• Begin by selecting a fly culture of the required type (wild, mutant or F₁) in which the pupae are beginning to hatch.
   

• Remove all adult flies from the vial.  These adults might be used for subcultures or crosses, as appropriate, or terminated if not needed.
   

• Using a dissecting needle carefully remove several mature pupae from the wall of the culture vial and place them on a glass microscope slide.
  
  Note: 1). Mature pupae have dark features (e.g. the wing buds and the eyes) that can be seen through the pupa case.  Young pupae or empty pupa cases do not have these characteristics.  Mature pupae are within a day or two of hatching.  2). Be sure not to puncture or fracture the pupa cases with the dissecting needle.  Use the side of the needle, not the point, when loosening and removing the pupae from the wall of the vial.  Usually a pupa will stick to the side of the needle if the needle is carefully slid between the pupa and the wall of the vial.  The pupa can then be transferred to the glass slide.
   

• Examine the ventral side of one of the mature pupae on the glass slide using a monocular compound microscope set at 100X (10X objective and 10X eyepiece) with the diaphragm fully open so the maximum amount of light will pass through the pupa. 
   
  Note: If you inspect the ventral side of a mature pupa, you should see the eyes, wing buds and legs of the developing fly inside the pupa case (see fig. 13 on p. 12 of the Carolina Drosophila Manual * ).  But, if the pupa is dorsal side up you will only see black bristles and hairs.
   

• Identify a foreleg (front leg) of the developing fly within the pupa case and determine if a sex comb is present.  Male fruit flies have a sex comb on each of their two forelegs, but females do not.
  
  Note: 1). The front legs of the developing fly are the innermost of the three pairs of legs seen when examining the ventral side of the pupa.  2). A sex comb is a small, black, comb-like structure located about 1/3rd of the way up the foreleg from its distal end (see fig. 14 on p. 12 of the Carolina Drosophila Manual * ).
   

• Once the sex of a developing fly has been determined, place the pupa on an index card with a label.  Be sure to keep males and females clearly separated.
   

• Repeat the above process until you have sexed the number of flies in each sex that you require.

* The Carolina Drosophila Manual is available from Carolina Biological Supply Company.
 
 

CONTACT PERSON:  Robert Weibust - weibust@mnstate.edu

LAST UPDATE:  4/25/03

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