Insect Puparium Removal: A Method for Pupal Investigation

This protocol is an excerpt from Fukutomi et al., Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera, J. Vis. Exp. (2018).

1. Removing puparium

NOTE Pupae of Drosophila are covered by a structure called the puparium. An insect of Muscomorpha (flies) does not shed its larval cuticle at pupation; instead, it hardens the cuticle after apolysis, and uses it as a protective cover of the pupa, the puparium. A pupa residing inside a puparium has a true pupal cuticle, which is very soft and fragile. Before apolysis takes place around P4(ii), epithelia and puparium are attached together, and therefore removing the puparium without damage is very difficult. After P5, removing the puparium is laborious, but useful for morphological observation and definition of pupal stages. The process is carried out as follows.

1. Affix a piece of double-sided tape on a piece of paper towel.
2. Place a pupa on the double-sided tape ventral side up (Figure 1A).
3. Locate the space between the anterior side of the puparium and the internal pupa. Grasp and remove the puparium around this gap using forceps, and expose the anterior side of the head of the pupa.
4. Insert the tip of a forceps by moving it parallel to the anterior-posterior axis. Lift the tip of the forceps to locally break the puparium. Repeat this action until the breakage reaches the posterior part of the puparium. Ensure that a gap is also formed between the puparium and pupal legs, and break the ventral side of the puparium and minimize the damage to the internal pupa (Figure 1B).
5. After breaking the puparium as much as possible, take out the pupa using a fine paintbrush (#5/0) (Figure 1C).
6. Place the pupa on a piece of tissue paper that has been moistened with ddH₂O and placed in a plastic Petri dish (diameter 60 mm x height 15 mm). Take photographs as soon as possible because the exposed pupa is vulnerable and easily becomes dry.