Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

The overall goal of this experiment is to stage pupal periods and measure wing pigmentation of Drosophila guttifera. This method can help answer key questions in the evolutionary developmental biology field, such as how pigmentation patterns are developed in Drosophila. The main advantage of this technique is that we can obtain detailed information about morphological development and pigmentation process.

Though this method can provide insight into the morphological development and pigmentation process of Drosophila guttifera, it can be applied to other Drosophila species. To begin puparium removal, affix a piece of double-sided tape on piece of paper towel. Place a 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 paint brush.

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. Proceed with image preparation and remove the anterior portion of the puparium with forceps.

Take out the pupa and place it into phosphate-buffered saline, or PBS, in a plastic Petri dish. Next, cut the basal joint of a wing. As the wing is folded, place it into a plastic Petri dish filled with ddH2O to extend it by osmotic pressure.

Collect newly eclosed adults once every 10 minutes from a stock file. Anesthetize a fly using a CO2 anesthetizing pad. Confirm anesthetization by immobility and cut the basal joint of a wing.

Drop 10 microliters of PBS on a glass slide. Place the wing in the drop. And cover the drop with a cover slip.

Next, turn on the light of the stereo microscope and set it to maximum. Set the objective lens to 11.5x and set the diaphragm to the most open state. Then, turn on the camera and set the camera.

Focus on the sample by moving the focus knob of the microscope. Push the shutter button of the remote control unit to take an image. Take an image of the wing, centered a campaniform sensillum, positioning the distal part of the wing on the left side and the interior part of the wing on the upper side.

Open an image with campaniform sensillum spot at the center. Click Image, Type 1, 8-bit, to convert the image to an 8-bit image. Click rectangle in area selection tools and draw a rectangle.

Set the right side of the rectangle so that it is located to the right of the campaniform sensillum spot. Click Edit, Selection, Add to Manager, then check the Show All column. Next, click angle tool in the line selection tools.

Draw the first line on the posterior line of the third longitudinal vein. Set the left endpoints of the line on the vertex of the previously drawn rectangle. Draw the second line on the upper side of the rectangle.

Press the M key to measure the angle between the two lines drawn in this step. Click the window of the image on the screen of the computer. Click Edit, Selection, Add to Manager.

Click rectangle in area selection tools and draw a rectangle approximately 1/9th the size of the image window. Click Edit, Selection, Rotate. Write the minus degrees of the previously measured angle in the Angle column and click OK to rotate the rectangle drawn in this step.

Move the rectangle drawn by using the arrow keys and set the endpoint of the posterior line of the second longitudinal vein on the left side of the rectangle. And the lower side of the rectangle, attach to the posterior line of the third longitudinal vein. Confirm a perpendicular line from the endpoint of the second longitudinal vein to the posterior line of the third longitudinal vein was drawn.

Define the foot of the perpendicular line as point A.Record the X coordinate and the Y coordinate of point A, indicated below area selection tools when placing the cursor on point A.Open the image with point A in the center. Click Image, Type, 8-bit, to convert the image to an 8-bit image. Click rectangle in area selection tools and draw a rectangle of approximately 1/9th the size of the image window.

Click Edit, Selection, Specify. Check Oval and Centered column. Write 100 pixels in the width and height columns.

Write the X coordinates of point A in the X coordinate column, and write the Y coordinates of point A in the Y coordinate column, then click OK.Next, click Analyze and Measure. If the calibration has already been finished, ODs are indicated in Mean column. Comparing the data of multiple stages revealed that stage P12 is the timing of onset of pigmentation and that pigmentation is completed by 12 hours after eclosion.

After its development, this technique paves a way for researchers in the field of evolutionary developmental biology to explore differences in pupal development and pigmentation process among Drosophila species.