Methods in Anopheles Research - MR4

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Chapter 3 : Specific Anopheles Techniques3.1 Embryonic Techniques3.1.1 Microinjection Methods for Anopheles EmbryosPage 3 of 83. Harvest eggs 60-96 hours after females are bloodfed by placing 10 to 15 females in a transparentcylindrical container (~40 ml) open at one end and covered with rubber dental dam at the other(Figure 3.1.1.1). Alternatively, eggs may be collected on damp filter paper in a small Petri dish 4 .Figure 3.1.1.1. Cylindrical container (~40ml) containing 10-15 previously bloodfedfemales open at one end and covered withrubber dental dam at the otherFigure 3.1.1.2. Container with females resting overa single water-filled well on a cobalt blue ceramicdepression plate. Embryos deposited in singledepression shown (inset).4 Cooling the eggs will extend their useful time, but MQB’s experience has been that this also reducessurvival.
Chapter 3 : Specific Anopheles Techniques3.1 Embryonic Techniques3.1.1 Microinjection Methods for Anopheles EmbryosPage 4 of 8Figure 3.1.1.3. Egg darkening at 20 min intervals (top left to bottom right) beginningapproximately 15 m after oviposition and incubated at room temperature (~22°C). Most of thedarker eggs in the lower two left panels are suitable for injection. Note that one egg did notdarken. It will not hatch. When eggs have fully darkened (as in the lowest right panel) but areoften still at the blastoderm stage, they are more difficult to inject (photographs courtesy of G.Labbe, Oxitec, used with permission). Non-melanized eggs cannot be handled without sufferingmortality.4. Slide the container over a single water-filled well on a depression plate such that the females canaccess the water (see Figure 3.1.1.2). Cover to darken for 30 minutes and then remove the females.5. Age the new embryos for at least 30 minutes at insectary conditions (28°C; 80% humidity) or at roomtemperature. After this time, the eggs should be medium gray (Figure 3.1.1.3).Method 1:The distinctive feature is that embryos are injected under saline, the concentration of which is determinedempirically to balance internal pressure. The balance pressure must be such that the turgidity is sufficientfor needle penetration yet low enough that oozing and needle backflow are minimized. The saltconcentration can be adjusted as needed to achieve this for each species.1. At room temperature (~ 24 o C), transfer eggs from the depressions to a glass slide with a finepaintbrush and align with the dorsal (flattened, concave) surface facing up.2. Align the anterior ends of 25-35 eggs in 25 mM NaCl against a strip of reduced-fiber filter paper.3. Remove the filter paper by tugging it away sharply, so as not to disturb the alignment of the embryos.4. Allow eggs to desiccate slightly.5. Press a taped coverslip gently against the eggs’ dorsal surface and immediately invert. Getting theembryos to stick to the tape is the most difficult part of this procedure. They cannot be too wet - theywill not stick - or too dry - in which case they die.6. Cover with a solution of 25 mM NaCl (or Halocarbon oil) to prevent drying and place eggs in a humidbox at room temperature until injection. Eggs are appropriate for injecting around 2 hours afterdeposition when they have are medium dark. Choosing eggs for injection is discussed in Figure3.1.1.3.7. Immediately prior to injection, add more 25 mM NaCl to the coverslip. A large volume surrounding theembryos is desirable as it reduces distortion of the image. Attach the coverslip to a glass slide with abit of double-stick tape along the edge and place on the stage.8. Inject embryos on the ventral surface, near the posterior end, with the embryo turned at an angle ofabout 15-25 degrees. The horizontal angle of the needle can vary, but should be roughly within 30degrees vertical from the plane of the stage. Take care to avoid injection into the periplasmic space.Instead, inject immediately anterior to the periplasmic space and posterior to the egg floats. Injectionsshould be carried out at 100X magnification.9. Immediately after all of the embryos on a slide are injected, remove the slide from the scope and andplace the coverslip carrying the eggs into a cup of reverse-osmosis/deionized sterilized (RO/DI) H 2 0at room temperature to recover.10. When all injected coverslips from a cohort have been aligned and injected, placed all coverslips in acup of 50 ml RO/DI H 2 0 under insectary conditions to hatch. It is not necessary that the eggs float forhatching.11. Hatching will begin in approximately 48 hours after which the larvae can be handled as described inthe chapter on family culture.
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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Methods in Anopheles Research - MR4

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