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 remove the adults.&#160;Alternatively, quickly put a vessel of collected adults on ice and, then, deposit them on the leaf. Limit the time between oviposition and egg fixation to no more than 60 min, to ensure the observation of the sperm transition into the paternal pronucleus in eggs. 
	NOTE: Eggs not removed from the leaf can be reared to adulthood to record adult sex ratios.</li>
	<li>Before or during whitefly oviposition, prepare a microscope slide by cleaning it with soap and water, drying it well, and then stretching a piece of paraffin film over one end, making sure the paraffin film surface does not break (Figure 1). 
	NOTE: The paraffin film is hydrophobic and semi-opaque, allowing liquids to form drops and eggs to be more easily seen.</li>
</ol>
2. Dechorionation
<ol>
	<li>With a glass Pasteur pipette, add drops of bleach solution (0.83% sodium hypochlorite) to the paraffin film. 
	NOTE: It is easier to keep track of eggs if only 2&#8211;3 eggs are in each drop. Alternatively, to manage a large egg number, collect the eggs in drops of 1x phosphate-buffered saline (PBS). 
	CAUTION: Bleach is corrosive; wear gloves when handling bleach and do not inhale.</li>
	<li>Remove the adult whiteflies from the leaf.</li>
	<li>Place the leaf under a microscope to see the eggs clearly and collect the eggs singly and carefully with a thin probe. To make a probe, insert a minuten nadel&#160;pin at 45&#176; or at a comfortable working angle into a melted pipette tip (Figure 2).</li>
	<li>Transfer the eggs to bleach or 1x PBS. If using 1x PBS, remove the 1x PBS with a glass Pasteur pipette once all the eggs are collected and, then, add bleach to the eggs. 
	NOTE: When collecting eggs, slowly lift the egg from its base until the pedicel is removed from the leaf. The pedicel is sticky, and the egg will commonly stick to the probe tip until it is dipped into the bleach. 
	NOTE:&#160;It is recommended to use separate glass Pasteur pipettes for all reagents, and the pipettes may be modified with heat to make the tips narrower and reduce the risk of accidentally aspirating whitefly eggs. To prevent residue and contamination, clean the pipettes with deionized water after every use.</li>
	<li>Wait for 10 min. If there is an interest in embryogenesis in eggs older than 1 h, leave the eggs in bleach for up to 15 min. 
	NOTE: For eggs that are up to 1 h old, 10 min is sufficient.</li>
</ol>
3. Fixation
NOTE: These steps are taken from a Hymenopteran protocol17.
<ol>
	<li>Remove the bleach (containing the chorion fragments) with a glass Pasteur pipette and discard it. Add drops of glacial acetic acid with a glass Pasteur pipette and wait 3 min. 
	CAUTION: Proceed with this step under a fume hood. Glacial acetic acid is corrosive; wear gloves when handling glacial acetic acid and do not inhale, especially in combination with the residual bleach.</li>
	<li>Remove the glacial acetic acid with a glass Pasteur pipette and add drops of Clarke&#8217;s solution (3:1 of absolute ethanol:glacial acetic acid) with a glass Pasteur pipette. Wait until most of the solution has evaporated (or 10 min max).</li>
	<li>Add drops of 70% ethanol to the eggs with a glass Pasteur pipette and wait until most of the ethanol has evaporated (or 10 min max).</li>
</ol>
4. Staining
<ol>
	<li>Remove any residual ethanol with a glass Pasteur pipette and add drops of 1x PBS to the eggs with a glass Pasteur pipette to get the pH close to 7.0. Set the microscope slide in a humidity chamber to prevent desiccation, for example, in an empty pipette tip box with a wet paper towel inside (Figure 3). Wait at least 30 min. 
	NOTE: This is the best point if a pause is needed, as long as the humidity chamber can prevent desiccation.</li>
	<li>Remove the 1x PBS with a glass Pasteur pipette, and add drops of 0.1 &#956;g/mL DAPI, a DNA fluorescent stain, with a glass Pasteur pipette. Set the microscope slide in a dark humidity chamber and wait at least 15 min. 
	CAUTION: DAPI is an irritant, so handle it with gloves.</li>
</ol>
5. Washing
<ol>
	<li>Remove the DAPI with a glass Pasteur pipette.</li>
	<li>Add drops of 1x TBST (5x solution made from 30 g of Tris, 43.8 g of NaCl, 5 mL of polysorbate 20, and 1.0 g of NaN3 [pH 7.5], and brought to 1 L with PCR grade water) to the eggs with a glass Pasteur pipette. Wait 5 min before removing the 1x TBST with a glass Pasteur pipette. Repeat this step 2 times.</li>
</ol>
6. Mounting
<ol>
	<li>After the final wash, carefully pipette all the eggs from the paraffin film onto a clean part of the microscope slide. Remove excess 1x TBST; then, add 20 &#181;L of mounting media (80% glycerol and 20% 1x TBST with 2% n-propyl-gallate) and place a clean cover slide on top of the eggs.</li>
	<li>For long-term storage, seal the cover slide with clear nail polish and, then, either store the slide in the dark at 2 &#176;C or immediately view it under a fluorescent microscope.</li>
</ol>

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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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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. 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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. -. 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The authors have nothing to disclose.

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 ...

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JoVE Journal

Genetics

5.3K Views.  University of Arizona. 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.

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

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

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. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. An approach to assist in determining the likelihood of pathogenicity is signal-to-noise analysis at the protein sequence level. This protocol describes a method for amino acid-level signal-to-noise analysis that leverages variant frequency at each amino acid position of the protein with known protein topology to identify areas of the primary sequence with elevated likelihood of pathologic variation (relative to population &#34;background&#34; variation). This method can identify amino acid residue location &#34;hotspots&#34; of high pathologic signal, which can be used to refine the diagnostic weight of VUSs such as those identified by next generation genetic testing.

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant &#34;hotspots&#34; within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

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 is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

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 reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

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