BACKGROUND:

Drosophila melanogaster, or fruit flies, are exemplary model organisms in the field of genetics. They are small, cheap, easy to maintain, and have a very short generation time—yielding ample offspring (Flinn Scientific). Drosophila’s life cycle is divided by distinctly visible stages. Normally, one day after a male and female Drosophila reproduce, an embryo is produced in the form of an egg (Griffiths et al., 2000). Then, the offspring goes into its first, second, and third instar stages, which are larval stages that usually last 2 to 3 days (Griffiths et al., 2000). The Drosophila then enters the pupa stage, where it transforms from its immature to mature stage over 3 to 5 days (Griffiths et al., 2000). Finally, the Drosophila’s mature stage (adult) occurs after eclosure from the pupa. During the adult stage, the fly has reached sexual maturity and is able to move and fly around. Once they are in their adult stage, Drosophila usually have a median lifespan of 70 days and a maximum lifespan of 90 days (Piper & Partridge, 2020). This relatively short and simple life cycle allows for genetic experiments that would normally take months or even longer in vertebrate organisms to be completed in only a matter of weeks (Jennings, 2011).

It is not just their rapid life cycle and reproduction rate that make Drosophila ideal for genetic experiments, but also their less redundant, 60% homologous genome to that of humans (Griffiths et al., 2000). Interestingly enough, 75% of the genes that cause human diseases also have homologs in Drosophila (Mirzoyan et al., 2019). With these commonalities to humans, and their general genetic tractability, Drosophila are frequently used as models of human genetics and diseases. Amodio et al., for example, used the UAS/GAL4 system to study common human cardiac transcription factors (TFs) in Drosophila (2011). These cellular components control gene expression and are central to normal human heart development. Naturally their malfunctioning can be central to human heart diseases (CHDs). The researchers, by over-expressing GATA4, NKX2.5, and TBX5 TFs in the Drosophila cardiac tube, eyes, and wings, found that the TFs up-regulate their natural target enhancers and cause defects in the Drosophila’s eyes and wings (Amodio et al., 2011; Lambert et al., 2018). This genetic model could lead to the development of future assays to study the interactions between human TFs and their natural target promoters (which are not easily done in human tissue cultures). This model can also be used to discover the genetic nature of human TFs, which could help investigate the genetic mechanisms that cause CHDs (Amodio et al., 2011).

Others, like Pham et al., have also used Drosophila to evaluate the effects of cinnamaldehyde (a compound in cinnamon) on Drosophila with and without Alzheimer’s disease (AD and non-AD, respectively) (2018). The goal of their research was to see if cinnamaldehyde is an effective method to treat humans with Alzheimer's Disease. The researchers evaluated Drosophila via a rapid iterative negative geotaxis (RNG), and a courtship conditioning test, and found that Drosophila in both the AD and non-AD groups portrayed an improved climbing ability, and a better short-term memory and locomotion thanks to the cinnamaldehyde (Pham et al., 2018). Now, future studies can be carried out to track the safety and efficiency of cinnamaldehyde as a potential therapy in humans (Pham et al., 2018). Many researchers also use Drosophila as simplified models of cancer, tissue regulation, wound healing, and as tools to discover many important genes and proteins (including but not limited to: the first potassium and transient receptor potential (TRP) channels, circadian clock genes, and genes required for memory and learning) (Mirzoyan et al., 2019) (Jan & Jan 2008) (Jennings, 2011). Overall, Drosophila have affected and enhanced many areas of biology—specifically medical genetics and treatment biology—thanks to their homology to humans and genetic efficiency (Jan & Jan 2008).

Individually, Drosophila have numerous visually identifiable traits & sex-distinct characteristics that contribute to their utility as model organisms. Among Drosophila, the females have cream-colored, peach shaped abdomens, and the males have pointed, darker, and more bristled abdomens (these characteristics can be viewed without the aid of high magnification). Both male and female Drosophila have multiple genes that affect their body color, size, bristles, wing size, wing vein patterns, wing angles, and more (Sturtevant, 1921). Researchers are still exploring the specific inheritance patterns for each of these traits, thus a knowledge gap is present regarding the mechanisms involved with inheriting various features. Out of all the known Drosophila traits, eye color is by far the most well understood and studied (Griffiths et al., 2000).

Drosophila are commonly used in both classroom lab settings and professional lab settings to investigate eye color—genetics, inheritance, and evolution. Interestingly enough, there is ample eye color variation in the fruit fly. Eye color is linked to the X-chromosome, with the Wild-Type (WT) eye color allele being dominant (and located on the CG12207 gene), and the white eye color (w) allele, for example, being recessive (and located on the CG2759 gene) (Grant et al., 2016) (Takahashi et al., 2003) (Xiao et al., 2017) (Heil et al., 2012). Thomas Hunt Morgan discovered the white-eye mutation in 1910 (Miko, 2008). During his research, he found that among fruit flies, eye color genes form linkage groups on chromosomes, which are inherited through Mendelian genetics (Bellen, 2015).

Mendelian inheritance is a broad topic that includes the principles of heredity first discovered by Gregor Mendel in 1865. This model of genetic inheritance includes the Law of Dominance, the Law of Segregation, and Law of Independent Assortment (Bellen, 2015). First, the Law of Dominance states that some alleles that code for a particular gene are dominant over other alleles that code for that same gene, which are termed recessive (Lewis & Simpson, 2020). If an organism contains a dominant variant, then it will display the phenotype, or effect, of the dominant allele (Lewis & Simpson, 2020). Next, the Law of Segregation states that, during gametogenesis (the production of gametes, or sex cells), the parents’ alleles separate from each other, allowing each parent to pass down one allele to their offspring. Thus, the progeny inherit one allele from each parent (Britannica) (Lewis & Simpson, 2020). Finally, the Law of Independent Assortment states that during gametogenesis, the alleles of a gene pair located on a pair of chromosomes are inherited independently of the alleles of a gene pair located on another chromosome pair. In addition, they are distributed independently of one another in the next generation (Lewis & Simpson, 2020).

As a result of Mendelian inheritance patterns, when a WT male mates with a w female, their offspring have a 50% chance of being female (100% of which will have the WT eye color) and a 50% chance of being male (100% of which will have the w eye color). In the next several generations, as a result of the dominant inheritance pattern of the WT allele, there is a greater increase in the frequency of WT eye color as males in the population inherit the WT allele from their heterozygous mothers. This inheritance pattern is portrayed below in figure 3 of Heil et al.’s study. It is important to note that there are various assumptions of Mendelian inheritance that allow the allelic frequency of this cross to be predicted - including random mating, and that genes from the maternal and paternal line are equally expressed in the offspring. In a natural setting, however, the inheritance of genes is not solely based on these assumptions: courtship and mating behavior also play an important role.

(Heil et al., 2012)

Among Drosophila, males perform courtship rituals. A male’s courtship involves a series of actions that include auditory, visual, and chemosensory signals (Mackay et al., 2005). Using visual and olfactory signals for orientation, the male aligns himself with the female, taps the female with his foreleg, uses pheromonal cues, and vibrates his wings to produce a courtship song (Mackay et al., 2005). The female then decides whether to accept or reject the males’ copulation (Mackay et al., 2005). A number of scientists have found that, due to a mating behavior impairment, mutant Drosophila are at a disadvantage when mating compared to WT Drosophila (Connolly et al., 1969). For example, when studying the mating rates of WT Drosophila versus mutant, yellow-eyed, white-eyed, and bar-eyed Drosophila, researchers like Backstock et al. and Petit et al. found that the mutants’ reduction in mean wing vibration (relative to the WT) resulted in less mating success. Connolly et al.’s study confirmed this phenomena, specifically indicating that the reduced mean period length of wing vibration among brown-eyed male drosophila during courtship impaired their mating success with WT females.

This year, with Backstock et al.’s, Petit et al.’s, and Connolly et al.’s findings in mind, I plan on investigating whether the phenotypic frequency of WT eye color increases over generations in a population of mutant Drosophila as a result of mating preference. This will be done by introducing a WT fly to a group of mutant, Sepia (Se) flies (which is located on the CG6781 gene) (FlyBase). I hypothesize that the phenotypic frequency of the WT eye color will increase in the population faster than predicted by the assumptions of Mendelian Inheritance as a result of the Se flies’ less desirable wing vibration; thus, resulting in WT’s mating attempts to be preferred. If this occurs, and the male WT’s courtship is accepted by the Se females, their offspring would be heterozygous and have WT eyes. The second generation would also be affected by mating preference, but the gene pool would be more diverse (as WT flies could now mate with homozygous mutants or heterozygous WT).

To my knowledge this future study has not been conducted, and, thus, my results could potentially support previous findings about mutant Drosophila mating behavior, and provide us with evidence that Se Drosophila are also at a courtship disadvantage compared to WT. In general, Se Drosophila’s specific courtship and mating behavior have not been heavily researched, so my results could deepen our understanding of the mutation’s effects on sexual fitness. Perhaps the results and observations I gather from this research on eye color and its effect on mating success, can be leveraged to investigate the effects other understudied phenotypes (eg. body color, wing type, and bristles, etc.) have on courtship.

METHODS & PROCEDURE:

Preliminary Study (11th grade):

Overview:

Preliminary research was based on Heil et al.’s 2012 work that investigated how the phenotypic frequency of the Wild-type (WT) eye color changed in a population of fruit flies when a WT fruit fly was added to a group of mutant, White-eyed (w) flies. Although the positive control (WT only) or negative control (w only) was not completed due to time constraints, the experimental arm (ie. adding a WT fly to a group of w mutants) was completed. This was done by setting up three crosses (eg. Experimental Cross #1, #2, and #3) each consisting of three female and three male w Drosophila, and one WT male. In Heil et al.’s study, researchers used five female and five male w per cross. However, due to a lack of w subjects available, this study only utilized three subjects of each sex per cross.

Senior Study:

Overview:

The Senior Study was similar to preliminary research however the experimental question focused on whether the phenotypic frequency of WT eye color increases over multiple generations in a population of mutant Drosophila due to mating preference. This was done by introducing a male, WT fly to a group of five male and five female mutant, Sepia (se) flies. Researchers have found that, relative to WT, the reduced mean period length of wing vibration among mutant Drosophila impairs their mating success with females (Bastock et al., 1956) (Connolly et al., 1969) (Petit et al., 1959). The following methods aim to find data to support or refute these findings.

General Materials & Care:

Before all research sessions, hands were thoroughly washed with soap, and safety equipment including lab coats, gloves, and goggles were put on. The se (Item # 172634) and WT (Item # 172100) Drosophila, FlyNap (Item # 173010, MSDS), vial set & vial labels (Items # 173076 and # 173190), sorting brushes (Item # 173094), and Drosophila medium (Item # 173210) were purchased from Carolina Biosciences, as recommended by Heil et al. (2013). FlyNap was always used inside a hood to prevent inhalation, as it can cause damage to respiratory organs. Drosophila arrived in their respective vials, which contained food, medium, and mesh. Throughout the experiments, knocked out flies were manipulated using a sorting brush, and vials with sedated flies were kept sideways until they woke up. In Chen et al.’s work, they found the best time to revert the vials back to an upright positioning was usually 50 minutes to several hours post FlyNap (2013). Thus, vials were turned upright after this period to ensure the medium did not drown the flies. Horváth et al.’s research indicated that fly cultures are considered overcrowded when there are approximately 50+ flies in one vial (2016). When this was the case, new vials of medium and mesh were prepared (following the modes described in the “Initial Experimental Cross Housing Preparation” section), and the flies in the overcrowded vials were anesthetized using FlyNap (following the safety precautions explained earlier in this section). The anesthetized flies were then divided up (~15 flies per vial) into the newly created vials. When a vial of Drosophila contained mostly dead flies and the medium was depleted, or when a vial was no longer needed, flies were anesthetized (following the safety precautions explained earlier in this section) and killed. The morgue vial where flies were killed and disposed of was supplied by the FlyNap kit and filled with ethanol (Carolina Biosciences, Item # 861261, MSDS) up to the line drawn on the morgue. As recommended by its MSDS, ethanol was always stored in a secure fire safety cabinet, away from flammable substances. The vials were then filled with a water, bleach, and dish soap mixture (25% bleach solution in water, Clorox Bleach, MSDS) to kill off any remaining larvae. The contents of the culture tube were then rinsed out and discarded into a secure trash bag.

First Experimental Arm:

Initial Experimental Cross Housing Preparation:

Seven vials were made and used for the Experimental Cross. They were labeled, “Male 1”, “Male 2”, “Male 3”, “Mixed”, “Female 1”, “Female 2”, and “Female 3”. Approximately 2 cm of medium formula and 5 mL of water was placed in each vial, followed by a folded piece of mesh. Then the vials were plugged with their respective foam caps, and labeled with one of the names specified above.

Anesthesia, Gender Differentiation, and Separation:

First, a vial of se flies was placed on a Drosophila stand (Carolina Biosciences, Item # 173030) in a turned-on hood. Flies were then sedated using the anesthetic wands included in the FlyNap kit for 3 minutes (following the safety precautions outlined in the “General Materials and Care” section). After the anesthetization was complete, the hood was turned off, and the sedated flies were then transported to a plastic weigh dish. Using a sorting brush and magnifying glass, the flies were then divided by sex based on the appearance of their abdomens. Among Drosophila, the females have cream-colored, peach shaped abdomens, and the males have pointed, darker, and more bristled abdomens (these attributes can be viewed without the aid of high magnification) (Asahina, 2018) (Ziabari & Shingleton, 2017). Males were placed on one side of the dish, and females were placed on the other. A Flinn Scientific Dissection Microscope (Model # 59-1815) was then used to verify the sorting. Next, the male flies were divided into three groups of five, and each group was placed into the “Male 1”, “Male 2”, and “Male 3” vials. The same process was done with the females, except each group of five was placed into the “Female 1”, “Female 2”, and “Female 3” vials. Only flies that had intact body parts were chosen as experimental subjects, and if flies appeared to have a disability they were not chosen. This was done to limit confounding variables associated with physical deformities that could impede sexual fitness. The extra, leftover se flies that were not used as experimental subjects and were placed in the “Mixed” vial for continued propagation.

Crossing of the Flies:

2-3 days later, once the female flies had time in isolation to lay any eggs from previous copulations, a vial of WT Drosophila were also anesthetized (using the methods and safety precautions previously described in the “Anesthesia, Gender Differentiation, and Separation” section). Once the WT flies were sedated, and the hood was turned off, they were then placed on a plastic weigh dish, and three physically intact WT males were identified and verified using the sorting brush, magnifying glass, and microscope (using the sex-identification methods previously described in the “Anesthesia, Gender Differentiation, and Separation” section). Next, a single WT male was guided into the “Male 1”, “Male 2”, and “Male 3” vials. At this point, each male vial contained five se males and one WT male. All the remaining, unused WT Drosophila were placed in the “Mixed” vial for continued propagation. The male and female experimental subjects were then crossed. This was done by using a sorting brush to guide the sedated Drosophila from the “Female 1” vial into the “Male 1” vial—while a peer held the male vial in place. The same was done for the “Female 2” and “Male 2” vials, and the “Female 3” and “Male 3” vials. Once this process was complete, and the three crossed vials of ten se and one WT Drosophila were established, the vials were re-labeled, “Experimental Cross #1”, “Experimental Cross #2”, and “Experimental Cross #3”, respectively.

Check-in Days for the P1 and F1 Generations:

After all three experimental crosses were set up, it was necessary to wait about 7-10 days for larvae to appear in the experimental cross vials (as recommended by Griffiths et al.) (2000). Thus, vials were checked every 2-3 days, and monitored for the development of the F1 (first filial) generation, and the health and quantity of the pre-existing P1 (first Parental) flies. During this time, photographic, qualitative data was also gathered.

Anesthetization and Extermination of the P1 Generations:

Once the larvae appeared in the experimental cross vials, each vial was anesthetized (using the modes and safety precautions previously described in the “Anesthesia, Gender Differentiation, and Separation” section), the hood was turned off, and all the P1 flies were placed on a plastic weigh dish. Ethanol was then added to the morgue (following the safety precautions outlined in the “General Materials and Care” section), and the P1 flies were extinguished.

P2/F2 Housing Preparation:

Right before the F1 generations reached adulthood, three vials were made (using the modes previously described in the “Initial Experimental Cross Housing Preparation” section). The vials were labeled “F2 Experimental Cross #1”, “F2 Experimental Cross #2”, and “F2 Experimental Cross #3”. These vials were made so that once the F1 generations were phenotyped, they—who would then become the P2 generation—could court in a fresh vial. And, subsequently, their F2 generations would develop in a fresh vial without eggs or larvae from previous generations.

Data Observation and Recording of the F1 Generations:

Once all the F1 larvae developed into adult flies (which, following Griffith’s et al. 's recommended timeline, took an additional 3-4 days following the appearance of larvae), they were phenotyped (2000). First, the “Experimental Cross #1” vial was anesthetized (using the modes and safety precautions previously described in the “Anesthesia, Gender Differentiation, and Separation” section), the hood was turned off, and the F1 flies were put on a plastic weigh dish for phenotyping. The number of WT and se flies were counted and phenotyped using a sorting brush, magnifying glass, and microscope. Using the sorting brush, the entire F1 generation was then moved to the fresh “F2 Experimental Cross #1” vial. The original “Experimental Cross #1” vial was discarded in the manner previously outlined in the “General Materials and Care” section. This procedure was repeated, respectively, for the rest of the experimental cross vials.

Check-in Days for the P2 and F2 Generations:

Verbatim procedure to that of the Check-in Days for the P1 and F1 generations.

Anesthetization and Extermination of the P2 generations:

Once the larvae appeared in the F2 experimental cross vials, each vial was anesthetized (using the modes and safety precautions previously described in the “Anesthesia, Gender Differentiation, and Separation” section), the hood was turned off, and all P2 flies were placed on a plastic weigh dish. Ethanol was then added (following the safety precautions included in the “General Materials and Care” section) to the morgue included in the FlyNap kit - and the flies were extinguished.

Data Observation and Recording of the F2 Generations:

Once all the F1 larvae developed into adult flies (which, following Griffith’s et al. 's recommended timeline, took an additional 3-4 days following the appearance of larvae), they were phenotyped (2000). First, the “F2 Experimental Cross #1” vial was anesthetized (using the modes and safety precautions previously described in the “Anesthesia, Gender Differentiation, and Separation” section), the hood was turned off, and the F2 flies were put on a plastic weigh dish for phenotyping. The number of WT and se flies were counted and phenotyped using a sorting brush, magnifying glass, and microscope. Ethanol was then added to the morgue (following the safety precautions included in the “General Materials and Care” section), and all the F2 flies were extinguished. The “F2 Experimental Cross #1” vial was discarded in the manner previously outlined in the “General Materials and Care” section. This procedure was repeated, respectively, for the rest of the experimental cross vials.

Second Experimental Arm:

Verbatim procedure to that of the “First Experimental Arm” however five male and five female WT were placed in each experimental cross vial instead of five male and five female se drosophila. In addition, instead of adding a single WT male to each experimental cross vial, a single se male was added.

Data Analysis:

For the first experimental arm, assuming random mating, calculations predicting the likelihood that the F1 flies would have se eyes versus WT eyes was conducted. Phenotypic data that was recorded during experimentation was then compared to the predicted outcomes to asses whether the female flies had a phenotypic preference when mating. Phenotypic results of the F2 generation was then compared to the F1 and P1 generations. This prediction and comparison process was repeated, respectively, for the second experimental arm.

RESULTS AND DISCUSSION:

As represented in Figure 1, male drosophila have darker, pointed, more bristled abdomens, while females have rounder, more cream colored, peached shaped ones. In addition, the Wild-type (WT) and Sepia (se) eye-colors contain overt differences: the se eye-color is more grayish black, while the WT’s is more red. These distinct visual differences allow drosophila to be great model organisms for controlled genetic crosses as mix-ups are infrequent. Sepia drosophila’s specific courtship and mating behavior has not been heavily researched in comparison to Wild-types’. With this, researchers have found that mutant, yellow-eyed, white-eyed, and brown-eyed drosophila have a deviation in wing vibration when mating, resulting in the WT’s more desirable mating attempts to be preferred by females (Connolly et al., 1969). With this knowledge in mind, for my research, I hypothesized that the phenotypic frequency of the WT eye color would increase in a population of se flies faster than predicted by the assumptions of Mendelian Inheritance, assuming se flies also contain a less desirable wing vibration.

Figure 2 contains the first filial (F1) results of three experimental crosses (1 male WT in a population of 5 male and 5 female se flies). Assuming random mating, there’s a ⅚ chance that the F1 flies would have sepia eyes (se/se) and a ⅙ chance that they would have Wild-type eyes (WT/se). In this figure we can see that the phenotypic frequency of the WT eye color increased in each population—and was greater than the 1:6 ratio of WT to se predicted by random mating. Specifically, the ratio of WT to se increased to 13:18, 23:29, and 47:12, in crosses 1-3 respectively. These results support that WT males have more desirable mating attempts than se males, which are at a courtship disadvantage. However, the ratio of WT to se flies varies widely across each replicate cross, suggesting something was unaccounted for in the experimental design that affected mating across each replicate. This variability could be due to replicate crosses containing fertile se females, who already carried eggs from previous se matings, thus impacting the number of se offspring recorded per vial. In order to account for this confounding variable, female flies were isolated for 48 hours to lay eggs from previous copulations before being crossed in the second phase of the experimental arm.

The proportion of WT flies among the second filial (F2) generation greatly decreased from the F1 generation (as displayed in Figure 3). Predicting the expected outcome assuming random mating for the F2 generation is much more complex than the F1 generation because the F1/second parental (P2) generation consists of homozygous se or heterozygous WT flies. In addition, the sexes of the F1/P2 flies were not accounted for. With this being said, even if WT is still preferred over se, we would expect to see a resurgence in the se in the F2 generation because 50% of the WT P2’s gametes carry the se gene. Among the first and second F2 replicates, the ratio of WT:se flies was 7:32 and 10:30 respectively. Lastly, for F2 Experimental Cross #3, there was a ratio of 11:7 WT:se. Overall, although the phenotypic frequency of the WT phenotype increased for F1, , the frequency of the WT phenotype generally decreased in F2. And, as predicted, we do see a resurgence in se flies in F2 compared to F1. However, it is important to note that the variability across replicates could have resulted from sedated F1/P2 flies being moved into culture vials before they were conscious again, causing many to drown in the liquid media and ultimately die. It’s also possible that inconsistent temperatures among the replicates crosses lead to different growth rates of eggs and larvae into adult flies.

After the first experimental arm was complete, the second experimental arm was conducted (1 male se in a population of 5 male and 5 female WT flies). By recording the number of se phenotypes in the F2 generation of this arm, we are able to make an enhanced inference on whether the se flies had a selective disadvantage when mating. Assuming random mating, if one se male mated with a WT female, then their progeny would be heterozygous WT. Likewise, if two WT mated their offspring would be homozygous WT. Thus, there is no possibility of having se offspring in the F1 generation of the cross—however they could potentially appear in F2 generation (by two heterozygous flies mating). The adaptations made to this arm of research included, first, allowing the WT females 48 hours of isolation to lay any eggs from prior matings. And, second, the number of first parental (P1) flies was recorded every two days to ensure they were living for the entirety of the mating period. Unfortunately, when the number of living P1 flies was first recorded it was noted that all the se males had died (likely from being over-anesthetized and then drowning), thus the experiment was unable to continue, and only F1 phenotypic data was collected. The number of WT F1 flies were 42, 48, and 63 in replicate crosses 1-3 respectively.

Overall, my research sought to better understand whether se drosophila males are at a courtship disadvantage compared to their WT counterparts. To my knowledge, the first experimental arm I conducted was unprecedented, and supported that mutant drosophila are less sexually fit than WT. However, the second experimental arm of research, which was intended to further validate or refute these results, was unable to be completed. Thus, future experimentation should focus on repeating both the first and second experimental arms of this study in their entirety—including adaptions made along the way such as: allowing the female flies 48 hours to lay any eggs from previous matings before being crossed, tracking the number of living P1 flies, recording the sex of the F1 flies, and using a less harsh anesthesia procedure (ie. shorter time or ether instead of FlyNap) to prevent over-sedation and drowning. The results of these future assays would allow us to advance and solidify our knowledge on the se mutation’s effects on sexual fitness. ​​

Both my and this proposed future research investigating the se eye color and its effect on mating success can be leveraged to investigate the effects of other drosophila eye colors & phenotypes—e.g., body color, wing type, and bristles, etc.—on courtship. Together, these assays would allow us to deepen our understanding of the complexities and consequences of drosophila mutations on mating, which is currently understudied in the field, and, overall, broaden our understanding on how mating preferences affect the persistence of certain mutations in the wild and the course of evolutionary change.

Lab Notebook:

https://docs.google.com/document/d/1LhiiV7sn_irPQlId_VuD_UdzzXpUyHCowcVqdVBLJE0/edit?usp=sharing

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