TITLE: Protocol for fixing and peeling Aedes aegypti embryos
AUTHORS: William Reid, Geyenna Sterling-Lentsch, Marc S. Halfon

[DESCRIPTION]
The collection, fixing, and immunohistochemical staining of Aedes aegypti embryos is challenging in comparison to D. melanogaster since the vitelline membrane of Ae. aegypti must be manually removed. Herein we report on an improvement for the methods to prepare Ae. aegypti embryos. The adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to process an average of 100-150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and in situ RNA hybridization studies. Critical to the success of the optimized protocol is the selection and description of the tools required, and differing approaches for younger and older embryos.

[BEFORE_START]
This protocol was developed using the Liverpool strain of Ae. aegypti reared under optimal conditions at 27°C, 80% RH and a 12:12 (L:D) photoperiod. Larvae were reared in 14 x 8 x 5 “ containers and fed a slurry of Tetramin fish food (Spectrum Brands Pet, LLC, Blacksburg, VA) ad libitum. Adults were provided with raisins as a sugar source.

[STEPS]
SECTION: Preparation of an aspirator
1.  

 
Cut both ends from a 10 mL serological pipette. Save the end with the cotton plug.
SECTION: Preparation of an aspirator
2. Trim the tapered tip and remove any sharp edges.
SECTION: Preparation of an aspirator
3. Whittle the thinner plastic end of the serological pipette where the cotton plug is.
SECTION: Preparation of an aspirator
4. Fix two layers of chiffon fabric as a blocking filter by inserting the shaved end of the pipette tip into the pipette. 
SECTION: Preparation of an aspirator
5. Attach a length of tygon tubing for mouth aspiration.
 

SECTION: Preparation of an egging chamber
6. 
 

 Remove the threads from the cap and tube of a 50 mL conical tube.
 

SECTION: Preparation of an egging chamber
7. Create an entry port for mosquitoes by drilling a hole that is slightly larger than the outer diameter of a 10 mL serological pipette into the 50 mL conical tube.
SECTION: Preparation of an egging chamber
8. Use a strip of laboratory tape to create a removable closure over the entry port.
SECTION: Preparation of an egging chamber
9. Place a second smaller section of tape on the back of the tape closure so that when the mosquito entry port is closed, there is no sticky side of the tape facing the inside of the 50 mL conical tube.
 

SECTION: Electrosharpen a tungsten wire
10. 
 

 Cut a 3-4 cm length of 0.005” tungsten wire.
SECTION: Electrosharpen a tungsten wire
11. Secure the tungsten wire in an inoculating loop holder.
SECTION: Electrosharpen a tungsten wire
12. Connect the anode (negative terminal) of a 9V battery to a 1.0M solution of KOH or NaOH.
SECTION: Electrosharpen a tungsten wire
13. Connect the cathode (positive terminal) of the 9V battery to the metallic end of the inoculating loop holder.
SECTION: Electrosharpen a tungsten wire
14. Repeatedly dip the end of the tungsten wire into the 1.0M solution of KOH (or NaOH) to electrosharpen the tungsten wire.
 

SECTION: Collection of embryos
15. 
 

 
Place 250-300 Ae. aegypti pupae into a cage at a sex ratio ranging from 1:1 to 2:1 (F:M).
SECTION: Collection of embryos
16. Provide the mosquitoes with a fresh blood meal three to five days post adult emergence.
 

SECTION: Collection of embryos
17. Four days post blood meal, use the aspirator to transfer 20-50 females into an egging chamber lined with three overlapping strips of wetted Whatman #1 filter paper.
 

SECTION: Collection of embryos
18. Darken the conical tube by covering the tube with aluminum foil. Allow the mosquitoes to lay eggs undisturbed for 60 min.

SECTION: Collection of embryos
19. Transfer the egg paper to a plastic petri dish lid lined with two layers of wetted Whatman #1 filter paper and close the lid with the bottom of the dish.
SECTION: Collection of embryos
20. Incubate the embryos at 27 °C for the desired duration of time. Make sure the embryos are positioned on a slight incline so that they are not submerged in water.

SECTION: Fixation of embryos
21. 
 

 

 Prepare the following solutions for fixing embryos: 20 mL 50% bleach (Final NaClO = 3%), 10 mL PEM-F (prepared fresh), 10 mL MeOH, 10 mL heptane.

SECTION: Fixation of embryos
22. Set up an egg basket (i.e., 100 µm cell strainer) in a 9-cm-diam Petri dish bottom and fill the dish bottom with distilled water.

SECTION: Fixation of embryos
23. Dislodge the embryos from the edges of the filter papers with a fine tipped paintbrush.
SECTION: Fixation of embryos
24. Rinse embryos into the egg basket with distilled water.
SECTION: Fixation of embryos
25. Dechorionate embryos in 50% bleach (3% NaClO) for 30 s.
 

SECTION: Fixation of embryos
26. Generously rinse embryos in distilled water three times.
 

SECTION: Fixation of embryos
27. Rinse embryos from the egg basket into a scintillation vial.
SECTION: Fixation of embryos
28. Remove excess water from the scintillation vial and add 10 mL of PEM-F.
 

SECTION: Fixation of embryos
29. Incubate embryos in PEM-F at 60 °C for 30 min.

SECTION: Fixation of embryos
30. Remove the PEM-F and add 10 mL of heptane.
 

SECTION: Fixation of embryos
31. Transfer embryos to a -70 °C freezer for 5 min.

SECTION: Fixation of embryos
32. Add 10 mL methanol and shake the vial under running hot (60 °C) water for 30 s.
 


SECTION: Fixation of embryos
33. Remove the upper heptane layer and most of the methanol from the embryos.
 

SECTION: Fixation of embryos
34. Add an additional 10 mL methanol to the scintillation vial.
 


SECTION: Fixation of embryos
35. Transfer embryos to a 1.5 mL tube.
SECTION: Fixation of embryos
36. Wash embryos with three changes of 1 mL of 100% methanol. Embryos can be stored in methanol at -20 °C. Alternatively proceed with the rehydration step.
 



SECTION: Removal of the vitelline membrane (“embryo peeling”)
37. 
 

 Rehydrate the embryos using a methanol/PBS(P) dilution series (1 mL and 10 min each): 75%, 50%, 25%, 100% PBS(P), PT.
 

SECTION: Removal of the vitelline membrane (“embryo peeling”)
38. Replace the final rehydration with a new 1 mL aliquot of PT and store embryos 30 s at 4 °C prior to peeling.
 




SECTION: Removal of the vitelline membrane (“embryo peeling”)
39. Wet a 1”x2” square of Whatman #1 filter paper with PBS(P) and place on a smooth glass surface.
SECTION: Removal of the vitelline membrane (“embryo peeling”)
40. Add a glass coverslip so that it slightly overlaps onto the filter paper.
 

SECTION: Removal of the vitelline membrane (“embryo peeling”)
41. Flood the underneath of the coverslip with PBS(P) so that a complete “water bridge” is formed. Wick off any excess PBS(P).
SECTION: Removal of the vitelline membrane (“embryo peeling”)
42. Affix an equal-length strip of double-faced tape to the edge (1-2 mm overlapping) of a plastic coverslip.
SECTION: Removal of the vitelline membrane (“embryo peeling”)
43. Trim the other edge of the double-faced tape to leave a clean edge.
SECTION: Removal of the vitelline membrane (“embryo peeling”)
44. Peel back a corner of the tape backing and place the prepared transfer tape aside.
SECTION: Removal of the vitelline membrane (“embryo peeling”)
45. Add 50-100 fixed embryos along the edge of the glass coverslide overlapping the wetted filter paper.
SECTION: Removal of the vitelline membrane (“embryo peeling”)
46. Gently brush embryos to the edge glass coverslip.
 

SECTION: Removal of the vitelline membrane (“embryo peeling”)
47. Use a fine tipped paintbrush to position the posterior (thinner end) of the embryo towards the edge of the glass slide and the anterior (wider end) away from the glass slide.
SECTION: Removal of the vitelline membrane (“embryo peeling”)
48. Continue to align embryos tightly against each other until all embryos have been aligned.
 

 

SECTION: Removal of the vitelline membrane (“embryo peeling”)
49. Remove the PBS from underneath the glass coverslip by pressing paper toweling onto the filter paper.
SECTION: Removal of the vitelline membrane (“embryo peeling”)
50. Remove the glass coverslip and gently move the line of embryos towards the edge of the glass plate by sliding the paper across the glass surface without lifting.
 

SECTION: Removal of the vitelline membrane (“embryo peeling”)
51. Transfer the embryos to the double faced tape so that the anterior of the embryos slightly overhangs the outer edge of the tape. Gently press on the backing of the double faced tape to ensure that the embryos are affixed to the tape.
 

SECTION: Removal of the vitelline membrane (“embryo peeling”)
52. Remove the tape backing and place the tape into the well of a glass well slide (embryo side up).
SECTION: Removal of the vitelline membrane (“embryo peeling”)
53. Flood the well with PBS(P). Add PBS(P) as needed to prevent the embryos from drying out.
SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
54. 
 

     
 
Make a small incision at the anterior of each embryo using a fine stab knife.
 

SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
55. Carefully slip the anterior end of the vitelline membrane off of the embryos by gently inserting the tungsten wire underneath the membrane at the hole/seam and pushing forward with a very slight upward pressure. The main pressure of the tungsten wire should be forward with a very slight upward pressure.
 

SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
56. Remove the anterior end of the vitelline membranes from all of the embryos.
 

SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
57. Rinse the loose vitelline membrane fragments from the slide with a gentle stream of PBS(P), then refill the reservoir with clean PBS(P).
 

SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
58. Using the blunt Roboz probe, gently push on the posterior end of the embryo until it extrudes out of the membrane.
 

SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
59. Move and press any extruded embryos retaining a serosal cuticle into the sticky surface of the double faced tape.
SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
60. Tear the serosal cuticle at the anterior end of the embryo.
SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
61. Extrude the embryo from the serosal cuticle by pushing on the posterior end of the embryo with the blunt side of the tungsten wire probe.
 

SECTION: Peeling older embryos (&gt;20h AEL at 27°C)
62. Collect embryos into a pile and transfer into a 1.5 mL tube using a Pasteur pipette.
 

SECTION: Peeling younger embryos (&lt;20h AEL at 27°C)
63. 
 

 
  

 
Tear a small opening in the posterior of the embryo using the tungsten wire probe.
SECTION: Peeling younger embryos (&lt;20h AEL at 27°C)
64. Insert the tungsten wire probe into the posterior opening of the vitelline membrane and carefully tear upwards in a stepwise fashion until the entire vitelline membrane has been dissected along the sagittal plane.
 

SECTION: Peeling younger embryos (&lt;20h AEL at 27°C)
65. Press the edges of the vitelline membrane into the tape adhesive to free the embryo.
 

SECTION: Peeling younger embryos (&lt;20h AEL at 27°C)
66. Collect embryos into a pile and transfer into a 1.5 mL tube using a Pasteur pipette.
 

SECTION: Peeling younger embryos (&lt;20h AEL at 27°C)
67. Store all peeled embryos at 4 °C and process for staining within one day for best results.