BIO306 Drosophila melanogaster Experiment

Overview

You will be randomly assigned two vials of homozygous mutant flies (males and females mixed) along with all of the tools needed to perform the crosses. Using these flies, you will set up the following crosses:

  • Parental cross: two virgin mutant 1 females x two mutant 2 males (two vials)
  • F1 cross: two F1 females x two F1 males (three vials)
  • F2s will be counted and scored for phenotypefor up to 12 days and the results compared to the expected ratio using a chi square analysis.

Life Cycle

The entire life cycle (see figure next page) takes about two weeks. Fertilized eggs too tiny to see are laid in the medium by females. These hatch into tiny larvae that will eat and eat until they grow large enough to pupate. They grow fast. One day you will see nothing in the vial, and the next it will be teeming with larvae. Any time you see larvae in a vial after setting up a cross, this is your sign that the cross was a success (at least in terms of getting male and female flies together in the same vial).

After about a week or so, larvae will crawl to a dry place to pupate. This dry place is usually the side of the vial and you will see yellowish-brown “cases” on the sides of the vial. These will darken as the time of adult flies hatching out of them approaches. Sometimes, if your medium is somewhat dry, the larvae will simply pupate in the medium. If so, you will not see the yellowish-brown pupae on the sides, but then suddenly one day, you will have adult flies in your vial! Either way, you now have adults to either cross (if they are F1s) or count (if they are F2s).

Once adults hatch, note that they have a limited life in this stage, sometimes just a few days. Therefore, if you set up a cross, as long as you see the flies awake and moving around after the anesthetization, they have mated, laid eggs, and produced the next generation for you. Even if they are dead after a day, that is OK. Rather than panic and work at setting up another cross (or 10), just be patient and wait to see if larvae emerge. (This usually takes about a week from the day that you set up the cross).

NOTE: Once you see larvae from the parental cross, this is your sign that all is well. Now you must remove any remaining adult flies. If you leave the adult parental flies in the vial, once the larvae pupate and hatch into the next generation of adults, these adults will back-cross to their parents. Not only is this ewwwwwwwwwwwwwww!, but it will screw up your experiment since you want an F1 x F1 cross, not an F1 x P cross.

General Procedures

Sexing flies (three methods)

  • Females are generally larger than males (not always the case though due to normal variation)
  • Females have V-shaped abdomens and black stripes while males have rounded abdomens, a black patch at the tip, and then black stripes (not always clear in body color mutants or newly hatched flies though)
  • Males have a shiny black spot on each front leg (a sex comb) while females do not (the most accurate way to distinguish sex but takes a microscope to see)

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Selecting virgin females

A female fly stores sperm internally from prior “encounters” in order to fertilize eggs produced later (in case she never finds another male fly on the banana…). Therefore, for the parental cross, you must obtain (virgin) mutant 1 females before they have copulated with their mutant 1 male vial mates because you only want them to fertilize their eggs with sperm from mutant 2 males. How do you isolate virgin females? We take advantage of the fact that after female flies hatch from the puparium, they are not ready for mating for about eight hours, so as long as you find female flies that are less than “eight hours old”, these females will be virgins.

Use the following procedure to isolate virgin female flies:

  • At some point during the day, preferably morning because more flies tend to hatch in the morning, remove all of the adults from the mutant vial from which you are collecting females by anesthetization and placement into the morgue (vegetable oil). This is called “clearing the vial”.
  • Return any time within eight hours from that time. Any female flies that have hatched out within that eight hours are virgins and can be used to set up the first Parental cross with any males from the other mutant vial.

NOTE: You need a total of four females for your first cross, but use whatever females you have to set up what you can. In other words, do not throw perfectly good virgin female flies away and start over just because you didn’t get four! For example, if you have one mutant 1 female, put her in a vial with two mutant 2 males (she can handle it) and try again to get another female to add later. Remember, you really only need one male and one female for this to work. We are using extras in case something goes wrong.

NOTE:The F1 cross doesn't require virgins so do not waste your time acquiring them for that second cross. Even if your F1 females have already mated with F1 males so that they have stored “F1 sperm”, aren’t you going to be crossing them to F1 males anyway? So the second cross is easy: just wait until a bunch of F1 flies have hatched from your parental cross (and you can use F1 flies that are all from one vial or mix and match from your two parental cross vials) and then isolate six females and six males and divide them into three vials. Done! 

Preparing a vial

  • A scoop of medium (food) is added to a clean vial
  • One squirt of water is added and the vial is mixed until the medium turns uniformly blue or until it solidifies
  • A pinch of 3-5 grains of yeast are sprinkled onto the surface of the medium (the fly larvae will eat the yeast)
  • The vial is plugged with a sponge

Hints:

  • medium should not be dry and flakey or sloppy and runny
  • more than 6 grains of yeast is too much as the yeast will use up the oxygen in the stoppered vial
  • use vials the same day they are made
  • if mold develops in the vial, the flies will die
  • you are crossing flies in multiple vials to improve the odds of one working; remember that you only need one to work!

Anesthetizing flies

  • “FlyNap” (a proprietary solution that causes the flies to pass out)--- Tap the flies down to the bottom of the vial and away from the sponge. Dip the little brush into the FlyNap to dampen the brush, then insert the brush past the sponge of the vial. Leave it in place with the vial sideways until the flies pass out. Try not to touch the sides of the vial with the brush.

Advantages:

  • good technique for runny medium or if a chunk of medium dislodges
  • FlyNap does not kill flies

Disadvantage:

  • takes longer than ether to knock flies out
  • Ether--- Squirt several drops of ether onto the sponge found at the bottom of the anesthetizer. Tap the flies to the bottom of the vial and away from the sponge. Invert vial and hold it over the well of the anesthetizer, pull out the sponge, and immediately place the open mouth of the vial over the well of the anesthetizer. Tap the vial to “help” the flies fall into the well of the anesthetizer. When the vial is empty, place the stopper onto the well of the anesthetizer and put the sponge back into the vial. Leave the flies in the well for a minute initially, then look inside the well to check for twitching. Continue checking every 30 seconds or so until they are not moving at all.

Advantage:

  • faster than FlyNap

Disadvantages:

  • if the fly medium is too wet or dry, pieces may fall into the well that you will have to clean out
  • ether can kill if you treat the flies for too long
  • ether is flammable so very dangerous

The Experiment

All equipment and media will be housed in the “fly room” to which access will be made available to you 24/7. The door is always unlocked from the hallway, and beginning Monday of the second week of classes, your name will be on a list sent to the University Police. If the building is locked, call them and they will let you in. Be sure you have your student ID in case they ask for that.

Your goal is to set up a parental cross between virgin females of one mutant type (mutant 1) and males of a different mutant type (mutant 2) using the procedures outlined above. Once you have placed anesthetized flies into your vials, leave the vials on their sides in your basket. This way, the anesthetized flies will not be “face-planted” into the medium. Come in the next day to see that they are still alive and then place the vials in an upright position. Now you wait, often the most difficult part. 

After you see larvae in your vial (check after about a week), remove any surviving parental adult flies to prevent back-crossing later. Then check every couple of days for pupae, and finally, hatching adult F1 flies. Once the F1 flies hatch, wait until there are enough to get the six females and six males needed for the second F1 cross. Don’t wait too long though, because these F1s are mating with each other, and females are laying their eggs in this vial. Their fly life goes on; they won’t wait for you!

Once you have your F1 cross completed, wait another week for the larvae to be visible, and then remove the F1 adult flies. After these F2 larvae pupate, they will hatch into F2 adults and you will be counting and scoring their phenotypes from all three vials, and comparing your observed results to your expected using a chi square analysis.

You must be vigilant to catch the first day of hatching for each vial. This means that once you see pupae, you should check on your flies once a day, if possible. Why do you need to know the first day of hatching? Well, remember that the entire life cycle is about two weeks (which we are going to underestimate to 12 days, just to be safe). Once the adult F2 flies hatch, you simply want to count them. However, they are flies! They will mate, lay eggs, and F3 larvae will emerge, pupate, and F3 adults will hatch. If you don’t keep track of when the F2s hatch, then you run the risk of counting and scoring F2s and F3s. (Go ahead and try to predict your F3 results given the variety of potential crosses from an F2 population. Then agree that we do not want to have to do that.) You want to avoid having F3 flies mixed in with your F2 population.

Therefore, by keeping track of “Day 1” of hatching per vial, you will know when “Day 12” is and when you should stop counting. You do not have to count your flies every day, but you should avoid counting them all on Day 12 to prevent insanity. There will be a lot of flies!! A good approach is to simply note Day 1 for each vial, and then count the flies on Days 3, 6, 9, and 12. Each time, anesthetize the flies, sort and count, then throw in the morgue.

NOTE: If one or both of your mutations is on the X chromosome (chromosome 1 in flies) then you must also record your results in terms of how many of each phenotype are male and female. If neither of your mutations is not on chromosome 1, then you do not need to record sex in your final count.

After recording your numbers, throw the bodies in the morgue and wait for more adults to hatch, up to and including Day 12. If you reach a count of 500 flies total from all of your vials before Day 12, you can stop early. If you never reach 500 flies even after 12 days of counting, then that is your destiny.

Community Issues (i.e. DO NOT BE A JERK IN THE FLY ROOM!!)

  • There are about 160 students all needing to work in this tiny space this semester, along with students from other biology lab courses. Share the materials, clean up after yourself, put things back where you found them, and keep your hands off other people’s baskets. This is something you should have learned in Kindergarten. If not, you can learn it now.
  • Do not clear your vial of adult flies by removing the sponge and letting them loose into Cowley Hall. (There will be enough escapees flying around the building by accidental release). Anesthetize them and place them into the morgue. If you don’t have enough time to do that extra 5-minute step, come back later and learn to plan your time better.
  • If you mess up a vial (too much yeast, medium is too runny, etc.) or get mold in one, or if no larvae ever appear, or if for any reason you must discard a vial, remove and throw away any tape from the outside of it, and place the vial into the special discard bin, but keep the sponge in it (or put one in it). This will keep any flies inside the vial that you may not have seen, and will keep other loose flies from having a nice place to hang out.

Finally, hang on to all of your vials (except the messed up ones) until the official fly basket ceremony near the end of the semester. Instructors may need to borrow your vials once you are done to provide for students who have tried hard to get things to work with no success. Instructors will always leave a note that we have taken something if this is the case, so if something disappears without a note, it probably means there was a jerk in the fly room (see first bullet point under “Community Issues”).