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ATP6 in Drosophila Melanogaster: Mechanical Stress Sensitivity & Motility
The mutation ATP6was able to cause effects within the Drosophila Melanogaster through mechanical stress induced paralysis. The focus of this study was to be able to identify exactly what the effects of the ATP6mutation were that occurred when the Drosophila Melanogaster were put through different sensitivity tests; one being the bang sensitivity test and the other being vortexed. The bang sensitivity test showed the effects of running time in both the mutated and wildtype Drosophila Melanogaster, while the vortex test showed the effects of recovery time after being vortexed for 20 seconds. It was found that it took longer for the Drosophila Melanogaster with the ATP6 mutation to recover after being vortexed for 20 seconds, and it was also shown that the Drosophila Melanogaster with the ATP6 mutation also had a longer run time compared to the Drosophila Melanogaster that did not have the mutation (wildtype).
The ATP6 gene is what provides the vital information to make a protein which is essential for typical mitochondrial function. The function of the mitochondria is to convert energy from food into energy that the cells can utilize to function. Mitochondria produce energy through the process oxidative phosphorylation which is the use of oxygen and sugar to make ATP which is the main source of energy for the cells. ATP6 only forms one part of an even larger whole enzyme, ATP synthase. ATP synthase is the last step during oxidative phosphorylation. Protons are allowed to flow across a membrane during ATP synthase to get inside of the mitochondria. On the inside of the mitochondria is where the proton is used to convert adenosine diphosphate into ATP (MT-ATP6 Gene). When there are mutations within the ATP6 gene, complications can arise with health problems such as maternally inherited Leigh syndrome (MILS) and neuropathy, ataxia and retinitis pigmentosa (NARP). NARP and MILS are genetic disorders caused by abnormalities which are affecting the energy production within the mitochondria. NARP can be characterized as an affected ability to coordinate movements while MILS can be characterized as a mitochondrial disorder with increased levels of lactic acid found throughout the body. The Drosophila Melanogaster made for the perfect model organism in this experiment due to its short lifespan, easy to manipulate genome, and for the fact that is shares most many of the genes found in humans that contribute to disease (Dautant et al., 2018). Studies that have been done prior to this experiment showed that mutations within the Drosophila Melanogaster as well as mutations in humans proved to reveal common phenotypes such as paralysis, a much shorter lifespan, as well as neuromuscular degeneration. The study also showed that the locomotor impairment, neural dysfunction, and myodegeneration found within the mutated Drosophila Melanogaster were also found in humans with the MILS disease (Celotto et al., 2006). Given this information, the hypothesis that was created for this experiment was that it was going to take longer for the mutated ATP6 Drosophila Melanogaster’s to recover or get up from being vortexed for 20 seconds compared to the wildtype of Drosophila Melanogaster that does not have the mutated gene. This was hypothesized due to the mutated flies having the same mutated gene that causes MILS and NARP in humans. One symptom of the diseases is seizures, meaning the flies are going to be bang sensitive making them more susceptible to seizures causing the time for recovery to become much longer than that of the wildtype fly who will get up with ease since it is not susceptible to seizures because of neurological deterioration (DiMauro, 2005). Also, it was hypothesized that the mutated Drosophila Melanogaster will run slower than the wildtype flies (have decreased motility) after receiving mechanical stress. This was hypothesized because the Drosophila Melanogaster that have the mutated gene will show the same symptoms of a human with MILS disease which include locomotor impairment and neural dysfunction. Locomotor impairment is the inability to move efficiently which will cause the Drosophila Melanogaster to move at a slower rate than the wildtype flies.
Methods and Materials:
45 Drosophila Melanogaster (age 8-10 days) were used to determine the motility in seconds to reach a certain point, as well as the recovery time from mechanical stress after being vortexed. 23 Drosophila Melanogaster were wildtype which indicated that they did not possess the mutated ATP6 gene while the remaining 22 Drosophila Melanogaster had the mutated gene. The flies were first put into a tub which was covered with a cotton swab to ensure the flies would remain inside. The flies were then placed on the Vortex Genie and vortexed for 20 seconds which would allow the opportunity to test the recovery time of wildtype flies versus mutant flies. After the 20 seconds of vortexing, the flies in the tube were set down and the time it took for the flies to recover and get back on their feet and move around was recorded in seconds. After this was recorded, the flies were then tapped to the bottom of the tube and the time it took for them to climb back up the tube past a set marking was recorded in seconds. The averages for each test was then found (AVG recovery time/AVG running time), as well as the standard deviation.
For the first experiment, time to recover from mechanical stress, it was conducted to see if the mutated gene ATP6 caused the recovery time in Drosophila Melanogaster to lower. As shown in figure 1, the time to recover from mechanical stress was significantly higher in the mutated flies compared to the time to recover for the wildtype flies. The average time it took for the wildtype fly to recover was approximately one second, while the time it took for the mutated fly to recover was around 69 seconds or one minute and nine seconds. The longest time it took a single mutated fly to recover was 208 seconds while the maximum time it took for a wildtype fly was only one second. The standard deviation for the wildtype fly was approximately 0 while the mutated fly was about 61. There were a few outliers found within the results of the time to recovery for the mutated flies, for example one mutated fly took 208 seconds while another only took one second. Compared to the average that was found for the mutated fly, these data points were way off and could be considered outliers to the data. Looking at the figure and the results, it can be concluded that the hypothesis was correct when determining that the recovery time for the mutated Drosophila Melanogaster would be significantly higher due to the flies having the same condition that a human would have if they had the MILS disease, neurological deterioration which allows for seizures to become much more common (DiMauro, 2005).
For the second experiment, running time to asses motility, it was performed to see which genotype took the longest time to make it across the set mark in the tube. As shown in figure 2, the mutated Drosophila Melanogaster took longer to reach the mark in the experiment. The average time it took for the wildtype fly to cross the line was about 10 seconds, while it took the mutated Drosophila Melanogaster 27 seconds to cross the mark. The longest time it took for a mutated fly to cross the line was 100 seconds or one minute and 40 seconds, while the wildtype fly’s longest time was 27 seconds. It can be noted that the average for the mutated was equal to that of the maximum for the wildtype, which shows right there that the mutated fly took more time than the wildtype. The standard deviation for the wildtype fly was calculated to be approximately 7, while the standard deviation for the mutated fly was approximately 27. One outlier in the data was in the mutated run time of 100 seconds while another mutated fly only took three seconds, compared to the average for the mutated fly these times were way off. Compared to the data and figure, the hypothesis of the mutated fly taking longer to cross the line was correct. The hypothesis was supported due to the mutated flies having the same symptoms of a human with MILS disease which include locomotor impairment and neural dysfunction.
In conclusion, the hypothesis of the mutated Drosophila Melanogaster taking longer to both recover from mechanical stress and taking longer to asses motility was correct. The hypothesis for time to recover from mechanical stress was supported due to the facts that the mutated gene found within the mutated fruit fly caused the fly to be more prone to seizures as well as having fast neurological deterioration. The hypothesis was correct for the mutated flies also having a slower running speed was supported through the evidence that shows that the mutated ATP6 gene causes locomotor impairment which had caused the flies to move at much slower speeds than the wildtypes. Looking at the mutated gene in humans, the hypothesis makes sense because humans begin to have trouble moving due to the neuromuscular deterioration and have a harder time recovering and acting based on the neurological deterioration in the brain (Sauvanet et al., 2012).
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The data fit well with previously published data that had been taken before this experiment. The results of the previously done experiment had stated that the mutated flies had become paralyzed after being administered the mechanical stress which is exactly what happened in this experiment which allowed for the time to recover to be taken. The reason for this was stated due to neural dysfunction that slows down the brains thinking processes which ultimately causes the mutated flies to take longer to respond to stimuli or in the experiments case, respond time. Previously published data also concluded that the reason why mutated flies move slower than the wildtype flies is because there are holes found in the muscle tissue of the mutated flies that make it harder for them to move as easily as the wildtype. This is what caused the wildtype flies to move at a faster speed than the mutated flies making the mutated flies the last to reach the set point in the experiment (Celotto et al., 2006). Outliers could have been present in this experiment for many different occurring reasons, one being that the deterioration of the mutated flies muscles and brain were not fully developed yet which would have caused them to both run as fast as the wildtype flies and even recover nearly as fast as them causing outliers. The same thing goes the other way as well. There may have been flies well into their deterioration stages and it may have taken them much longer to recover or regain their motility which shows the cause for such high outliers.
In the future, the experiment could be done differently in numerous ways to ensure more accurate and precise data. One thing that could be altered in the future is to use more flies. Using more flies and having more data will ensure that the average is closer to what it is expected to be even with outliers. The more data you have, the less room for error there is. Another thing that could be done differently is having some sort of goal for the flies to move to reach the line inside of their tube. One point of error could have been that they simply did not want to crawl to the top of the tube after being tapped down which would allow the seconds to build up giving them a running time that is too high. Future suggestions for the experiment are to look at the time to take flight for the flies, or even the age for which they live.
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