Introduction

Drosophila melanogaster, the common fruit fly belonging in the Order Diptera, has been used for lab experiments since T.H. Morgan started his experiments in 1907. Drosophila make good genetic specimens, because they are small, easy to culture in the laboratory, and produce many offspring. It is easy to anesthetize them, and they have a short generation time (Geiger, 2002). Differences in body features help distinguish between male and female flies. Females are slightly larger and have a light-colored, pointed abdomen. The abdomen of the male fly is dark and blunt. The male flies also have dark bristles and sex combs on the upper portion of their forelegs(Flagg, 2005).
Drosophila melanogaster use hydrophobic odorants called pheromones in the ritualized process of mating. Our hypothesis states that blind and wild type male flies will mate at equal rates, because they use their pheromones to find a mate regardless of whether or not they can see. These are picked up by sensory neurons located on their antenna, maxillary palps and most of the surface area of the fly (Galindo, 2001). They use these for the regulation of sexual behaviors. In Drosophila this can be shown by the modulation of the males’ courtship behaviors. Males use the receptors in their legs, wings, and antenna when pursuing a female. It has been determined that if the necessary pheromones are absent, there will be no male courtship response to the female (Lin, 2005).
The purpose of this investigation is to study the mating behavior between wild-type males and wild-type females, and white-eyed males with wild-type females to see if these flies use their vision or their pheromones to mate. Conversely, behavior between normal and white-eyed males mated with white-eyed females was also observed (Flagg, 2005). There are several steps which are followed to achieve copulation between a male and a female fly detailed below:

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Materials and Methods

Bottles were used to prepare crosses between male and female Drosophila. Instant Drosophila Medium Formula 4-24 and distilled water were added to the clean bottles. Fleischmann’s Active Dry Yeast were sprinkled on top of the medium. Virgin male wild-type Drosophila were carefully added to two of the bottles. Then, wild-type females were added. The last two bottles were filled with male and female white-eyed Drosophila. The bottles were set aside for ten days to ensure that the mating cycles completed a supplemental amount of virgin Drosophila in various life stages for collection.
In order to collect virgins, the adult Drosophila were disposed of in the morning via dumping them into Fly Morgue. Those that remained were pushed into the medium so they would not affect the outcome of the virgins later. Smaller amounts of food and distilled water, along with yeast, were put in small, clean vials. These vials were labeled male w+, male w-, female w+, and female w-. After 8 hours, the adult virgins that hatched were put to sleep using FlyNap, collected and separated between both sexes of all types using a microscope. Curly flies were removed and disposed of in the Fly Morgue, then males and females were separated and placed in their appropriate vials. Collections were made three days each week for two weeks.

Three weeks later, mating assays were observed by crossing male w+ with female w+, male w+ with female w-, male w- with female w+, and male w- with female w- and recording the time it took for them to copulate. Time was recorded in intervals of 20 minutes, and the Drosophila were disposed of in the Fly Morgue afterward. Multiple assays were performed throughout a time period of two weeks when adequate amounts of Drosophila for each type were present.

**This entire procedure was conducted a total of two times, having four weeks apart, in order to accumulate a reasonable amount of Drosophila for the experimental data.

Results

Table 1 gives the time it took for a wild type male and a wild type female to mate in each particular cross. Table 2 gives the time it took for a blind male and wild type female to mate in each cross. Table 3 gives the time it took for a blind male and a blind female to mate in each cross. And, Table 4 gives the time it took for a wild type male and a blind female to mate in each cross. Table 1 has an average copulation time of 6:31, Table 2 has an average copulation of 8:37, Table 3 has an average copulation of 6:34, and Table 4 has an average copulation of 7:12. The copulation times that are recorded as 20:00 minutes are the assays that did not initiate mating behavior during the allotted time period. Because all of the averages are relatively close to each other, and the cross containing both blind sexes is not the highest, there must be something other than sight that allows them to mate. Normal-eyed flies are denoted as W+, and white-eyed flies are denoted as W-. Time of copulation was measured by minutes:seconds.

**Note that the Table's are denoted as follows:

Crosses:

W+ f X W+ m (Table 1): 15:36, 9:56, 20:00, 1:21, 2:41, 9:27, 3:16, 3:18, 8:42, 1:29, 3:02, 4:28, 1:35

W+ f X W- m (Table 2) : 12:05, 10:56, 1:48, 2:27, 6:38, 9:56, 6:32, 6:50, 20:00, 6:14, 7:08, 3:12, 2:05, 4:07, 20:00

W- f X W- m (Table 3): 1:02, 1:53, 6:37, 7:17, 9:31, 3:15, 14:17, 20:00, 8:24, 4:15, 2:45, 1:20

W- f X W+ m (Table 4): 5:28, 2:53, 11:27, 1:47, 4:27, 20:00, 9:51, 15:10, 7:28, 3:44

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Discussion

The results that are presented supports our hypothesis that pheromones, not vision, is the main element in Drosophila melanogaster mating. Overall, the white-eyed flies and the wild-type flies mated in about the same amount of time. There was no significant difference between the four crosses. The cross with the wild type male and females had the better average time, but only a couple seconds faster than the flies with the blind males and females. There is a slight increase in the time that it took for the flies to mate when one fly was blind and the other was wild type. Therefore, no significant difference can be made from this data. Hence, it can be interpreted that the flies are able to find a mate by such means other than vision. Flies most likely find their potential mate by the detection of pheromones produced by themselves and the other sex. However, it does seem that they use more than one technique to find a mate.

Based on the results from the crosses from Table 2 and Table 4, it seems to be more important for the males to be able to see. The cross with the wild type males and the blind females had a faster average time than the crosses with blind males and the wild type females. This is because the males initiate the behavior and need to be able to locate the female. The females just wait until the males find them. Therefore, when the males were blind it takes a little more time for the mating to occur. Yet, the overall results show that if a fly has a mutation such as blindness, they would have no disadvantage when finding a mate. When attempting to find a mate, there was no significant difference in the fitness of the flies.

Another way of explaining the slight difference in the data is the time of day the assays were done. Drosophila melanogaster releases their pheromones according to a circadian cycle (Stern, 2007). Since the assays were not done at the exact same time every day, there may have been more pheromones released at one particular time of day that helped guide the flies more so than any other time. There may have been contamination distributed in the collection of virgin fly vials, due to the variations in the larval stages of the flies’ maturation periods. There was also difficulty collecting adequate amounts of virgin flies, which could potentially change the results. One major variation could be the integration of non-mating assays recorded as twenty minutes, which were included in the average copulation times. All of these variations could be possible sources of error toward the experiment.

There were other similar experiments done by previous Shippensburg Students that presented conflicting data to our results. One experiment done by Nicole Thierwechter got results of blindness having a negative affect with mating (Barrett, 2008). Another experiment by Shanna Barrett had the opposite results of the males taking longer to mate in the presence of pheromones (Barrett, 2008). The results were observed in our experiment seem to be in the middle of these two experiments. In order to get a better idea of what really effects the mating of Drosophila melanogaster more experiments would need to be done.

From the experimental data, it can be concluded that there is no significant difference between the use of vision and pheromones in Drosophila during mating. Therefore, flies most likely find their potential mate by the detection of pheromones produced by themselves and the other sex. However, some changes could be made to better the experiment. These changes include collecting more virgin flies to prepare more mating assays, as well as conducting assays at the same time each day.

References

Barrett, Shana. 2008, Nov. 30. Effect of pheromones on mating behavior of Drosophila melanogaster.

Galino, Kathleen. 2001. A Large Family of Divergent Drosophila Odorant-Binding Proteins Expressed in Gustatory and Olfactory Sensilla. Genetics. 159; pp. 1059-1072.

Lin, Helping. 2005. A Drosophila DEG/ENaC channel subunit is required for male response to female pheromones. PNAS. 102:36; pp.831-836.

Flagg, Raymond. 2005. Carolina Drosophila Manual 45-2620. Carolina Bioligical Supply Company.

Stern, Peter S. Yu, Lian, Choi, Man-Yeon. Jurenka, Russell A. Becker, Liron. Rafaeli, Ada. 2007. Molecular modeling of the binding of pheromone biosynthesis activating neuropeptide to its receptor. Journal of Insect Physiology: Aug 2007; Vol. 53 Issue 8, pp. 803-818.