Pre-Trials:

As an overview of my methodology, I first completed population cultivation, which allowed me to have enough vials for virgin week. Fruit flies are considered to be virgins for 8 hours after hatching. In order to collect virgins, I would transfer out all adult flies in a vial eight hours prior to class (See image to the right). This way, any fly that hatched would be considered virgin. See my previous post for more details about this process. Virgin fruit flies were required in order to induce RNAi. To induce RNAi, I utilized the UAS/Gal4 system, which was used as a method in order to regulate genes. The UAS/Gal4 system works by using offspring crosses. In an offspring cross, the UAS flies have dsRNA that is responsible for gene silencing and the Gal4 flies have a promoter that expresses the dsRNA throughout the entire organism. When the two fly lines are crossed, the offspring will exhibit RNAi for the spookier gene. The female Gal4 flies must be virgin, so that when crossed with the UAS males, the offspring will form a genetic cross of only Gal4 and UAS flies. 

Expiremental-Trials:

Once virgins were collected, two male flies and six virgin female flies (eight total) were placed into a vial and will then be given six days to reproduce. After the six days, I performed the larvae quantity assay. These methods for counting the number of larvae involved adding water to the media, allowing many of the larvae to float. From here, I took the media solution under a microscope and sorted out the larvae from the media (See image to the right). Here I counted and recorded the number of larvae present. This process was repeated eight times (four experimental and four control). The only difference in the control was that the virgin females were bred with Gal4 males, instead of UAS males, which was used to ensure that the Gal4 promoter did not adversely impact the offspring. 

For data collection, I have quantified data from eight vials (four control and four experimental). For the control group, the average number of larvae offspring is ​​34.75, with a standard deviation of 18.28. For the vials where flies exhibited RNAi, the average number of larvae offspring was 12.75 with a standard deviation of  10.779. The graph to the left depicts these values.

Findings:

In this research project, I explored the role of the Spookier gene in the development of fruit fly. Specifically, I used a technique called RNA interference (RNAi) to reduce the expression of the Spookier gene and observed the effects on the number of larvae offspring. My findings suggest that when the activity of the Spookier gene was limited in expression, there was a statistically significant decrease in the number of larvae. However, this reduction in gene activity did not result in loss of embryonic viability, meaning that the embryos could still develop into larvae despite the reduced activity of the Spookier gene.

These results are particularly interesting when we consider the work of Dr. Ono and his team, who found that the Spookier gene is primarily expressed during the larvae stage of development, although it begins to be expressed in the later stages of embryonic development. This suggests that the Spookier gene might be involved in producing a growth hormone that aids in embryo development, but is not absolutely necessary for the embryo to develop into a larva. Dr. Ono’s findings support mine, as it could explain why there was only a reduction and not an elimination of larvae present. While outside the scope of my lab, I noticed that larvae failed to progress to the pupa stage and also tended to have a brown hue (See imgaes to the right).

My research contributes to the larger academic conversation by providing new insights into the role of the Spookier gene in fruit fly development. It suggests that while the Spookier gene plays a role in the number of larvae offspring, it is not essential for embryonic viability. This finding opens up new avenues for future research, as scientists can now investigate how the Spookier gene affects larval and embryo development in more detail, as well as explore the functions of other genes during this critical stage of development.

Conclusion:

In conclusion, my  study highlighted the importance of understanding the genetic factors that influence the development of organisms. By uncovering the role of the Spookier gene in fruit fly larvae, I hope to contribute to a broader understanding of developmental biology, which is essential for advancing our knowledge of genetics. This research helps understand the complex processes that govern fruit fly development. Soon, I will be sharing my results with the University of Indiana Bloomington, so they can record the results of RNAi of the spookier gene in their databases.

Limitations:

Given the nature of my lab, there are a few limitations that could have impacted the results of my study. First, I was unable to ensure that every offspring in the experimental group exhibited RNAi for the Spookier gene. Ensuring the presence of RNAi was outside the scope of this lab and would have required sequencing for the dsRNA, which could have interfered with the larvae. Additionally, I was unable to control for the health and age of the male flies before breeding. Research from the Berg Lab shows that health and age both impact the sperm quality and count of flies, which could have impacted the number of larvae offspring. To limit the effects of these limitations, the same number of flies, of similar age were bred together, to help ensure similar egg quantities. These methods give me the best chance for success to get an insight into the development of the embryo and larva stages, without requiring additional equipment.

• Explored RNAi effects on the Spookier gene in fruit fly embryos.

• Found a significant decrease in larval offspring without affecting embryonic viability.

• Methodology inspired by Dr. Rockwell's larval counting technique.

• Results highlight the Spookier gene's role in larval development, not embryo survival.

• Research contributes to understanding genetic influences in developmental biology.

• Preparing to share findings with the University of Indiana Bloomington.

• See a recap in the presentation to the right.