Name: determining the egg fertilization rate of bemisia tabaci using a cytogenetic technique
Uploaded: 2019-04-01T15:00:00
Description: Watch this Scientific Journal Video about Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique at JoVE.com

This research was funded by an NSF grant (DEB-1020460) to M.S.H. and a USDA AFRI grant (2010-03752) to M.S.H. The authors thank Brennan Zehr for staining whitefly eggs with much skill and Zen. The authors thank Mike Riehle for allowing the use of his fluorescent microscope for imaging. The authors thank Suzanne Kelly and Marco Gebiola for the egg images. The authors thank Suzanne Kelly and Jimmy Conway for helping at crucial moments during the experiments.

NOTE: Ensure that all work is performed at room temperature in a well-ventilated area or under a fume hood. All &#8216;drops&#8217; in this protocol are defined as 5&#8211;20 &#181;L, depending on the operator&#8217;s preference.
1. Initial Setup
<ol>
	<li>Allow female whiteflies to oviposit on clean leaves. Examples for oviposition arenas include clip cages or leaves cut to fit on agar in a Petri dish. Make a coverable hole in the clip cage or Petri dish cover to insert and...

<ol>
	<li>Charnov, E. L., May, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+ratio+in+spatially+structured+populations.">Sex ratio in spatially structured populations.</a> The Theory of Sex Allocation. , 67-92 (1982).</li><li>Godfray, H. C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Parasitoids: behavioral and evolutionary ecology. , (1994).</li><li>Bourke, A. F. G., Franks, N. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+mating+interactions+drive+widespread+invasion+and+displacement+in+a+whitefly.">Asymmetric mating interactions drive widespread invasion and displacement in a whitefly.</a> Science. 318 (5857), 1769-1772 (2007).</li><li>Crowder, D. W., Sitvarin, M. I., Carri&#232;re, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+discrimination+in+invasive+whitefly+species.">Mate discrimination in invasive whitefly species.</a> Journal of Insect Behavior. 23 (5), 364-380 (2010).</li><li>Crowder, D. W., Sitvarin, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+mating+behavioural+interactions+and+life+history+traits+in+the+competition+between+alien+and+indigenous+whiteflies.">Roles of mating behavioural interactions and life history traits in the competition between alien and indigenous whiteflies.</a> Bulletin of Entomological Research. 102 (4), 395-405 (2012).</li><li>Sun, D. -. B., Li, J., Liu, Y. -. Q., Crowder, D. W., Liu, S. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reproductive+interference+on+the+competitive+displacement+between+two+invasive+whiteflies.">Effects of reproductive interference on the competitive displacement between two invasive whiteflies.</a> Bulletin of Entomological Research. 104 (3), 334-346 (2014).</li><li>Liu, S. -. S., Colvin, J., De Barro, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+concepts+as+applied+to+the+whitefly+Bemisia+tabaci+systematics:+how+many+species+are+there?.">Species concepts as applied to the whitefly Bemisia tabaci systematics: how many species are there?.</a> Journal of Integrative Agriculture. 11 (2), 176-186 (2012).</li><li>Gilbertson, R. L., Batuman, O., Webster, C. G., Adkins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+insect+supervectors+Bemisia+tabaci+and+Frankliniella+occidentalis+in+the+emergence+and+global+spread+of+plant+viruses.">Role of the insect supervectors Bemisia tabaci and Frankliniella occidentalis in the emergence and global spread of plant viruses.</a> Annual Review of Virology. 2 (1), 67-93 (2015).</li><li>Giorgini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rickettsia+symbionts+cause+parthenogenetic+reproduction+in+the+parasitoid+wasp+Pingala+soemius+(Hymenoptera:+Eulophidae).">Rickettsia symbionts cause parthenogenetic reproduction in the parasitoid wasp Pingala soemius (Hymenoptera: Eulophidae).</a> Applied and Environmental Microbiology. 76 (8), 2589-2599 (2010).</li><li>Himler, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+spread+of+a+bacterial+symbiont+in+an+invasive+whitefly+is+driven+by+fitness+benefits+and+female+bias.">Rapid spread of a bacterial symbiont in an invasive whitefly is driven by fitness benefits and female bias.</a> Science. 332 (6026), 254-256 (2011).</li><li>Cass, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+endosymbiont+Rickettsia+in+an+insect+pest.">Dynamics of the endosymbiont Rickettsia in an insect pest.</a> Microbial Ecology. 70 (1), 287-297 (2015).</li><li>Vavre, F., de Jong, J. H., Stouthamer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+mechanism+and+genetic+consequences+of+thelytoky+in+the+wasp+Trichogramma+cacoeciae.">Cytogenetic mechanism and genetic consequences of thelytoky in the wasp Trichogramma cacoeciae.</a> Heredity. 93 (6), 592-596 (2004).</li><li>Cass, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+fitness+benefits+of+the+Rickettsia+bacterial+symbiont+in+an+insect+pest.">Conditional fitness benefits of the Rickettsia bacterial symbiont in an insect pest.</a> Oecologia. 180 (1), 169-179 (2016).</li><li>Hunter, M. S., Asiimwe, P., Himler, A. G., Kelly, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+nuclear+genotype+influences+phenotype+of+a+conditional+mutualist+symbiont.">Host nuclear genotype influences phenotype of a conditional mutualist symbiont.</a> Journal of Evolutionary Biology. 30, 141-149 (2017).</li><li>Gottlieb, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+intracellular+ecosystem:+symbiotic+bacteria+share+bacteriocytes+in+whiteflies.">Inherited intracellular ecosystem: symbiotic bacteria share bacteriocytes in whiteflies.</a> FASEB Journal. 22 (7), 2591-2599 (2008).</li><li>Luan, J., Sun, X., Fei, Z., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+inheritance+of+a+single+somatic+animal+cell+displayed+by+the+bacteriocyte+in+the+whitefly+Bemisia+tabaci.">Maternal inheritance of a single somatic animal cell displayed by the bacteriocyte in the whitefly Bemisia tabaci.</a> Current Biology. 28 (3), 459-465 (2018).</li><li>Guo, J. -. Y., Cong, L., Wan, F. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+generation+effects+of+high+temperature+on+the+development+and+fecundity+of+Bemisia+tabaci+(Gennadius)+(Hemiptera:+Aleyrodidae)+biotype+B.">Multiple generation effects of high temperature on the development and fecundity of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B.</a> Insect Science. 20 (4), 541-549 (2013).</li><li>Cui, X., Wan, F., Xie, M., Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+heat+shock+on+survival+and+reproduction+of+two+whitefly+species,+Trialeurodes+vaporariorum+and+Bemisia+tabaci+biotype+B.">Effects of heat shock on survival and reproduction of two whitefly species, Trialeurodes vaporariorum and Bemisia tabaci biotype B.</a> Journal of Insect Science. 8 (24), (2008).</li><li>Boykin, L. 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This protocol is the first to capture the fertilization rate or primary sex ratio of B. tabaci. The challenge of this protocol is that it requires researchers to learn how to handle the whitefly eggs quickly, ensuring that not more than 1 h has passed since the eggs were oviposited until they are fixed. During preliminary experiments, eggs that were fixed at 3 h or more postoviposition were too old to observe fertilization, as syngamy had occurred and mitotic divisions were underway. Between 1 to 3 h, the pronuc...

The study of sex allocation, or the relative investment in male and female offspring, is a cornerstone of behavioral ecology1,2,3. In addition to its power for testing adaptive models of behavior, knowing the sex allocation strategy of an organism may improve models of its population dynamics. In many species, sex allocation is controlled by mothers. To determine sex allocation, it is important to determine the primary sex ratio or the proportion of females at the time of egg deposition. Although the sex ratio at adult emergence may provide clues to sex allocation, differential developmental mortality between male and female juveniles may commonly skew the adult sex ratio substantially. In some species of Hymenoptera, the order of insects that contains ants, bees, and wasps, the primary sex ratio has been determined with cytogenetic assays, staining the embryos to view genetic DNA. Because hymenopterans are haplodiploid, an incipient male egg is haploid and contains only the female pronucleus (n), while incipient female eggs are diploid and contain both male and female pronuclei (2n). Although Aleyrodidae, the sap-feeding family of true bugs (Hemiptera) known as whiteflies, are also haplodiploid, there has not been an established assay to find the primary sex ratio in these insects. This is perhaps surprising given the intensity of study of the few cosmopolitan serious pests in this family and the importance of sex ratios in competitive interactions of whiteflies4,5,6,7,8,9,10 and in population dynamics generally. In haplodiploid insects too, sex ratios are unconstrained by sex determination systems, allowing the possibility of selective fertilization and labile sex ratios that vary with the environment2. Here we present a technique to determine the primary sex ratio of the species complex of whiteflies known collectively as the sweetpotato whitefly, B. tabaci. This one species name encompasses more than 28 species worldwide11and includes some of the most damaging global invasive pests12,13. The application of this technique to determine sex allocation patterns in B. tabaci and other Aleyrodidae will allow a more rigorous investigation of variables, including temperature, host plant, endosymbiotic bacteria, or plant/whitefly pathogens, that may influence whitefly primary sex ratios and whitefly population dynamics.
We are unaware of any comparable egg-staining techniques for B. tabaci. The protocol is convenient in comparison with staining methods used for other insect eggs14 as it omits an overnight fixation step and, therefore, can be completed within 3 h. As one example of an application, an endosymbiotic bacterium, Rickettsia sp. nr. bellii, is associated with female bias in our laboratory lines of B. tabaci Middle East-Asia Minor 1 (MEAM1)15,16. In one B. tabaci MEAM1 laboratory line (&#34;MAC1,&#34; collected from the Maricopa Agricultural Center), we test whether Rickettsia-infected (R+) females fertilize more eggs than uninfected (R-) females.

<table>
	<tbody>
		<tr>
			<td>1XPBS</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1XTBST</td>
			<td>Any</td>
			<td></td>
			<td>5X solution made from 30g Tris, 43.8g NaCl, 5 mL Tween-20 and 1.0g NaN3 pH7.5, and brought to 1L with PCR grade water</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td></td>
			<td>Any household bleach will work as long as it can be diluted to 0.83% Sodium hypochlorite</td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI dilactate</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc300415</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Any</td>
			<td></td>
			<td>Dilute to 70% EtOH</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Nikon Eclipse 50i was used in this experiment, but any fluorescent microscope with 340/380 nm excitation filter and at least 4-10X magnification can be used</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Mallinckrodt</td>
			<td>UN2789</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Wild</td>
			<td></td>
			<td>A Wild M5A microscope was used for this experiment, but any microscope where the operator can clearly see the whitefly eggs can be used</td>
		</tr>
		<tr>
			<td>Microscope slide covers</td>
			<td>Any</td>
			<td></td>
			<td>Methods are for 18mm x 18mm sized slide covers. More mounting media will need to be added for larger slide covers.</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Minuten nadel pins</td>
			<td>BioQuip</td>
			<td>1208SA</td>
			<td>Minuten nadel pins are optional for fashioning as probes with pipette tips</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>n-propyl-gallate</td>
			<td>Sigma/Santa Cruz Biotechnology</td>
			<td>P3130/sc-250794</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>Fisherbrand Disposable Borosilicate glass Pasteur pipettes 5.75 in. A Bunsen burner may also be needed if operator would like to lengthen and narrow pipettes</td>
		</tr>
		<tr>
			<td>PCR grade water</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Any</td>
			<td></td>
			<td>Pipette tips are optional for fashioning as probes with minuten nadel pins</td>
		</tr>
		<tr>
			<td>Small dropper bulb</td>
			<td>Any</td>
			<td></td>
			<td>Must fit on Pasteur pipette</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

determining the egg fertilization rate of bemisia tabaci using a cytogenetic technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

To test whether Rickettsia affects the fertilization rate of B. tabaci MEAM1 females, we reared Rickettsia-infected (R+) or uninfected (R-) B. tabaci on cowpea plants (Vigna unguiculata) in separate cages at 27 &#176;C, 70% relative humidity, and a 16 h light/8 h dark photoperiod. R+ and R- fourth instar whiteflies were carefully removed from leaves and isolated in 200 &#181;L strip tubes. When adults...

Watch this Scientific Journal Video about Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique at JoVE.com

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

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

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

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

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.

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Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...