Abstract 

Organisms are constantly being bombarded with information that requires them to respond and make a choice. When faced with an impending decision an organism must process incoming information relying on neural circuits that arose through development, and were shaped by evolution, to induce a specific behavioral response. One of the most consequential decisions that organisms make is whether to mate or not, and if they do mate how to identify appropriate mates. In Drosophila melanogaster, mating decisions involve a series of interactions between potential partners. Females rely on fine-tuned processing of multiple sensory inputs to determine male identity and suitability, resulting in a decision to either repel or accept males. This accept vs repel behavior has been used to identify genes that control female mating including the gene Neuroglian (Nrg). Previous work has demonstrated that Nrg mutant females reject mating and suggested that this occurs through the role that Nrg plays in mushroom body (MB) formation. The mushroom body is a specific region of the brain that integrates and processes information. In this research we demonstrate that Nrg has additional roles outside of MB development that control female mating behavior.

Introduction 

Previous research on Neuroglian have made connections between the mushroom body, a critical brain region for information processing, and female behavior using mutants (Carhan et al 2005; Figure 1). More recent work focusing on the evolution of female mate behavior has identified Nrg as an important gene differentiating populations (Jin et al 2022). What remains unknown is how Nrg controls female mating behavior.

The mutant NrgI7 (top) has an anatomically normal brain and high mating rate. The mutant NrgI4 (bottom)  is missing its mushroom body and has a mating defect. It mates at a significantly lower rate. These data support a direct role of Nrg in mating behavior through developmental control. However, we can develop a second model based on knowledge that Nrg is expressed in adults. We now have two models we need to distinguish between. Does Nrg play a role in female behavior  through MB development, or is there an additional role in adult females?

The lack of wild-type flies in crosses with UAS and GAL4 demonstrate that our knockdown is very efficient.

From the previous experiment we get the result that GAL4/UAS can knockdown Nrg. We want to restrict Nrg to a specific life stage so we can test which model is most accurate. To do this we need to manipulate Nrg just in the adult stage after the mushroom body develops. The MB development occurs in the pupal stage.

Crosses

Parent

yv; {Nrg RNAi} ; +  X  + ;  tub-GAL80[ts] ; tub-GAL4/TM6b

Female flies to keep

yv/+ ; {Nrg RNAi}/tub-GAL80[ts] ; +/tub-GAL4 (Wild type bristles)

Female flies to discard

yv/+ ; {Nrg RNAi}/tub-GAL80[ts] ; +/TM6b (Hu, humeral)

Control cross

Parents

yv; {attp40} ; +  X  + ;  tub-GAL80[ts] ; tub-GAL4/TM6b

Female flies to keep

yv/+ ; {attp40}/tub-GAL80[ts] ; +/tub-GAL4 (Wild type bristles)

Female flies to discard

yv/+ ; {attp40}/tub-GAL80[ts] ; +/TM6b (Hu, humeral)

Collect virgins. Puting virgins at room temp for 1 day, then place them in 30 degrees Celsius for 4 days. Put males into vials and complete mating experiment. Let them mate for 40 minute. After a week, look for larvae. If their have larvae means they mated. 

After knockdown we exposed Nrg knockdown and control flies to males to determine if they would mate. We observed that Nrg knockdown flies mated at a significantly lower percentage compared to the controls. This tells us that Nrg is important for the adult female Drosophila mating behavior. 

Behavior Experiment

Future 

We will test the Nrg on different life stage so we will test them on pupa stage and larve stage.