Mantenimento di un<em> Drosophila melanogaster</em> Cage Popolazione

1. Starting the Cycle: Seeding Embryos NOTE: The cycle starts with 1.5 g of collected material (a mixture composed of embryos and/or some first instar larvae) from the previous cycle. This material will be placed in a plastic container (see table of specific materials/equipment) with an active yeast mixture until the pupal phase. The container will be closed after the introduction of the biological mixture of embryos and larvae with the lid to avoid allowing the larvae to escape. It is necessary to make holes into the lid in order to allow air circulation. To avoid having larvae escape through the holes, foam plugs are used. Finally, It is recommended to wear gloves and lab coat, not only in this section but also in the whole protocol, to avoid getting dirty or staining clothes with bleach. Set up the plastic container. Make three square holes with a razor blade in the plastic container lid of approximately 2.2 cm. Add tape (3/4 inch labeling tape) to the four corners of each hole to ensure a tight fit for the foam plugs (50 x 55 mm, d x l), and then place one foam plug in each hole. Cover the inside of the plastic container with plastic wrap. Over this film, add a layer of cotton to cover the bottom of the plastic container. Tear the cotton with your fingers. Prepare the fly food. Add 333 ml of deionized water in a 500 ml beaker. While stirring the beaker with a magnetic bar, add 167 μl of propionic acid and 1.08 ml of phosphoric acid, from the stock solutions (99.96% and 85%, respectively). Slowly add 77.5 g of active dry yeast, avoiding big clumps. After dissolving the dry yeast, add 38.8 g of sucrose. NOTE: Sucrose should be the last ingredient to be added because immediately after adding this component, fermentation will begin. Immediately after dissolving the sucrose, pour the food over the cotton, and make sure to cover the cotton evenly. Close the plastic container with the lid to avoid escaped flies within the laboratory contaminating the food. Resuspend 1.5 g of the harvested embryos (not dechorionated) from the previous cycle with 5 ml of 70% ethanol. Cut in half two filter papers and distribute the biological mixture evenly over the 4 pieces using a spatula or a transfer pipet with a wide tip (cut if necessary). Lay the filter papers on top of the soaked cotton and close the lid. Finally, incubate the plastic container at RT (24ºC) and humidity (35%) until the pupa stage. NOTE: The plastic container should not be placed in a humid chamber, otherwise this will encourage the growth of bacteria. 2. Continuation of the Cycle: From Embryos to Flies. NOTE: This part of the cycle goes from the embryos placed in the plastic container on day 1 until 9 days later when the adult flies will emerge from the pupae. During these 8 days nothing has to be done but monitoring that the embryos progress correctly to the next stages of the Drosophila life cycle until eclosion. If larvae start dying and turning black during this time, check that the foam plugs are not too tight and that adequate ventilation is provided. This period is a good time to clean the fly population cage from the previous cycle. Observe the embryos become 1st instar larvae around 24 hr after the adult female deposited them into the fly food (see step 3). The resulting larvae will feed from the fly food prepared in step 1 during 4 days, growing and molting twice into 2nd and 3rd instar larvae. Clean the fly population cage. Place the cage at 4 ºC for at least 30 min to slow down the activity of the flies. Remove the net that is covering one side of the cage, and with a fast movement, cap the open side with a large biohazard plastic bag. Pour the contents of the cage into the bag. With the biohazard bag still covering one side of the cage, position the cage vertically over a sink with the biohazard bag side down. Carefully remove the plastic bag to prevent flies escaping and discard it in a biohazard waste container. Carefully open the upper net enough to create a small hole, rinse the inside of the cage with water, which will wash the flies into the sink. Slowly increase the size of the hole and eliminate all the flies inside the cage. Clean the cage and both nets with water and soap. While the nets are still wet secure them on both sides of the dry clean cage, and finally put inside a lab soaker paper, which will prevent the plastic film prepared in step 1.1.2 from sticking to the cage. Now the cage is ready for the next cycle. Four days after the initial setup, observe the larvae pupate. The larvae will remain in this stage for 4 more days. NOTE: During this phase the pupae will cover the entire cotton surface inside the plastic container. During the pupal phase and before the first flies emerge, open the plastic container and place the plastic film containing the cotton with all the pupae over the lab soaker paper inside the clean population cage. Close the net with a double knot and clean the plastic container and the lid for the next cycle. NOTE: Pupae affixed to the lid should be discarded and destroyed by autoclaving or freezing before eclosion. 3. Adult Flies. NOTE: After the 4 days of the pupa stage the first flies will emerge from the pupae. Within 24 – 48 hr all flies should have eclosed. In this part of the cycle, it is important to provide them food with the aim of creating the right environment for reproduction. Prepare the molasses trays. Combine in a 1 L flask 556.25 ml of deionized H2O, 90 ml of molasses, and 22 g of agar. Autoclave with a stir bar for 30 min, cool and add 9.25 ml of 10% Tegosept in 95% EtOH. Pour the mixture into a meat tray of 21 x 14.5 cm. Ensure that the volume is enough to cover 15 – 17 trays. Wait 10 – 20 min to solidify and cover each tray with a plastic film to avoid drying. Several flasks of 1 L can be prepared at the same time. Store the molasses trays at 4 ºC wrapped in plastic until required. Add a molasses tray covered with wet yeast inside the cage with the adult flies. Before adding the plate, place the cage at 4 ºC in the cold room for 30 min. Add 200 ml of deionized H2O into a beaker and slowly pour dry yeast while stirring with a spoon. Stop adding dry yeast when the mixture becomes approximately the consistency of peanut butter to prevent flies getting stuck into the food. NOTE: It is important not to allow the food solution to solidify otherwise it will be very difficult to cover the molasses tray and extract the eggs during the harvesting process (see section 4.3). 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. Be sure that the entire surface is covered. While the cage is still in the cold room, open the net and carefully add the molasses tray with wet yeast into the cage. Close the net and place the cage at RT. NOTE: It may be useful to cover the plate with an empty meat tray during insertion, but be sure not to block access of the flies to the molasses tray. Change the food plate every 24 – 48 hr to avoid the yeast to get too dry. Adult flies are very sensitive to desiccation. 4. End of the Cycle: Harvesting the Embryos NOTE: The best fly fertility is 3 – 5 days after eclosion. Therefore, harvesting during this time will get the best embryo yield. After desired collections are completed, the cycle is over. The flies should be sacrificed and the cage cleaned as specified in step 2.2. Typically, 2 days after fly eclosion, introduce a new molasses tray with fresh wet food into the cage, as explained in section 3.2.5. To increase the yield of eggs deposition, with the help of the fingers, make the surface of the wet yeast as irregular as possible. If an old food tray is present in the cage, before adding the new plate, carefully remove it and discard it into a biohazard bag. Another tip for increasing the yield is to remove the cotton layer so all the eggs will be laid in the food. Maintain the cage at RT. Observe the embryos as small white dots. NOTE: The eggs are ready to be collected. For routine maintenance of the fly cage, 2 day collection is the recommended time. This collection will contain mostly embryos and few 1st instar larvae. See representative results for typical yields of harvested embryos for various collection time points. Harvest the embryos Place the cage in the cold room for about 30 min. In the mean time prepare the plastic container and the fly food for the next cycle as explained in section 1. In an 8 L autoclave tray add water to ¼ of the total capacity and then add 1 ml of 10% Triton X-100. While the cage is in the cold room remove carefully the molasses plate and place it inside a biohazard bag. Remove the tray from the bag and submerge it quickly (to prevent flies from escaping) in the detergent solution in the autoclave tray. Close the biohazard bag and put in a biohazard container. Flies will not drown in water that does not contain detergent. Use a large paintbrush and distilled water gun to wash embryos and yeast off of molasses. Discard plates and molasses into biohazard box. Pour solution carefully through a three sieve set (coarsest sieve 30 on top, 40 in the middle and 100 on bottom). Wash clumps on top level with distilled water gun until no clumps remain. NOTE: The adult flies and 3rd instar larvae will be retained in the sieve 30. Rinse the autoclave tray with distilled water and pour the rinse water through the sieves. Remove top sieve and repeat process if yeast clumps remain. Most of the 1st and all of the 2nd instar larvae will remain in the sieve 40. Remove the second sieve. The yellowish material in the third (bottom) sieve is the mixture of embryos and small 1st instar larvae. Use distilled water gun to move all the eggs to one side of the sieve. Collect them with a spatula and weigh them. During these cycles the collection yield of embryos/larvae ranges between 7 and 13 g. Use only 1.5 g of this material to start a new cycle (section 1). Use the rest of the embryos for further processing or can be discarded. 5. Further Processing NOTE: The embryos not utilized for continuation of the population can be used immediately for experiments or alternatively can be frozen at -80 ºC. For both options, the embryos may need to first be dechorionated. For dechorionation, wash the embryos for 2 min in 50% bleach solution and rinse thoroughly with distilled water. To freeze the embryos, first dry by pressing firmly with a paper towel, and then weigh and place them, with the help of a spatula, into a 15 or 50 ml conical tube. Finally submerge the conical tube in liquid N2 for a few seconds.