JMP-IN tutorial
Sex combs

Mutant phenotypes

Practical considerations:

• There are relatively few virgin mutant females available to start the crosses.  Therefore, you should practice anesthetizing and sexing the wild type cultures first and work with virgin females after you have gained some experience.
• When examining flies under the dissecting microscope, use the upper lamp for illumination only! The bottom lamp will cook your flies.
• There is a fine line between etherizing the flies too long and killing them, and too short and having them wake up before you are ready.  It is better to err on the side of too short because you can always use the re-etherizers (petrie plate lid with a wad of cotton taped inside) to knock them out again, although this process is annoying because it wastes so much time.  Dead flies develop "angel wings" (the wings stick up from the backs rather than lying flat), which makes them hard to roll over and observe (in addition to making further crosses impossible if that is what you want!).
• Ether is one of the three most flammable and explosive common laboratory reagents!  Do not make any spark or flame in the vicinity of the etherizers and keep the stock bottle in the fume hood.
• When you put anesthetized flies into a new vial, place the vial on its side during and immediately after the time when you add the flies. If you drop the anesthetized flies straight into the medium they will either drown or get stuck in the medium.

Sex Determination

It is imperative that you be able to reliably differentiate between male and female flies on the basis of sex combs!

In future phenotyping and crosses a mistake in sexing one fly can ruin the experiment!  Have your teaching assistant check your work before setting up any genetic crosses!
 

The fastest and most reliable criterion for differentiating sex of adult flies is the presence of sex combs on the first tarsal segment of the front legs of males. The sex combs are absent in female flies.

More on sexual differences in D. melanogaster.
 

Mutant Phenotypes

Wild-type phenotype

Triple mutant phenotype

The mutations that will be used in this experiment effect genes coding for eye color, wing shape and bristle morphology. Thus, we will be examining alleles at three different loci. Today, we will set up crosses between virgin triple mutant females and wild type males. We will then follow genetic crosses through the F₂ generation.

You must be able to reliably identify ALL the mutant phenotypes involved in the crosses, especially the forked bristle phenotype.

Although the mutant females used today carry all three mutations, this will not necessarily be the case in subsequent generations.  Thus, today will be your opportunity to learn to identify all of the mutations using individuals that are certain to have them. 
 

wild-type bristles forked bristles

Bristle morphology is the most difficult trait to identify.  Flies with the forked bristle phenotype ("f", top; compare with wild type "+" bottom) may have many normal looking bristles in addition to forked bristles.  Thus it is important to examine the bristles on each fly thoroughly when deciding on the bristle phenotype.  The presence of a SINGLE forked bristle indicates that the fly has that phenotype. The distribution of forked bristles on the flies is not random.  They occur most commonly at the posterior end of the dorsal side of the thorax (the position indicated by the arrow).    Although the phenotype is called "forked", the mutant bristles may actually be hooked or even just shortened rather than actually forked.  They are not long, slender, and gradually tapering.  Experience will be your best guide to identifying the bristle phenotype!

Eye color is the most easily identified mutation.  The top fly has the white eye ("w") phenotype and the bottom has the red eye (wild type or "+") phenotype.

Wing morphology is also fairly easy to identify.  The top fly has wild-type ("+") wings and the bottom has miniature ("m") wings.  Note: flies that have just eclosed (emerged) from their pupae may have wings that have not yet fully expanded.  In that situation, the folded wings will look like small black blobs on the backs of the flies.  Since it will be impossible to identify their phenotype, they should be excluded from use in crosses or analyses of phenotype frequencies.

Additional information

There are several other mutant phenotypes available for linkage mapping in Drosophila. Some of these mutant phenotypes are shown below:

• mutant eye phenotypes
• mutant body color phenotypes
• other mutant fruit fly phenotypes - (from the Exploratorium Science Museum)

• sex-linked crosses