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In this paper we describe a detailed and flexible protocol for the maintenance of a fly population cage. An efficient way to regularly produce milligrams to tens of grams of embryos, larvae, pubi and adult flies. Collected flies that just come in for the process to obtain large quantities of DNA, RNA, proteins and other similar components required for biochemistry and molecular biology experiments.

Population cage can be time consuming to setup. However, maintaining a cage over months saves time and enables rapid collection of biological material in a short period. To begin, setup the plastic container by cutting out three two-by-two centimeter squares in the plastic container lid.

Add three quarter inch labeling tape to the four corners of each hole to ensure a tight fit for the foam plugs. And then place one foam plug in each hole. Cover the inside of the container with plastic wrap.

Over this film, add a layer of cotton to cover the bottom of the plastic container and tear away the excess cotton. Next, move on to fly food preparation by adding 333 milliliters of deionized water to a 500ml beaker. In a chemical hood, stir the solution with a magnetic bar and add 167 microliters of propionic acid and 1.08 milliliters of phosphoric acid from stock solutions.

Slowly add 77.5 grams of active dry yeast, avoiding big clumps. After dissolving the dry yeast, add 38.8 grams of sucrose last. Immediately after dissolving the sucrose, pour the fresh food over the cotton.

Make sure to cover the cotton evenly, before closing the plastic container with the lid to prevent escaped flies from contaminating the food. We suspend 1.5 grams of the hardest id nondi coronated embryos from the previous cycle. Cut two filter papers in half and distribute the biological mixture evenly over the four pieces using a spatula.

Lay the filter papers on top of the soaked cotton and close the lid. Finally incubate the plastic container until the pupil stage. Monitor the embryos as they become first instar larvae.

The resulting larvae will feed on the yeast mixture, growing and molting twice into second and third instar larvae. During the growth period, clean the fly population cage as detailed in the accompanying text protocol. Four days after the initial setup, observe the larvae pupate.

The larvae will remain in this stage for four more days. Before the first flies emerge, open the plastic container and place the plastic film containing the cotton with all the pupi over the lab soaker paper inside the clean population cage. Autoplay the previously prepared molasses mixture with a stir bar for 30 minutes.

Cool the mixture and add Tegosept solution to the molasses mixture. Next, pour the mixture into a 21x14.5 centimeter meat tray. Ensure that the volume is enough to cover 16 trays.

Wait up to 20 minutes for the molasses to solidify, and cover each tray with plastic film to avoid drying. Then store the trays at 4 degrees Celsius until used. Add 200 milliliters of deionized water into a beaker, and slowly pour dry yeast while stirring with a spoon.

Stop adding dry yeast when the mixture reaches the consistency of peanut butter to prevent flies from sticking to the food. Remove the plastic film from one molasses tray, and cover it with a layer of the fresh prepared wet yeast using a spoon or spatula. Make sure the entire surface is covered.

Place the cage in the coldrum at 4 degrees Celsius for up to 30 minutes to ensure the flies have stopped flying. While the cage is in the coldrum, open the net and carefully add the molasses tray with wet yeast into the cage. Close the net and place the cage at room temperature.

Two days after eclosion, introduce a new molasses tray with fresh wet food into the cage. To increase the yield of egg deposition, make the surface of the wet yeast as irregular as possible. Maintain the cage at room temperature.

When the embryos can be visualized as small white dots, they are ready to be collected. To harvest the embryos, calm the flies by placing the cage in a cold room for up to 30 minutes. Prepare the plastic container and the fly food for the next cycle.

Next, in an eight liter autoclave tray, add water to one quarter of the total capactiy and add one milliliter of 10%Triton X-100. While the cage is in the coldrum, carefully remove the molasses plate and place it inside a biohazard bag. Remove the tray from the bag and submerge it quickly in the detergent solution in the autoclave tray, then close the biohazard bag and place the bag in a biohazard container.

Use a distilled water gun to wash embryos and yeast off of the molasses. Discard the plates and molasses into a biohazard box. Pour the solution carefully through a three sieve set.

Wash the clumps on the top level with the distilled water gun until no clumps remain. Rinse the autoclave tray with distilled water, and pour the rinse water through the sieves. Remove the top sieve.

Most of the first and all of the second instar larvae will remain in sieve 40. Remove the second sieve. The yellow material in the third sieve is the mixture of embryos and small first instar larvae.

Use the distilled water gun to move all the eggs to one side of the sieve. Then collect them with a spatula and weigh them. Use 1.5 grams to start a new cycle.

Use the rest of the embryos for further processing or discard them. Six consecutive cycles were collected from starting material of 1.5 grams. And yielded an average of 10 grams of embryos and larvae per cycle.

The yields of five consecutive collections one hour after adding new food, and three consecutive collections three hours after placing a new molasses plate are shown. After watching this video, you should have a good understanding of how to regulate a culture, a population of drosophila melanogaster, using a fly population cage.