Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4′,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

The protocol that Liz developed allows us to determine whitefly fertilization rate. Applying this technique will improve our understanding of the behavior and ecology of whiteflies. Whiteflies are haplodiploid, so the fertilization rate is equal to the sex ratio at oviposition.

This protocol is the first described for whiteflies, to our knowledge, and can be completed within three hours. To begin, make a coverable hole in the Petri dish cover to insert and to remove the adult whiteflies. Set up a humidity chamber and fashion a probe by inserting a thin pin at a comfortable working angle into a melted pipette tip to make a thin probe.

Optionally, elongate glass pipette tips with heat for easier handling of whitefly eggs. Then, allow female whiteflies to oviposit on a clean leaf, cut to fit on agar in a Petri dish. During the whitefly oviposition, clean a microscope slide with soap and water.

Dry it well and stretch a piece of paraffin film over one end to allow liquids to form drops and eggs to be more easily seen. To dechorionate eggs, use a glass Pasteur pipette to add drops of 83%sodium hypochlorite bleach solution to the paraffin film right before adult removal and egg collection. Bleach and glacial acetic acid are both corrosive.

They should be handled with gloves in a hood or well-ventilated area. DAPI is also an irritant and should be handled with gloves. Then, remove the adult whiteflies from the leaf and place the leaf under a microscope.

Carefully lift each egg from its base until the pedicel is removed from the leaf. Practice on many eggs before performing this protocol. Lift the eggs slowly from the base and fully lift it off the leaf once you can see that the egg pedicel is removed from the leaf.

Transfer two to three eggs to each drop of bleach and leave them for 10 minutes. To fix the eggs, use a glass pipette to discard the bleach. Then, add drops of glacial acetic acid and wait three minutes.

Next, remove the glacial acetic acid and add drops of Clarke's solution. Then, wait 10 minutes until most of the solution has evaporated. Add drops of 70%ethanol and wait again for 10 minutes.

To stain the eggs, first remove any residual ethanol. Then, add drops of 1xPBS to the eggs to get the pH close to seven. Put the microscope slide in a humidity chamber to prevent desiccation.

After 30 minutes, remove the 1xPBS and add drops of 1 micrograms per milliliter DAPI. Put the microscope slide in a dark humidity chamber and wait at least 15 minutes. To wash eggs, first remove the DAPI solution.

Then add drops of 1xTBST to the eggs. After five minutes, remove the 1xTBST. Repeat this wash two more times for a total of three washes.

After the final wash, carefully pipette all the eggs from the parafilm onto a clean part of the microscope slide. Remove excess TBST and add 20 microliters of mounting media. Finally, place a clean cover slide on top of the eggs, seal the cover slide with clear nail polish for long-term storage, and proceed to imaging using a fluorescent microscope.

Egg dechorionation followed by DAPI nuclear staining allowed the assignment of fertilization and embryo sex when observed with a fluorescent microscope. The female pronucleus is near the center of both the fertilized female egg and unfertilized male egg, and the sperm is visible as a bright streak near the apex of the female egg only. One application of this technique is to determine whether bacterial endosymbionts affect primary sex ratios of whiteflies.

For example, the primary sex ratios of eggs laid by Rickettsia-infected and uninfected B.tabici whiteflies showed no significant difference between sex ratio in the two treatments. As for eggs reared to adulthood, similar results were seen for Rickettsia-infected and uninfected adults, providing no evidence, at least in this study, for greater fertilization rates by Rickettsia-infected females. By using this protocol, one can determine whether interspecific sperm can fertilize eggs.

If no fertilization is observed, one could back up and look to see if sperm is transferred during mating by looking in the spermatheca of the female. If fertilization does occur, one could compare the sex ratio of adults with the fertilization rate to determine the viability of hybrid offspring development.