The Effects of Sleep Pattern and Chronotype Alterations on Addictive Behaviors in D. melanogaster

Drosophila Maintenance

Wild Type Drosophila

Clk Mutant Drosophila

Vials and plugs

Drosophila food

Nutri-Fly Drosophila Agar

Propionic acid and hydrochloric acid (preservative)

Embryo Collection Cages

Grape Agar Plates

Nutri-Fly Grape Agar Premix

Petri dishes

Yeast-paste

Drosophila Tissue Supernatant Collection

Dounce homogenizer

Liquid nitrogen

1X PBS

Centrifuge

General

Micropipettes

Multichannel micropipettes

Micropipette tips (for both types of micropipettes)

Electronic pipettors

Pipettes (for electronic pipettors)

Carbon Dioxide Drosophila Anesthetizer Stations

Laboratory freezer

Distilled water

Adult Drosophila (for CAFE, FRAPPE, Knockdown, and Alcohol Dehydrogenase assays)

Larvae (for Odor Preference assay)

Sleep Regulating Machine

3D-printed machine case

Servo motor

Microcontroller

Axle

Real Time Clock

Vial clamps

CAFE Assay

Five concentrations of ethanol (0%, 4.5%, 15%, 30%, 45%)

Powdered red pigment

Capillary tubes

Feeding tubes with caps

Caliper

FRAPPE Assay

Rhodamine B

Fluorescein Sodium Salt

Clear 24-well microplates

Black 96-well microplate

Fluorescence band pass filters

Rhodamine B

Excitation: 545/40

Emission: 620/40

Fluorescein

Excitation: 485/20

Emission: 528/20

Fluorometer

Knockdown Assay

Inebriometer

Cylindrical tube (length = 122 cm, diameter = 7.62 cm)

Eight 3D-printed diffusers

Ten 3D-printed baffles

Petri dish

Ethanol vaporizer

500mL Erlenmeyer Flask

Size #7 Rubber Stopper - Two Holes

Two 1mL glass pipettes

Bunsen Burner

Plastic tubing

Air pump

3D-printed funnel

Ring stand

Utility clamp

Alcohol Dehydrogenase Assay

Alcohol Dehydrogenase Assay Kit

Assay Buffer

NAD Solution

MTT Solution

Diaphorase

Substrate

Calibrator

Odor Preference Assay

3D-printed plate

Cotton

Five concentrations of ethanol (0%, 4.5%, 15%, 30%, 45%)

Procedures

Breed and maintain wild type Drosophila and Clk mutant Drosophila

Construct the Sleep Regulating Machine

Upload the program for one of the variables into the Sleep Regulating Machine

Insert ten vials of wild type Drosophila into the Sleep Regulating Machine vial clamps

Remove the vials from the Sleep Regulating Machine after four days of synchronization

Repeat steps #3-5 for each variable

Run each of the five assays (see graphics for further explanation of assay procedures)

CAFE assay

FRAPPE assay

Knockdown assay

Alcohol Dehydrogenase assay

Odor Preference assay

Data Collection

CAFE assay

Measures the amount of alcohol the drosophila drink

FRAPPE assay

Tests the preference of alcohol by taste

Knockdown assay

Tests the resistance and tolerance build to alcohol

Alcohol dehydrogenases assay

Measures the amount of alcohol dehydrogenases

Odor preference assay

Tests the preference of alcohol by smell

Hello, my name is Carla del Río, and my experiment was determining the effects of variations of sleep patterns and circadian clock chronotype alterations on the addictive behaviors of Drosophila melanogaster, commonly known as fruit flies. The purpose of this experiment was to induce various sleep patterns and circadian clock chronotype alterations in Drosophila in order to see which was the most susceptible to Alcohol Use Disorder. The hypothesis was that if Drosophila’s sleep patterns and circadian clock chronotypes are environmentally altered, then the Drosophila with the Evening chronotype will build the most resistance to ethanol, and have the highest alcohol dehydrogenase levels, and the Drosophila with the Sleep Deprived pattern will consume the highest percentages of ethanol, and have the highest odor and taste preferences to ethanol. The experiment consisted of breeding and maintaining wild type Drosophila and Clk mutant Drosophila, using the Sleep Regulating Machine to synchronize the wild type Drosophila to each sleep cycle, and testing each of the five assays (CAFE, FRAPPE, Knockdown, Alcohol Dehydrogenase, and Odor Preference) on each of the eight sleep cycles of adult Drosophila or larvae. The Evening chronotype built the most resistance to ethanol and showed the strongest natural odor preference for ethanol, which indicated a higher risk of Alcohol Use Disorder. On the other hand, the Sun and Morning chronotypes showed the lowest natural taste preference to ethanol and were the least resistant to ethanol, which indicated a lower risk of Alcohol Use Disorder. This experiment could be used to help the group of people that are aware of their genetic predisposition to drug abuse by prompting them to decrease their exposure to risk-factors of Alcohol Use Disorder, through the avoidance of certain sleep cycles.

The Effects of Variations of Sleep Patterns and Circadian Clock Chronotype Alterations on the Addictive Behaviors, Specifically the Consumption of High Percentages of Ethanol, Odor and Taste Preferences to Ethanol, Resistance Built Against Ethanol, and High Alcohol Dehydrogenase Levels, of Drosophila melanogaster

Carla del Río

Florida Academy of Science (FAS)

American Heritage School, Plantation, Florida

Leya Joykutty

Florida Academy of Science (FAS)

The purpose of this experiment was to induce various sleep patterns and circadian clock chronotype alterations in Drosophila melanogaster in order to see which was the most susceptible to Alcohol Use Disorder. The hypothesis was that if Drosophila’s sleep patterns and circadian clock chronotypes are environmentally altered, then the Drosophila with the Evening chronotype will build the most resistance to ethanol, and have the highest alcohol dehydrogenase levels, and the Drosophila with the Sleep Deprived pattern will consume the highest percentages of ethanol, and have the highest odor and taste preferences to ethanol. The experiment consisted of breeding and maintaining wild type Drosophila and Clk mutant Drosophila, using the Sleep Regulating Machine to synchronize the wild type Drosophila to each sleep cycle, and testing each of the five assays (CAFE, FRAPPE, Knockdown, Alcohol Dehydrogenase, and Odor Preference) on each of the eight sleep cycles of adult Drosophila or larvae. The Evening chronotype built the most resistance to ethanol and showed the strongest natural odor preference for ethanol, which indicated a higher risk of Alcohol Use Disorder. On the other hand, the Sun and Morning chronotypes showed the lowest natural taste preference to ethanol and were the least resistant to ethanol, which indicated a lower risk of Alcohol Use Disorder. This experiment could be used to help the group of people that are aware of their genetic predisposition to drug abuse by prompting them to decrease their exposure to risk-factors of Alcohol Use Disorder, through the avoidance of certain sleep cycles.

What effect do sleep pattern and circadian clock chronotype alterations have on the addictive behaviors (specifically the consumption of high percentages of ethanol, odor and taste preferences to ethanol, resistance built against ethanol, and high alcohol dehydrogenase levels) of Drosophila melanogaster?

Hypothesis

If Drosophila’s sleep patterns and circadian clock chronotypes are environmentally altered, then the Drosophila with the Evening chronotype will build the most resistance to ethanol, and have the highest alcohol dehydrogenase levels, and the Drosophila with the Sleep Deprived pattern will consume the highest percentages of ethanol, and have the highest odor and taste preferences to ethanol.

Variables

Sun Chronotype

Active: 6:30am - 12:00 pm

Regular: 12:00pm - 5:00pm

Slow: 5:00pm - 8:00pm

Sleep: 8:00pm - 6:30am

Moon Chronotype

Active: 8:00pm - 12:00am

Regular: 12:00am - 5:15am

Slow: 5:15am - 6:15am

Sleep: 6:30am - 8:00am

Morning Chronotype

Active: 6:00am - 6:00pm

Regular: 6:00pm - 9:00pm

Sleep: 9:00pm - 5:30am

Regular: 5:30am - 6:00am

Evening Chronotype

Active: 3:00pm - 8:00pm

Regular: 8:00pm - 12:00am

Sleep: 12:00am - 10:00am

Slow: 10:00am - 1:00pm

Regular: 1:00pm - 3:00pm

Disrupted Sleep Pattern

Alternate between the Sun, Moon, Morning, and Evening Chronotypes

Sleep Deprived Pattern

Active: 5:00am -2:00am

Sleep: 2:00am - 5:00am

Wild Type (Negative Control)

Diurnal

Sleep cycle is not altered

Clk Mutant (Positive Control)

Circadian clock mutant

Disrupted circadian rhythm and sleep cycle

Sleep cycle is not altered

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The purpose of this experiment was to induce various sleep patterns and circadian clock chronotype alterations in Drosophila in order to see which was the most susceptible to Alcohol Use Disorder. The hypothesis was that if Drosophila’s sleep patterns and circadian clock chronotypes are environmentally altered, then the Drosophila with the Evening chronotype will build the most resistance to ethanol, and have the highest alcohol dehydrogenase levels, and the Drosophila with the Sleep Deprived pattern will consume the highest percentages of ethanol, and have the highest odor and taste preferences to ethanol. The results of the experiment indicated that the Evening chronotype built the most resistance to ethanol and showed the strongest natural odor preference for ethanol, which indicated a higher risk of Alcohol Use Disorder. On the other hand, the Sun and Morning chronotypes showed the lowest natural taste preference to ethanol and were the least resistant to ethanol, which indicated a lower risk of Alcohol Use Disorder.

The hypothesis can be separated into five parts, one for each assay, respectively: Knockdown, Alcohol Dehydrogenase, CAFE, Odor Preference, and FRAPPE. The statistical analysis of the Knockdown assay indicates that the mean knockdown time for the Evening chronotype Drosophila is significantly higher than that of any other sleep cycle. Therefore, this data supported the part of the hypothesis corresponding to the Knockdown assay, which stated that the Drosophila with the Evening chronotype will build the most resistance to ethanol. The knockdown assay was run at the same time for each sleep cycle, approximately 20:00-23:30, since this is the peak time of alcohol consumption. The high levels of resistance built to ethanol vapor of the Evening chronotype Drosophila could be due to the fact that they had the longest combined period of non-Active (Slow and Regular) speed level. The data also shows that Sun and Morning chronotypes behave in a very similar manner, having almost identical means and very similar distribution of the data. The Drosophila with the Sleep Deprived pattern fell in the shortest period of time, which supports the correlation between lack of sleep and low ethanol resistance. After statistical analysis, it can be observed that the data from the Odor Preference assay is separated into three clusters of sleep cycles: one consisting of the Clk Mutant, Sleep Deprived pattern, and Disrupted Sleep pattern, another consisting of the Evening and Moon chronotypes, and the last consisting of the Wild Type, Sun chronotype, and Morning chronotype. Of these three clusters, the first two preferred higher concentrations of ethanol, where the first cluster showing the highest ethanol preference with at least 15 out of the 20 larvae preferring the 45% concentration, and the rest preferring the 30% concentration. Additionally, the fact that the Disrupted Sleep pattern and Sleep Deprived pattern were clustered with the Clk mutant, indicates that a circadian rhythm alteration can be induced without having a circadian clock mutation. On the other hand, the last cluster preferred the lower concentrations of ethanol, showing a clear preference for the 0% and 4.5% concentrations. We can also observe that third cluster is negatively correlated with the other sleep cycles, meaning that they have opposing preferences in regards to ethanol. The statistical analysis for both the CAFE and ADH assays led to the conclusion that the difference between the sleep cycles was not statistically significant. After statistical analysis, it can be observed that the data from the FRAPPE assay is separated into three clusters of sleep cycles: one consisting of the Evening chronotype, Morning chronotype, and Clk Mutants, another one consisting of the Moon chronotype, Sun chronotype and Disrupted Sleep pattern, and the last consisting of the Wild Type and Sleep Deprived pattern. The first cluster showed the highest preference for ethanol since the average percent of ethanol consumed was significantly higher, the second cluster showed equal preference for ethanol and water since an approximately equal amount of ethanol and water was consumed, and the third cluster showed the lowest preference for ethanol since the amount of water consumed was higher than that of ethanol.

Applications

The experiment was designed using Drosophila melanogaster since they are an ideal model organism, and have been used in behavioral scientific experiments for over a century. Drosophila constitute excellent model organisms for humans due to the fact that their behavioral responses and molecular pathways are remarkably similar to those of mammals. Another advantage of using Drosophila as a model organism would be the fact that around 98% of its genome is known and sequenced. Additionally, the majority of human genes have a Drosophila ortholog, which is extremely beneficial when working with genetic mutations, such as the Clk mutation, which occurs on an ortholog of the human CLOCK gene. Drosophila can also be adequately used for modeling disorders, such as Alcohol Use Disorder, for various reasons. For instance, Drosophila rapidly learn cues and contexts that predict the availability of highly rewarding drugs. Furthermore, Drosophila show a remarkable preference for ethanol after prolonged or repeated exposure. Approximately 25% of people around the world suffer from addictions and drug abuse, and almost 74% of these people suffer from Alcohol Use Disorder, making it one of the most widely abused drugs; therefore, getting a better understanding of the causes as well as ideas for prevention would be instrumental in decreasing its prevalence in today’s society. This experiment can help the group of people that are aware of their genetic predisposition to drug abuse by prompting them to decrease their exposure to risk-factors of Alcohol Use Disorder, through the avoidance of certain sleep cycles. The data in the experiment did not point to one specific cycle as being the most high-risk due to the fact that each assay was targeting a different aspect of the behaviors of Alcohol Use Disorder. The Evening chronotype built the most resistance to ethanol and show the strongest natural odor preference for the 45% ethanol, which indicates a high-risk of Alcohol Use Disorder. On the other hand, the Sun and Morning chronotypes show the lowest natural taste preference to ethanol and were the least resistant to the ethanol vapor, which indicates a low-risk of Alcohol Use Disorder.

Limitation

Like in any other scientific experiment, limitations were present. An example that exists in every experiment is the amount of trials conducted. Increasing the amount of trials will make the data more statistically robust, resulting in a better analysis of the data. Ten trials for each of the eight variables were conducted for the Odor Preference assay, where each trial consisted of twenty larvae; therefore, the sample size of the entire Odor Preference assay was one thousand six hundred larvae. Whilst an increase in the number of trials would have been beneficial to the statistical analysis, it would not have been feasible to collect a greater amount of larvae than that of which was collected in this experiment. Ten trials for each of the eight variables were conducted for the CAFE assay as well, however each trial consisted of five adult Drosophila; therefore, the sample size of the entire CAFE assay was four hundred adult Drosophila. Due to a limited number of Feeding Tubes and the length of the assay, increasing the number of trials was not possible, however, a more plausible solution would be to change the amount of adult Drosophila in each Feeding Tube from five to ten; therefore, the new sample size of the CAFE assay would be eight hundred adult Drosophila. Ten trials for each of the eight variables were conducted for the Knockdown assay, where each trial consisted of five adult drosophila; therefore the sample size of the entire Knockdown assay was four hundred adult Drosophila as well. Due to the fact that the Drosophila must be observed the entire duration of the assay, in order to record the knockdown times of each of the five Drosophila per trial, it would not have been managabled to increase the number of Drosophila per trial. It also would not have been reasonable to run more trials, as each took around thirty minutes, resulting in a total of forty hours for the eighty trials (excluding the fifteen minute cleaning period between each trial). Five trials of Working Reagent for each of the eight variables and five trials of Blank Working Reagent for each of the eight variables were conducted for the Alcohol Dehydrogenase assay, where each trial was one well on a clear 96-well microplate. The assay kit protocol only required one trial of Working Reagent for each variable and one trial of Blank Working Reagent for each variable, due to the fact that each well was loaded with the exact same solutions; however five trials were done of each Reagent for each variable in order to account for any errors with the microplate reader, or imprecise measurements. Ten trials for each of the eight variables were conducted for the FRAPPE assay, where each trial was one well on a black 96-well microplate. Similar to the Alcohol Dehydrogenase assay, since each well in the FRAPPE assay was loaded with the exact same solution, Drosophila tissue supernatant, it was only necessary to do one trial for each variable; however ten trials were done for each variable in order to account for any errors with the fluorometer, or imprecise measurements. A possible limitation in this experiment was that in the CAFE assay, the Drosophila were in the Feeding Tubes for a period of five days, and the Feeding Tubes were not on the Sleep Regulating Machine. Due to this, the Drosophila could have lost their acquired chronotype or sleep pattern, resulting in possibly skewed data. Lastly, in the FRAPPE assay, Drosophila were placed in Starvation Vials for a period of 12-18 hours, and then in Dehydration Vials for a period of 2-3 hours; however, the exact times were determined by lab hours. As a consequence of this, some variables were starved or dehydrated for longer periods than others which could have resulted in them being prone to consuming larger amounts of liquid. In order to account for this limitation, the data was converted from fluorescence intensity to nL consumed, and then the final data was recorded as ethanol consumed to total liquid consumed, written as a percentage.

Error Analysis

With any experiment, there is the possibly that numerous measurement errors occurred during data collection. For example, a majority of the experiment consisted of measuring or detecting extremely small quantities of a variety of substances, using equipment such as the fluorometer, microplate reader, centrifuge, caliper, or micropipette; however, regardless of how precise the instrument is, there will always be a lack of accuracy. Furthermore, in the CAFE assay, there were some instances in which bubbles formed within the capillary tubes. Since those bubbles can be considered solution that the Drosophila consumed, the size of any bubble or bubbles which formed within a capillary tube was estimated, and then added to the length of the solution consumed by the Drosophila. Additionally, human error is always present in every aspect of preparing and collecting data for each of the assays. For instance, in the CAFE assay, there is a slight possibility that the solutions within the capillary tubes evaporated slowly throughout the five day feeding period. Due to the fact that ethanol evaporates faster than water, the higher concentrations could have a greater amount of solution missing due to evaporation, although it appears to be due to the Drosophila’s consumption of the solution. Lastly, due to the fact that loading technique for the clear 96-well microplate in the Alcohol Dehydrogenase assay was not efficient, there was a difference of around ten minutes, the amount of time it took to complete the loading process, in the reaction time of the samples. Due to this, the reaction times for the samples from the initial reading would be anything between zero and ten minutes, depending on when they were loaded (ten minutes being the first and zero being the last), and the reaction times for the samples from the time thirty reading would be anything between thirty and forty minutes, depending on when they were loaded (forty being the first and thirty being the last). However, in order to convert the data from Optical Density, which is what the microplate reader uses, to enzyme activity using the Alcohol Dehydrogenase Activity Formula, the difference of the time thirty reading and time zero reading must be taken for each sample. Therefore, the inefficiency of the loading technique should not influence the results, unless the samples reach their peak level of enzyme activity within the thirty minutes.

Future Research

There are a variety of ways in which the present experiment can be modified. The majority of these alterations can be carried out by adjusting sections of each assay protocol. For example, in the Knockdown assay, instead of doing one pre-exposure to ethanol, the Drosophila could be recovered after the first run, and the assay could be run four more times with a one day recovery period between each of the five runs. This new data would be used to determine which chronotype builds the most resistance to ethanol over the course of five exposures. Furthermore, the data from the two versions of the Knockdown assay could be compared to see whether or not the chronotype which built the most resistance remained the same. Those results would determine whether or not one chronotype built the most resistance over the course of five exposures to ethanol and built the most resistance after one pre-exposure to ethanol. Another example of modifying an assay protocol would be measuring the capillaries in the CAFE assay every twelve to twenty-four hours within the five day period, since it could be observed that each variable consumed varied amounts of each concentration initially, but after the five days had passed, the Drosophila all developed a preference for the higher concentrations of ethanol. Rather than removing the capillary tubes after the five day period, measuring them more frequently can be achieved by taking photographs of the capillaries with a ruler for reference in the background. Additionally, the Alcohol Dehydrogenase assay could be run four more times, using Drosophila from each variable that were pre-exposed to ethanol one, two, three, and four times, in addition to using Drosophila that were never exposed to ethanol. This new data would determine if any variable or variables develop greater levels of Alcohol Dehydrogenase activity after repeated exposures to ethanol. Another example of an adjustment that could be made in the Alcohol Dehydrogenase assay would be to take readings at smaller intervals of time, rather than just at the initial time and after thirty minutes. After plotting the points for readings taken every five minutes, a standard curve could be made, which could be used to determine the Optical Density of the samples at any given time. Using this new data, any time between the initial time and time thirty (excluding the extremes) could be used as a replacement for the time thirty reading when the Alcohol Dehydrogenase activity is calculated. This would account for the possibility that the samples could have reached a peak level of enzyme activity during the thirty minute reaction period, since any value could be substituted for that of thirty minutes. An alternative form in which the experiment can be modified is through the creation of completely separate experiments, which have branched off of its central concept. For example, the experiment could be repeated using second, third, fourth, and fifth generation of Drosophila placed in the Sleep Regulating Machine, in addition to using the first generation Drosophila. In order to do this, the Drosophila would be transferred into the Sleep Regulating Machine for four days, the adult Drosophila would be transferred to a new vial, as they would have already attained their chronotype or sleep pattern, and the larvae would be left to hatch while on the Sleep Regulating Machine. This would be done to observe if any behaviors, such as those of Alcohol Use Disorder, would be amplified in any future generation. Furthermore, the experiment could be repeated using the second generation Drosophila whose sleep cycles will remain unaltered. This new data could be compared to the current data collected, in an effort to observe if the data from a variable of the second generation matches that of the same variable or another variable in the first generation, which would determine whether or not the chronotype or sleep pattern maintained the same from one generation to another, meaning that they are or are not hereditary. Lastly, the Clk mutant Drosophila could be placed in the Sleep Regulating Machine, in addition to placing the wild type Drosophila in the Sleep Regulating Machine, resulting in fourteen variables, rather than eight. The seven variables that used wild type Drosophila were the four chronotypes, two sleep patterns, and one group of unaltered wild type Drosophila (negative control). The only variable that used Clk mutant Drosophila was the group of unaltered Clk mutant Drosophila (positive control). The variables using Clk mutant Drosophila could be expanded to include the four chronotypes and two sleep patterns. The data collected from this new experiment would show whether or not the circadian mutation can be corrected through sleep cycle alterations.

Kepa Oyarbide (supervisor/mentor)

American Heritage School, Plantation Florida

Luisa Stelling (mother)

Heberto del Río (father)

Sofia del Río (sister)

When I began drafting topic ideas for potential research projects, I was initially interested in reading scientific papers which studied epigenetics as a form of gene expression and gene silencing. After analyzing papers regarding the various forms of applying this gene regulation, I began researching diseases or disorders that could potentially be correlated to a gene mutation. This lead me to read many papers which discussed the correlation of Alcohol Use Disorder (AUD) and gene mutations, along with the use of epigenetics to ameliorate the effects of AUD. Due to limitations caused by my age and grade level, I was not able to incorporate epigenetics into my experiment. I did, however, remain interested in AUD, and then began reading about its correlation with circadian rhythm. After drafting many experimental proposals, I was finally able to synthesize the idea for the project I ended up conducting. This project took me from school fair to county fair, and additionally qualified for state fair, which was unfortunately canceled due to COVID-19. I am elated that I now have the opportunity to present my project before a scientific group as well known as AJAS.