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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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 pronuclei took on a rounder shape. While the early presence of two nuclei indicates a fertilized egg, a bit later, the apposition of the two nuclei in preparation for syngamycan appear to be one nucleus, and later again, the two products of the first mitotic division are found in both male and female eggs. Therefore, the distinction between the sexes is not clear at these later time points, and we advise limiting the interval from oviposition to fixation to 1 h as a conservative measure. It is also challenging to learn how to be gentle with the eggs with each transfer of liquid so that they are not accidentally aspirated into the pipette. At the time of viewing the eggs under a fluorescent microscope, a few of the eggs may have broken during the protocol, so those eggs cannot be sexed and counted as the pronuclei and yolk may have escaped. Otherwise, once the operator becomes comfortable with these steps, the protocol can be completed conveniently within 3 h, as it does not require an overnight fixation step. It is also flexible in that it can be modified to stain older eggs, for those researchers interested in capturing development.
An application of this protocol includes research on sex allocation. Although many dozens of studies of whitefly biology have reported adult sex ratios in different environments, adult sex ratios confound sex allocation of the mother with sex-specific developmental mortality of nymphs and make it impossible to determine the cause of any sex ratio pattern. The cytogenetic technique described here allows the possibility of understanding whitefly sex allocation patterns more generally. While the large dispersive populations of B. tabaci and other pests such as the greenhouse whitefly, Trialeurodes vaporariorum, might be predicted to result in 1:1 sex ratios, as often exhibited in laboratory settings22,23, we also predict that reproductive interference, endosymbionts, and potentially host plant quality could influence primary sex ratios. That these same factors could also influence sex-specific mortality patterns during development, systematically skewing sex ratio estimates, underscores the need for a direct measure of the primary sex ratio. 
While female bias associated with Rickettsia infection has been consistently found in the genetic line "MAC1"15,18,19, the adult sex ratios were not significantly female biased in the current study. The primary sex ratios observed with the cytogenetics technique were not significantly biased either. We censused the primary and adult sex ratios of B. tabaci females on just 1 day, at the beginning of an oviposition bout, when the females were 4 to 5 days old, so the sex ratios studied may not have represented the sex ratios that we observed over longer periods. Nonetheless, the technique made it possible to determine the fertilization rate or primary sex ratio and, in this instance, showed a correspondence between the primary and adult sex ratios. 
Determining fertilization may also be valuable in instances in which researchers might want to determine the type of reproductive isolation among whitefly populations. While it has been a matter of some controversy12,24,25, it is now generally accepted that the name B. tabaci refers to tens of cryptic species, with evidence being provided by both genetic divergence and crossing tests in which few females are produced12,25. In these studies, it would be of interest to know where the isolation occurs. Is sperm transferred, and does heterospecific sperm fertilize eggs, or are attempted matings unsuccessful? Regarding the determination of the primary sex ratio, this technique can determine whether the primary sex ratio is influenced by endosymbionts, selfish genetic elements, or one of many other factors, including conspecifics, competitors, predators, parasitoids, pathogens, the host plant, or abiotic effects.

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>1x PBS</td><td>Any</td><td></td><td></td></tr><tr><td>1x TBST</td><td>Any</td><td></td><td>5x solution made from 30 g Tris, 43.8 g NaCl, 5 mL Tween-20 and 1.0 g NaN&lt;sub&gt;3&lt;/sub&gt; pH 7.5, and brought to 1 L 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 18 mm x 18 mm 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>NaN&lt;sub&gt;3&lt;/sub&gt;</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 emerged, they were collected in groups of 50% females and transferred to clean leaves in Petri dishes (n = ~50-100/leaf) for 4 days of mating. Groups of approximately 20 females and several males were then transferred to a clean leaf disk (one for R- adults, one for R+ adults) in 35 mm Petri dishes resting on 1% agar. The Petri dish lid had been cut out and the fine fabric mesh used for containment also prevented excess condensation. After approximately 45 min, all adults were removed, some of the eggs were harvested to determine the fertilization rate, and the eggs that were not collected were reared to adulthood to calculate the adult sex ratio. One cohort for a single day, for both R+ and R- whiteflies, was defined as a block. There were seven blocks for calculating the fertilization rate or primary sex ratio, while there were six blocks for calculating the adult sex ratio, as there were not enough leftover eggs from one block to rear to adulthood. A generalized linear model was used in the statistical package R to determine whether the fertilization rates or adult sex ratios were significantly influenced by Rickettsia infection and/or block. The response variables were the proportion of fertilized eggs/all eggs, or the proportion of female adults/all adults, respectively, while explanatory variables were the block and Rickettsia infection status.
Egg dechorionation followed by DAPI nuclear staining allowed the unambiguous assignment of fertilization (and embryo sex) when observed with a fluorescent microscope (Figure 4). For this experiment, 90 eggs laid by R-B. tabaci MEAM1 females and 82 eggs by R+B. tabaci MEAM1 females were scored. As for eggs reared to adulthood, 60 R- and 95 R+ adults were scored. While a female bias in adult sex ratios has been shown consistently in earlier studies15,18,19, in the current study, adult R+ sex ratios (69% females, median) were female-biased compared to R- females (50% females, median), but the sex ratios in the two treatments were not significantly different (&#967;2 = 1.02, df = 1, p = 0.31; Figure 5). The primary R- sex ratios (60% fertilized eggs, median) were female-biased compared to R+ sex ratios (44% fertilized eggs, median) but were also not significantly different (&#967;2 = 0.51, df = 1, p = 0.47), providing no evidence for greater fertilization rates by R+ females (Figure 5). Block also did not have a significant effect on the primary (&#967;2 = 0.29, df = 1, p = 0.59) or adult (&#967;2 = 1.20, df = 1, p = 0.27) sex ratio.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59213/59213fig1.jpg" /> 
Figure 1: Photo of a microscope slide with paraffin film and drops of bleach. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59213/59213fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59213/59213fig2.jpg" /> 
Figure 2: An example of a probe, fashioned with heat, from a pipette tip and a minuten nadeln pin. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59213/59213fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59213/59213fig3.jpg" /> 
Figure 3: An example of a humidity chamber, fashioned from an empty pipette tip box and a wet paper towel. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59213/59213fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59213/59213fig4.jpg" /> 
Figure 4: Fluorescent microscope images of B. tabaci eggs. (a) B. tabaci MEAM1 fertilized, incipient female egg. (b) Unfertilized, incipient male egg. The eggs were fixed at less than 1 h after oviposition and stained with DAPI. The base of each egg is fluorescing due to bacterial DNA (Portiera, Hamiltonella,&#160;and possibly Rickettsia) in the bacteriome progenitor cell, which is included in the laid egg20,21. In each egg, the female pronucleus is near the center of the egg, and in the female egg only, the sperm is visible as a bright streak near the apex of the egg near what is presumably an autofluorescing micropyle. The images are screenshots taken from a Z-stack generated video, produced by a laser scanning confocal inverted microscope. The scale bars were estimated from previous microscope images. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59213/59213fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59213/59213fig5.jpg" /> 
Figure 5: Primary and adult sex ratios of Rickettsia-infected or uninfected B. tabaci. Box and whisker plots of the primary sex ratio (percentage of fertilized eggs, or percentage of female zygotes) in black compared to the adult sex ratio (percentage of adult females) in grey, of the Rickettsia-infected (R+)&#160;and uninfected (R-) B. tabaci MEAM1, &#34;MAC1&#34; genetic line. The box and whisker plots show the median as the middle line, the mean as a plus sign, and upper and lower quartiles as the lines that make the ends of the box, and the range is represented in the outer lines extending from the box. For R- eggs scored: n = 90; for R+ eggs scored: n = 82. For R- adults counted: n = 60; for R+ adults counted: n = 95. In a logistical analysis of the primary sex ratio (proportion of fertilized eggs) performed in the statistical package R, no significant effects were found for block (n = 7, &#967;2 = 0.29, df = 1, p = 0.59) or for Rickettsia infection (&#967;2 = 0.51, df = 1, p = 0.47). Adult sex ratios, as influenced by block (n = 6) and Rickettsia infection status, were similarly analyzed. Here as well, no significant effects were found for block (&#967;2 = 1.20, df = 1, p = 0.27) or for Rickettsia infection (&#967;2 = 1.02, df = 1, p = 0.31). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59213/59213fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

determining-egg-fertilization-rate-bemisia-tabaci-using-cytogenetic

Nanyang Technological University

Research

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

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Environment

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

Biochemistry

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Immunology and Infection

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species&#8217; unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

Developmental Biology

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Biology

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain&#8217;s fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.&#160;
Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.&#160;

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Here, we present a protocol for studying mating competitiveness of male Aedes aegypti using fluorescent dye as a marker. Female mosquitoes are exposed to both marked and unmarked males for copulation. Post mating, their spermathecae are examined under a fluorescence microscope to determine their mating partner.

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for ...

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

Summary

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Chapters in this video

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes