect on Drosophila melanogaster. When the flies were put on the diet their morality decreased significantly; however, shortly after they were removed from the diet the mortality of the flies began to resemble that of the control group. It was also shown that the flies that were put on the CR diet had approximately the same lower morality all around, without regard to time of placement. Whether the flies were put on the diet for the entirety of their lives, or if they were put on the diet near the end of their lives, their morality still decreased by the same amount (Mair, Goymer, Pletcher, & Partridge, 2003).
Caloric and Protein Restriction 10
Figure 1. Male mortality rates based on varying diets. In figure 1A the mortality rate of flies with a diet restriction is compared to that of those that have a full diet. After eighteen days, a portion of the fully fed flies were moved to the diet restriction and shortly after their mortality rates dropped down to m atch that of those already on the restrictive diet. In figure 1B the mortality rate of flies with a diet restriction is compared to that of those that have a full diet. After eighteen days, a portion of the dietary restricted flies were moved to a full diet and shortly after their mortality rates rose to match that of the fully fed flies (Mair, Goymer, Pletcher, & Partridge, 2003).
Research Proposal Researchable question: How do culture media with varying protein concentrations affect Drosophila lifespan versus culture media with varying caloric concentrations? Hypothesis: Drosophila cultured on media with caloric or protein restriction will have extended lifespans, and flies cultured on protein-‐restricted media will live longer than flies cultured on caloric restricted media. Procedure: To test how yeast and caloric diets affect the longevity of Drosophila there will be many different environments created for the fruit flies. The base environment will comprise 55.059 grams of water, 0.952941 grams of yeast, 0.550588 grams of soy flour, 4.02353 grams of yellow cornmeal, about 0.317647 grams of agar, 4.235 grams of light corn syrup, 0.265412 grams of mold inhibitor,
Caloric and Protein Restriction 11 and 0.521394 grams of sugar (Leora, 2011). The environments are made up of a control media that will contain regular the previously stated amounts, two control media without any yeast, two variable media with 30% higher yeast content, and two variable media with 30% lower yeast content. In one set of these environments, caloric content will remain constant through the addition or subtraction of sugar; in the other set of environments, the sugar content will remain constant. The media were created using a standard 250mL beaker. Each of the ingredients was then measured out to two times their original recipe in a standard plastic cup. The soy flour, baker’s yeast, light corn syrup, and sugar were then combined in a single cup with 18 grams of water and stirred continuously until all clumps were removed. Afterwards, the yellow cornmeal, agar, and mold inhibitor were combined into a single solo cup with 28 grams of water and were stirred continuously until all clumps were removed. The beaker was then filled with 20 grams of water and was placed on the stove inside a pot, to evenly distribute heat, until it boiled. Once the water began to boil, the yellow cornmeal mixture was added to the boiling water and was continuously stirred until the combined mixture began to boil again. Upon second boil, the soy flour mixture was added and was continuously stirred until it began to boil. The mixture was then allowed to boil with continuous stirring for 10 minutes and was then separated equally into three vials. This process was repeated for all seven separate environments. The flies will then be allowed to mature into their imago stage and will be separated by sex. After the flies are separated, their reaction times will be recorded by a simple test during which the vial is tapped repeatedly to move the flies to the bottom of the vial. Once the flies are at the
Caloric and Protein Restriction 12 bottom of the vial, their reaction time will be measured by the amount of time taken for the final fly to reach the top of the vial due to geotaxis. This process shall be repeated each day for two weeks and the reaction time of the control will be tested against the variable environments. Viability of diet will be assessed through the lowest reaction time. The controls of the experiment, aside from those mentioned above, included fly strain and temperature.
Caloric and Protein Restriction 13 Methodology To test how yeast and caloric diets affect the longevity of Drosophila many different environments were constructed for the fruit flies. The base environment comprised 110 grams of water (tap water, H2O), two grams of baker’s yeast (Saccharomyces cerevisiae, store brand), one gram of soy flour (store brand), eight grams of yellow cornmeal (store brand), about 0.3 grams of agar (purchased from Carolina Biological), eight grams of light corn syrup (store brand), 0.3 grams of mold inhibitor (purchased from Flinn), and one gram of sugar (store brand, C12H22O11) following the methodology of Leora (2011). The environments were made up of a control media that contained the previously stated amounts of ingredients, two control media without any yeast, two variable media with 30% higher yeast content, and two variable media with 30% lower yeast content. In one set of these environments, caloric content was kept constant through the addition or subtraction of the sugar. In the other set of environments, the sugar content remained constant. The media were created using a standard 250mL beaker. Each of the ingredients was then measured out to two times their original recipe in a standard 8.89 centimeter plastic cup. The soy flour, baker’s yeast, light corn syrup, and sugar were then combined in a single cup with 18 grams of water and stirred continuously until all clumps were removed. Afterwards, the yellow cornmeal, agar, and mold inhibitor were combined into a single plastic cup with 28 grams of water and were stirred continuously until all clumps were removed. The beaker was then filled with 20 grams of water and was placed on the stove inside a small pan to evenly distribute heat until it boiled. Once the water began to boil, the yellow
Caloric and Protein Restriction 14 cornmeal mixture was added to the boiling water and was continuously stirred until the combined mixture began to boil again (see figure 2). Upon second boil, the soy flour mixture was added and was continuously stirred until it began to boil. The mixture was then allowed to boil with continuous stirring for 10 minutes and was then separated equally into three vials (see figure 3). This process was repeated for all seven separate environments. Five to ten flies of each gender were placed in each vial and were allowed to reproduce. The parental generation was then flipped into a new vial, of the same type of environment, after four days. The eggs left in the original vials were then allowed to mature into their imago stage and were separated by sex into vials of control media (see figure 4). After the flies were separated, they were left alone to complete their life cycle. Viability of diet was assessed through the length of the life of the fly. The controls of the experiment, aside from those mentioned above, included fly strain (wild type Drosophila melanogaster) and temperature (27 degrees Celsius).
Caloric and Protein Restriction 15
Figure 2. Production of media for D. melanogaster. The media must be allowed to boil over a stove with the correct ingredients and constant stirring to avoid burning.
Figure 3. Media for D. melanogaster poured into vials. After being cooked, the media is then poured into three separate vials and is labeled according to the type of m edia.
Figure 4. Drosophila separated by sex. After the flies reach the imago stage, they are all separated by sex into control medias. Each vial is labeled with the date of transfer, type of media the flies were cultured in, and gender.
Caloric and Protein Restriction 16 Results
Table 3. The number of flies living per day of female Drosophila.
Lifespan
The Effects of Dietary Restriction on Drosophila (Alive, Females).
Days
Control
30% less with controlled calories
30% less
30% more with controlled calories
30% more
1
72
13
44
41
28
2
72
13
44
41
28
3
72
13
44
41
28
4
70
13
44
41
28
5
70
13
44
41
28
6
65
12
43
41
23
7
62
12
43
41
23
8
54
12
41
38
23
9
40
11
35
38
13
10
35
9
33
37
7
11
29
8
22
29
4
12
16
7
16
25
3
Caloric and Protein Restriction 17
Percent of female D. melanogaster alive
1.2
1 Control 0.8 30% less yeast with controlled calories
0.6
30% less yeast 0.4
30% more yeast with controlled calories 30% more yeast
0.2
0 1
2
3
4
5 6
7 8
9 10 11 12
Days Figure 5. Graph of the percent of alive female D. melanogaster in response to each diet. The above shows that the diet with 30% more yeast was detrimental to the lifespan of the fruit flies. It also shows that the diets with the highest lifespan were those that were restricted or supplemented with a constant caloric content.
Caloric and Protein Restriction 18 Table 4. The amount of flies living per day of m ale Drosophila.
Lifespan
The Effects of Dietary Restriction on Drosophila (Alive, Males).
Days
Control
30% less with controlled calories
30% less
30% more with controlled calories
30% more
1
80
21
37
36
34
2
80
21
37
36
34
3
80
21
37
36
34
4
80
21
35
35
34
5
73
21
35
35
34
6
73
21
35
35
34
7
64
21
33
35
34
8
54
17
32
30
31
9
34
17
31
29
30
10
21
17
22
23
28
11
10
15
21
21
25
12
10
13
17
20
17
Caloric and Protein Restriction 19
Percent of male D. melanogaster alive
1.2
1 Control
0.8
30% less yeast with controlled calories
0.6
30% less yeast 0.4
30% more yeast with controlled calories
0.2
30% more yeast
0 1
2
3
4
5
6
7
8
9 10 11 12
Days Figure 6. Graph of the percent of m ale D. melanogaster alive in response to each diet. The above shows that the control group seemed to have the shortest lifespan while all other diets seemed to prolong lifespan.
Viability of D. melanogaster 90
Number of Progeny
80 70 60 50 Male
40
Female
30 20 10 0 Control
30% more
30% more with control
30% less 30% less with control
Figure 7. The number of progeny in each type of vial of Drosophila medium. This graph shows that the control group had the most progeny compared to all the other diets.
Caloric and Protein Restriction 20 Data Analysis and Discussion Based on the trends in the data, nutritional variances in the media do affect the longevity of Drosophila melanogaster. In the population of males, only twelve percent survived after a period of twelve days while each other diet had a survival rate above 45 percent. These results were found to be significant at an alpha of 0.05 because the chi-‐square value was calculated to be 55.2521 with three degrees of freedom. This means that because 55.2521 is greater than 2.366, the expected chi-‐ square for three degrees of freedom, it is safe to reject the null hypothesis that type of diet does not affect the lifespan of D. melanogaster; however, the results of this test may be inconclusive because more than twenty percent of the expected cells were below a count of five. The females, on the other hand, exhibited exceedingly different results. Based on the data, the diet with 30 percent more yeast was the least beneficial with a survival rate of eleven percent. The control group, however, had the next lowest survival rate of 22 percent. Each of the other diets had a survival rate above 36 percent. These results were shown to be significant at an alpha of 0.05; however, this is much less significant than that of the males. The resulting chi-‐square value was 12.061 with three degrees of freedom; therefore, because 12.061 is greater than 2.366 it is significant at the 0.05 level. The caveat regarding the conclusiveness of the data must be applied here as well because 25 percent of the expected cells were below a count of five. The flies that were cultivated in the media with an absence of yeast did hatch; however, the time spent in the egg stage was nearly two times greater than
Caloric and Protein Restriction 21 those in all other environments. Unfortunately, these flies did not hatch in time to produce viable results.
Caloric and Protein Restriction 22 Conclusions
The data partially supported the original hypothesis because it was shown
that there is a relationship between the lifespan of Drosophila and the supplied diets that is possibly statistically significant. The data suggested that there might be a difference in the way diets affect the longevity of different genders. The data for the male fruit flies suggested that any type of diet, reduced or supplemented, would increase their overall lifetime. The data for the female flies, on the contrary, proposed that diets with supplementation or reduction in yeast with a controlled amount of calories would be more beneficial than those without a caloric control. In one case where calories were not controlled, the diet was even shown to be detrimental to the lifespan of the female flies. These results cannot effectively be extrapolated to humans; however, aspects of these results pose possible areas of research for human development.
Caloric and Protein Restriction 23 Limitations and Assumptions
Some of the limitations of experimentation were the time and monetary
constraints, which allowed for fewer trials than desired as well as the inability to test the yeast absent vials of flies, and scale and timing limitations, which allowed for less precision than preferred. Along with these restrictions were several assumptions including that all equipment used was properly working, Drosophila melanogaster raised in captivity are representative of those raised in the wild, the physiology of fruit flies is indeed similar to that of human physiology, and the control group is representative of the population of Drosophila melanogaster.
Some possible sources of error include minute differences in growth media,
slight differences in media consistency due to variations in the water content of the media and how much evaporated, the measurement of the reaction time measured by counting the number of flies that did not reach the top of the vial in the five second interval, the possible damage to nervous system due to the use of FlyNap, and the accuracy of the standard stop watch app on an Iphone.
Caloric and Protein Restriction 24 Applications and Future Experiments The overlaying applications of the project are based on the ability to suggest directions for future experiments. At this time, it is impossible to experiment on humans directly, thus Drosophila melanogaster were used as physiological models. The main idea behind the project was to determine which type of diet would be more beneficial to undergo, in reference to lifespan, a reduction in calories, a reduction in protein, or both. There are many possible extensions to this project including testing the viability of other dietary concerns such as fats and experimenting on vertebrate such as mice. By experimenting on vertebrate that are more similar to humans physiologically, it becomes easier to extrapolate possible correlations between dietary intake and reaction time near death (ultimately longevity). Another area of research that would be beneficial to study would be the use of the information obtained to repel Drosophila from plant species. Because it was found that any change in diet reduced the number of progeny, a spray could be developed with trace amounts of protein to reduce the deterioration of plant life. (G. Stoltz, personal communication, February 2013). In extensions of this project, results would be much more conclusive due to the experience gained and due to the removal of high school student restrictions. If it were possible to replicate this experiment, the number of trials would be increased, humidity would be controlled, number of flies in each vial would be increased, devices used would be more precise, the media and fly environments would be set up much earlier, and the media would be slightly more consistent. New work in the field of protein versus caloric restriction opens
Caloric and Protein Restriction 25 the door to many experiments to extend and understand lifespan. The eventual goal of this project is to be able to aid in the overwhelming problem of obesity in the world and also to understand what types of dietary restriction allow for the healthiest and ultimately longest lives.
Caloric and Protein Restriction 26 Literature Cited Chapman, T., & Partridge, L. (1996). Female fitness in Drosophila melanogaster: an Interaction between the effect of nutrition and of encounter rate with males. Biological Sciences, 263(1371), 755-‐759. Retrieved December 17, 2012, from http://rspb.royalsocietypublishing.org Chippindale, A., Leroi, S., Kim, A., & Rose, M. (1993). Phenotypic plasticity and selection in Drosophila life-‐history evolution. i. nutrition and the cost of reproduction. Evolutionary Biology, 6(2), 171-‐193. Retrieved December 17, 2012, from www-‐scopus-‐com Demerec, M., & Kaufmann, B. P. (1957). Drosophila guide. Washington: Carnegie Institution of Washington. Guarente, L., & Picard, F. (2005). Calorie restriction-‐-‐the sir2 connection. Cell, 120(4), 473-‐482. Retrieved from http://www.sciencedirect.com Loera, J. (2011, May 20). Current bloomington recipe for drosophila media. Retrieved from http://flystocks.bio.indiana.edu/Fly_Work/media-‐ recipes/bloomfood.htm Mair, W., Goymer, P., Pletcher, S., & Partridge, L. (2003). Demography of dietary restriction and death in Drosophila. Science, 301, 1731-‐1733. Retrieved November 15, 2012, from http://www.sciencemag.org Northrop, J. (1917). The role of yeast in the nutrition of an insect (drosophila). Biological Chemistry, 30, 181-‐187. Retrieved November 14, 2012, from http://www.jbc.org Partridge, L., Green, A., & Fowler, K. (1987). Effects of egg-‐production and of exposure to males on female survival in Drosophila melanogaster. Insect Physiology, 33(10), 745–749. Retrieved December 17, 2012, from www.sciencedirect.com Partridge, L., Pletcher, S., & Mair, W. (2005). Dietary restriction, mortality trajectories, risk and damage. Mechanisms of Ageing and Development, 126, 35-‐41. Retrieved November 15, 2012, from http://www.sciencedirect.com Wixon, J., & O'Kane, C. (2000). Featured organism Drosophila melanogaster. Yeast, 17, 146-‐153. Retrieved November 15, 2012, from http://www.ncbi.nlm.nih.gov
Caloric and Protein Restriction 27 Acknowledgments
The author wishes to thank his parents for their ongoing support, from
providing moral support to laboratory space. Annabel Consilvio was also a great help to the author because of the hands on experience she was able to pass on regarding basic culturing needs for Drosophila. The author would also like to thank Dr. Sumner for providing help in experimental design ideas. She also provided an answer to any questions asked and overall aid in the creation of this paper.
