… and all you ever wanted to know about fruit flies!

Have you ever wondered how fruit fly scientists perform experiments with these tiny insects every day? Since I work with fruit flies, I want to provide a glimpse of the day-to-day life of a fly researcher (imagine you’re a “fly on the wall” in our lab!). My project is to figure out how neurons in the memory system communicate with each other to store long-term memories. I dissect fruit fly brains and use fluorescence imaging to watch what neurons are doing in real-time, and I collaborate with other graduate students and postdocs in the lab who perform behavioral experiments. But what are the day-to-day activities that every fly scientist has to take care of?

Our stocks of flies are kept in incubators at a stable temperature and light cycle.

My day usually doesn’t start until 10am when the lights in our flies’ incubators turn on. Most of our flies are kept in these refrigerator-sized incubators at a tightly-controlled temperature of 25°C (77°F), which is a fruit fly’s favorite temperature. They’re also kept on a strict light cycle—12 hours of light starting at 10am followed by 12 hours of darkness, which is important for reducing variation in behavioral experiments.

Fly lines are kept in vials, which are stored on racks for easy stacking.

There are thousands of different fly lines, but I only have about 200 of them. Each fly line is kept in a small vial with food at the bottom (the yellow stuff, which is a thick gel of yeast, sugar, and cornmeal) and a cotton plug at the top to keep them from escaping. Each fly line has to be “flipped” to a new vial every couple of weeks after the hundreds/thousands of flies have finished making a mess of their current home.

A fly vial in use.

If you look closely, you can see pupal cases stuck to the walls of the vials. An adult fly can live for about a month at its optimal temperature, during which time a female can lay hundreds of eggs. The eggs hatch into larvae after approximately 24 hours, which spend about 6 days chewing through food and gaining strength. When the time is right, the larvae climb the sides of the vials and turn into pupae. After 4 motionless days, a new

Fruit flies go through four life stages: egg, larva, pupa, and adult. source

fly emerges from its pupal case. In total, it takes about 10 days after an egg is laid before a fly emerges. We can tell when a fly is about to come out because the pupa gets darker as the fly develops inside it. Within hours after emerging, an adult female fly is ready to mate and lay eggs.

Knocking out flies with CO₂ in the vial prevents them from escaping

Flies are dumped onto a CO₂ pad to keep them unconscious while they are being sorted.

Now I need to get flies out of some of those vials so I can use them for my experiments. Carbon dioxide (CO₂) knocks flies unconscious, so we slide a syringe needle in through the cotton top of the vial and pump in some CO₂ to knock them out. Then we can dump the flies out onto CO₂ pads, which have a porous styrofoam top and CO₂ flow underneath to keep the flies unconscious.

Wild flies (bottom) have bright red eyes, but lab flies (top) usually have white eyes until a gene with an eye color feature is added. source

For our experiments, we need to make sure our flies have the right genes or mutations. In my case, I need flies with a fluorescent marker in memory-specific regions of the brain so I can see them under a microscope. This usually means I need to collect flies with two added genes in their DNA: a “marker” gene and a “location” gene. I will need to sort through the flies to make sure they have both genes. Luckily, when a scientist makes a fly line with a new gene, they attach some DNA code for a physical feature to the gene. Usually, the feature is a change in the flies’ eye color. Although wild flies have bright red eyes, our lab flies are bred to have a mutation in the eye color gene so that they have white eyes.

Lab flies with an extra gene inserted into their DNA usually have orange eyes. The shade of orange varies, but darker colors can often mean more than one new gene is present. Photo by Weiwu Xie, source

Because our flies have no eye color, adding a gene with an eye-color feature makes these flies stand out among their white-eyed brethren. This way, we can sort through the flies and pick out the ones with colored eyes, most commonly orange. Flies with two genes have darker orange eyes because the color adds up, which is great for me.

We use a fine-tipped paintbrush to carefully sort through the flies without harming them.

I sort through the flies under a microscope using a paintbrush, and then brush the flies I need for my experiments into smaller vials to separate them.

“Fly morgues” are used to dispose of flies. They also conveniently double as fly traps.

There are, of course, tons of flies I don’t need. We can’t possibly save all the unnecessary flies, so they get dumped into a “fly morgue”. These are bottles filled with a mixture of water, ethanol, apple cider vinegar, and a drop of dish detergent. The funnel keeps them from escaping after we dump them in, and the detergent breaks the surface tension of the water so the flies can’t just skim across the surface. It’s a quick and painless death for our noble subjects, and at least they die swimming in their beloved vinegar. These also make great traps in the home to take care of fruit fly infestations. If you don’t have any vinegar, a splash of wine should do it.

A dissection microscope. A dish with wells for dissecting is shown underneath.

Now it’s time to prepare the flies for experiments. For some people in our lab, this means loading the live flies into machines that track their activity for sleep or memory studies. For others, such as myself, it means dissecting out the brains from individual flies (or in some studies, the larvae instead), and performing experiments on the brains instead of live animals. As you can imagine, it takes a pretty long time to learn how to dissect out the tiny brain from these insects. We use a microscope to see what we’re doing, and very carefully remove the shell of the fly head using fine-tipped tweezers.

A close-up “action shot” of a fly head being dissected. The head is being held with a pair of fine-tipped tweezers.

If you’re interested in seeing a dissection in action, check out this video from Jove, the Journal of Visualized Experiments.

A graphical representation of an adult fruit fly brain. Modified from Chiang et al, 2011

The final brain is too small to see by eye (though the most experienced among us can point out the tiny white dot in a drop of water). Fun fact: did you know that fruit flies don’t have blood like we do? They have an open circulatory system filled with “hemolymph”, which is a clear liquid filled

Click on the picture for a bigger version. The brain (circled in red) is floating in a solution. Can you spot it?

with nutrients for cells. We dissect brains in a solution that’s designed to mimic this hemolymph and keep the brain alive as though it was still inside the fly.

Close-up of the imaging set-up for dissected fly brains. Fresh hemolymph-like solution is being perfused in from the right to replace the old solution being vacuumed out on the left. A 60x magnification objective is shining a tiny beam of fluorescent light into the dish.

A structure in the fly brain called the “mushroom bodies” is fluorescing under a microscope. Photo by Jenett et al / CC-BY-2.0

Once I’ve dissected my brain, I pin it down in a dish for imaging. The brain-in-a-dish is placed under a microscope with a fluorescent light so that the fluorescent marker in the brain glows. During the experiments, I make sure that fresh hemolymph-like solution is flowing over the brain and being vacuumed out on the other side to keep the brain healthy, and sometimes I use this system to add in drugs which can affect activity in the neurons. This allows me to manipulate specific neurons and study how it affects communication between them.

In future “Fly Life” articles, I will go into detail about how specific experiments are performed, such as my imaging technique or the behavioral experiments used by my labmates. Until then…

Got any questions or want more details? Feel free to ask in the comments section!