My name is Sarah Ertel and I'm a postdoc in Ed Kravitz's Lab here in the Department of Neurobiology at Harvard Medical School. My name is Sibu. I am a medical student from Germany and I'm doing a research rotation here at the Kravitz Lab.
And we want to show you how we set up fights and study the aggression and fruit flies. The first thing we do is prepare isolation vials, which consists of microwaving fly food and adding 1.5 mils to a culture tube when the food has solidified. Then we trap it with cotton.
First step is to get socially naive flies, flies. I haven't seen any other fly. And so to do that we take late stage pupi from the kinds of flies we're interested in, and we remove the pupi from the vial carefully with the forceps.
Take the prepared isolation vial, remove the cotton and gently place the puy inside, recap it and label the vial. These puy are placed back into the day night cycle climate controlled incubator. And then the next day we check if they've enclosed and then we write the date on which the flies have closed.
Then we age them for three to five days, and during that time then we apply paint to the back of the abdomens of the flies so we can identify them. And so now we're making chambers for the, for the actual fights. And to do that we use plain microscope slides, two of them, and we'll measure the middle and make marks with a diamond cutter pin, scratch the surface, and then if find an edge and the microscope slide should just break easily in half.Okay.
Then we glue the edges of the slides together with adhesive glue. Then the two halves are glued together to make a box. We can clean it with 70%ethanol and then some water to remove our finger residues.
Fill the bottom of the chamber with 1%agros and stick the chamber inside. And that provides humidity for the flies. And then we wanna give the flies something to fight over.
And so we, even though the actual size of the chamber can be considered a territory, we provide them with food. And we put that food in a scintillation vial. And this provides an additional level of possible territoriality that one fly might wanna defend.
So I'll now take the freshly microwave food and fill it into the food cups, which are actually scintillation caps, which will be the fighting arena on which the flies will fight later. It's important to make it a little bit convex so that you can actually see the whole surface. You also want to avoid to have air bubbles, cause those will be an obstruction for the flies when they walk on the food cup, Which ones In order to attract the flies to the food cup.
We will also add a drop of yeast paste in the middle of the food cup. And we first have to prepare the yeast paste. So we just take live yeast paste, put it in a food cup, then we will add a drop of water and mix it until it gets a paste.
Once the food cups have cooled down, you put the yeast paste or just a little drop of it in the middle of the food cup. So we are in the behavior room right now, one of our two behavior rooms. And this room is completely dark if we turn on all the lights and it's humidity controlled and temperature controlled.
So we keep the temperature at 25 degrees and try to have the humidity over 40%so that the flies that we control for as many conditions as we can. And what I'm gonna do now is set up the fights. So we brought the food cups that we prepared prepared earlier are here, and I'm gonna insert them in those chambers.
Try to place them in the middle of the chamber. We put the lid back on.Okay. And I'm gonna use this aspirator to aspirate the flies exactly a pair of flies.
So I'm gonna use take two flies, aspirate them in here, and then insert them into the chamber. That's what I'm gonna do. Now, I will hold the vial like this because flies are known to go against gravity and towards the light, so they tend to go upwards.
So it's easier for me to catch them when I remove the cotton. As you can see, I have both flies in the ator right now. I also have to make sure that as these are painted flies, that I take one that is painted and one that is not painted.
Or if we use two colors, one that is painted in one color and one in the other color, but they are the same genotype. Now I insert them in the chamber for that. I remove the wax ball.
There is a little hole through which I will insert the flies, and I try to have the lid in a position so that the flies can't escape when I tap. They will fall down a little bit and then I just blow, just softly so I don't have to blow em in really hard because that will disturb them. It takes them between three and let's say 40 minutes till they settle down and they had a look at the environment in the chamber.
And then they will start occupying the food cup and the fight will begin. Now I will take the whole chamber and place them in front of the camera and I have to adjust the height so that I film the food cup from a slight angle so that we can see the whole food cup. And the whole food cup is in focus.
And the room in which these aggression assays are set up is important. It's a isolated room, kept free of traffic and noise. It's kept at 25 degrees and we try to keep the humidity between 45 and 55%to keep the flies happy and and healthy in that situation.
And also, we dim the overhead lights so that the light on the food cup should also be an attraction to the flies. Using DFA as a model system and using this type of setup allows us to look not only at like aggression intensities, but also the the specific patterns of aggression that we see. The specific behaviors we're very interested in knowing how the brain is wired during development and then, and then how that is manifested as a adult that hasn't learned these behaviors.
So they just come out immediately when they see the start interacting with the other flies. They know how to lunge, they know how to be aggressive. And so we wanna know how these behaviors are wired into the nervous system of the flies and how then they're controlled.
