This protocol is an excerpt from&#160;Fukutomi&#160;et al.,&#160;Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of&#160;Drosophila guttifera,&#160;J. Vis. Exp.&#160;(2018).
1. Removing puparium
NOTE Pupae of&#160;Drosophila&#160;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.
<ol>
	<li>Affix a piece of double-sided tape on a piece of paper towel.</li>
	<li>Place a pupa on the double-sided tape ventral side up (Figure 1A).</li>
	<li>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.</li>
	<li>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).</li>
	<li>After breaking the puparium as much as possible, take out the pupa using a fine paintbrush (#5/0) (Figure 1C).</li>
	<li>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. 
	&#160;</li>
</ol>

insect puparium removal: a method for pupal investigation

Source: Fukutomi, Y., et al. <a href="https://www.jove.com/video/56935/methods-for-staging-pupal-periods-measurement-wing-pigmentation">Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of&#160;Drosophila guttifera.</a>&#160;J. Vis. Exp. (2018).
This article describes a technique to remove an insect&#8217;s puparium while keeping the inside pupa intact and therefore fit for further investigation. The example protocol shows the procedure with Drosophila guttifera; however, this technique is applicable to other Drosophila species.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20048/20048fig1.jpg" /> 
Figure 1. Illustration of removing puparium.&#160;(A) Place a pupa ventral side up on a piece of double-sided tape. Remove the anterior part of the puparium. (B) Break the puparium with forceps from the ventral side. (C) After breaking the puparium, take out the pupa using a paintbrush.

Insect Puparium Removal: A Method for Pupal Investigation

Source: Fukutomi, Y., et al. Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of&#160;Drosophila guttifera.&#160;J. Vis. Exp. ...

-To gain access to the developing pupa, remove the puparium after the pupal epithelium has separated from the cuticle or after pupal stage P5. To begin, place the intact pupa with its ventral side facing towards you. Use forceps to hold the puparium at the empty space between the anterior end of the casing and the pupa's head. Then, gently break and remove the anterior piece of the puparium to expose the head. While keeping the ventral orientation, use the forceps to create small breaks along the anterior-posterior axis of the puparium and ensure a continuous gap is formed in the process. Be careful not to damage any of the pupa tissue directly underneath. Then continue to break the puparium from the gap outward, one portion at a time, and spread out the torn casing in the process. Using a fine wet paintbrush, pick up the exposed pupa for further investigation. In the example protocol, we will see how to remove the puparium of Drosophila guttifera and prepare the pupa for imaging. -To begin puparium removal, affix a piece of double-sided tape on a piece of paper towel. Place the pupa on the double-sided tape, ventral side up. 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. 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 formed between the puparium and pupal legs, and break the ventral side of the puparium while minimizing damage to the internal pupa. After breaking the puparium as much as possible, take out the pupa using a fine paintbrush. Place the pupa on a piece of tissue paper that has been moistened with ddH2O and placed in a plastic Petri dish. Immediately take photographs as the exposed pupa is vulnerable and dries easily.

insect-puparium-removal-a-method-for-pupal-investigation

Research

JoVE Encyclopedia of Experiments

Biology

Drosophila melanogaster (fruit fly)

Pupa

This article describes a technique to remove an insect&#8217;s puparium while keeping the inside pupa intact and therefore fit for further investigation. The example protocol shows the procedure with Drosophila guttifera; however, this technique is applicable to other Drosophila species.

Overview

Protocol

<img title="figure-representative results-25" alt="figure-representative results-25" class="xfigimg" src="/files/ftp_upload/20048/20048fig1.jpg" /> 
Figure 1. Illustration of removing puparium.&#160;(A) Place a pupa ventral side up on a piece of double-sided tape. Remove the anterior part of the puparium. (B) Break the puparium with forceps from the ventral side. (C) After breaking the puparium, take out the pupa using a paintbrush.

Insect Puparium Removal: A Method for Pupal Investigation

In This Article

Overview

Protocol

Representative Results

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