There are several foods that contain polyphenols, which when administered through the diet can reverse or prevent behavioral and cognitive dysfunction. Some of these foods are strawberries, blueberries, blackberries, and walnuts. Walnuts contain numerous antioxidant polyphenols, as well as various vitamins and healthy acids, all of which have been demonstrated to improve motor and cognitive abilities. Past experiments have demonstrated the effect of walnuts on the motor and cognitive behavior of caged rats. Other model organisms were used finding the effect of walnuts on the motor functions, but one that has rarely been used is the Drosophila melanogaster, or the fruit fly. The Drosophila melanogaster is used as a popular organism for understanding how motor functions could be affected by the genes and the diet, but there is a lack of research done to see how the motor functions of the Drosophila will be affected by a diet of walnuts. Therefore, the purpose of the study is to determine if the climbing ability of the Drosophila will be affected by walnuts. In the study, there were three control groups. In the first control group, Drosophila were fed Drosophila medium laced with 1g of walnuts. The second group were fed medium with 0.5g of walnuts. The third group was medium without walnuts. The Drosophila then fed on the medium for around two days, before the Drosophila climbing assay was performed on them. In the climbing assay, the Drosophila had a ten second time frame to climb past a marker that had been set 5.5 cm above the starting line. The data was recorded and analyzed in the statistical package for the social sciences through the use of a one way ANOVA. There was no significant difference found on the mean percentage of flies that crossed the 5.5cm marker (p-value=0.122).
Determining Whether Walnuts Affects the Climbing Ability of Drosophila Melanogaster
There are several foods that contain polyphenols, which when administered through the diet can reverse or prevent behavioral and cognitive dysfunction (Joseph, Shukitt-Hale & Lau 2007). Walnuts contain various vitamins and healthy acids, such as n-6 and n-3 PUFA linoleic acid, α-linolenic acid, vitamin E, folate, melatonin and numerous antioxidant polyphenolics (Crews et al. 2005). Past experiments have demonstrated the effect of walnuts on the motor and cognitive behavior of caged rats, where the rats that ate the walnuts demonstrated a improved cognitive and motor abilities when compared to the rats that ate no walnuts. (Willis et al. 2009). In the study, seventy-three Fischer 344 rats were fed a controlled diet of two percent, six percent and nine percent walnuts. In study one, a control and two percent walnut diets were fed (fourteen rats per group). In study two, control and six and nine percent walnut diets were fed (fifteen rats per group). Diets were provided ad libitum for 8 weeks. In each of the studies, the diets had an appropriate amount of maize in the diet to compensate for the walnuts. For example, the six percent walnut diet contained 60 g walnuts/kg and 40 g maize/kg; the control diet contained 100 g/kg maize. In order to test the motor abilities in rats, tests were conducted on the rats during the eighth week of the treatment. Examples of the tests are rod walking, and different sized plank walks. The result of the tests showed that the 2% walnut diet improved performance in the rod walking test, and the 6% walnut group did signiﬁcantly better than the other groups in the medium plank walk. The 9% walnut group fell in the large plank walk quicker than the control and the 6% group, which shows impaired motor performance. Other model organisms were used finding the effect of walnuts on the motor behavior, but one that has rarely been used is the Drosophila melanogaster, or the fruit fly.
There are many different assays used to measure Drosophila motor behavior, such as the larval crawling assay, mating assay, and the climbing assay (Nichols, Becnel, & Pandey 2012). The Drosophila climbing assay is based on the natural tendency of flies to climb, known as negative geotaxis. In order to perform the Drosophila climbing assay, place a known number of flies in a vial and tap the vial against a hard surface, causing the flies to fall to the bottom of the vial. The flies will then attempt to climb to the top of the vial, as it is an innate behavior of the Drosophila melanogaster (Madabattula et al. 2015). The Drosophila melanogaster is widely used in biological studies (Staats et al., 2018), but there is a lack of research done to see how the motor functions of the Drosophila will be affected by a diet of walnuts. Therefore, the purpose of the study is to determine if the climbing ability of the Drosophila will be affected by walnuts.
The wild type Drosophila melanogaster was obtained from Carolina Biological Supply Company.
The diet of the Drosophila melanogaster was Formula 4-24® Instant Drosophila Medium, which was purchased plain from Carolina Biological Supply Company. In the separate vials, crushed walnuts was added. In group 1, there was no walnuts mixed into 4g of medium, in group 2, 0.5g of walnuts was mixed in, and in group 3, 1g walnuts was mixed in. The flies in the groups fed on the medium mixtures roughly 2 days before the assay was to take place.
After the Drosophila melanogaster fed on the diet of walnuts and medium, the climbing assay was performed on the Drosophila melanogaster. In order to perform the assay, the groups of the Drosophila were transferred from the vials with the medium into the testing vial. The vial was then tapped against a hard surface, causing the flies to fall to the bottom of the vial. The flies then attempt to climb to the top of the vial. The results of how many flies climbed past 5.5cm marker on the vial during an allotted time period of 10 seconds were taken. The no walnut group was tested 6 times, and the 1 g of walnut group and 0.5 g of walnut group were tested 4 times each. All the tests were performed on the same group of flies. The assay was videotaped on a IPad for analysis in SPSS. In Figure 1, it can be seen in the first picture that all the flies are at the bottom of the vial. In the second picture, at eight seconds in, three flies are over the marker.
Figure 1. A vial of flies in the testing tube is shown here. In the first picture, the time of the assay was zero seconds with all the flies on the bottom of the vial, and in the second picture, the time was eight seconds and there were three flies above the marker. Graphic by Author
The mean percentage of Drosophila melanogaster that climbed over the 5.5cm in 10 seconds was analyzed using an one way ANOVA.
Results and Discussion
Walnuts vs No Walnuts
The statistical software SPSS was used in order to analyze the results and generate visual representations of the data. The climbing ability for the Drosophila melanogaster group that ate no walnuts had a greater mean percentage of flies that climbed over the 5.5 cm marker, which had a mean of .25 ± 0.06 (SE), compared to the Drosophila melanogaster group that ate 1 g of walnuts, which had a mean of 0.075 ± 0.048, and the Drosophila melanogaster group that ate .5 g of walnuts, which had a mean 0.225 ± 0.048. This is most likely because the ethanol in the walnuts possibly slowed down the reaction and climbing ability within the Drosophila melanogaster. A one way ANOVA was conducted in SPSS on climbing ability. Figure 4 shows the results for this test. The p-value of the experiment is 0.122, which signifies no significant differences. These results suggest that the walnuts consumed had little to no effect on the climbing ability of the Drosophila melanogaster. This may be because that the flies that were exposed to walnuts in their environment and diet on a daily basis slowly adapted to the ethanol and became more adjusted to the walnut based diet.
Figure 4. A one way ANOVA was conducted to compare the mean percentage of the flies that climbed past the 5.5cm line compared to the 3 groups of no walnuts, 1g of walnuts, and 0.5g of walnuts. The group that ate no walnuts had a higher mean percentage than the .5 g walnuts, which in turn had a higher mean percentage than the 1 g of walnuts. The p-value is 0.122. Graphic by Author
There were some limitations to the experiment. Since the Drosophila melanogaster were not monitored constantly and the behavior of the flies were not observed carefully, it is possible that the assay did not accurately reflect the true climbing ability of subjects, as factors such as sleep schedule and mating behaviors could affect the climbing ability. Secondly, only three groups of flies were used in the experiment, which reduced the statistical power of the study and increased the margin of error. Finally, the environment in which the assay was taken could have been improved by making sure that the vial used for testing was clean after every test, as a dirty vial could impact the climbing ability for some flies.
Relation to Previous Literature
The results presented do not support the previous literature about the effects of ethanol on the motor or functions of the Drosophila melanogaster (Rodan & Rothenfluh et al., 2010). It does not support the previous literature suggesting that walnuts improve the motor functions in caged rats (Willis et al., 2008). This might be because the size of the subject determines how a diet of walnuts will impact the motor functions, as both in both humans and flies, the walnuts had no significant effects on the subject, but in rats, there was a significant difference in those who ate walnuts and those who did not.
These results suggest that the consumption of walnuts had little to no relationship with the difference climbing ability in the Drosophila melanogaster. This may be due to the fact that the flies that were exposed to walnuts in their environment and diet on a daily basis slowly adapted to the ethanol and became more adjusted to the walnut based diet, or that the antioxidants in walnuts are stronger than the healthy acids and oils in walnuts, and would impact the motor functions of the subject more depending on the size of the subject.
This experiment could lend itself to interesting follow up studies. To start out with, some of the limitations in the experiment could be improved by adding more groups into the experiment. A major limitation in the experiment was that all the flies came from the same group, which lowered the statistical power and raised the margin for error.
A different potential follow up experiment could be to include different groups into the experiment, such as only walnut based. This could be interesting, as a larger range of groups could more detailed results.
A third follow up experiment could be to include different diets for the Drosophila melanogaster. By including groups with different diets, the experiment would become more varied, and it would be based on how the diet would affect the climbing ability of the Drosophila melanogaster.
Overall, this experiment aimed to see whether a diet of walnuts would affect the climbing ability in the Drosophila melanogaster. The results showed that this is not the case, as no relationship was found the mean percentage of flies over the marker in the groups.
- Staats, S., Wagner, A. E., Rimbach, G., & Kai Lüersen. (2018). Drosophila melanogaster as a versatile model organism in food and nutrition research. Journal of Agricultural and Food Chemistry, 66(15), 3737-3753. doi:http://dx.doi.org/10.1021/acs.jafc.7b05900
- Nichols, C. D., Becnel, J., & Pandey, U. B. (2012). Methods to assay Drosophila behavior. Journal of visualized experiments : JoVE, (61), 3795. doi:10.3791/3795
- Joseph JA, Shukitt-Hale B & Lau FC (2007) Fruit polyphenols and their effects on neuronal signaling and behavior in senescence. Ann N Y Acad Sci 1100, 470 – 485.
- Madabattula, S. T., Strautman, J. C., Bysice, A. M., O’Sullivan, J. A., Androschuk, A., Rosenfelt, C., … Bolduc, F. (2015). Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders. Journal of visualized experiments : JoVE, (100), e52741. doi:10.3791/52741
- Tuzcu M & Baydas G (2006) Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. Eur J Pharmacol 537, 106 –110.
- Shukitt-Hale B, Mouzakis G & Joseph JA (1998) Psychomotor and spatial memory performance in aging male Fischer 344 rats. Exp Gerontol 33, 615 –624.
- Rodan, A. R., & Rothenfluh, A. (2010). The genetics of behavioral alcohol responses in Drosophila. International review of neurobiology, 91, 25-51.