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

Summary

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.

Abstract

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′,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.

Introduction

The study of sex allocation, or the relative investment in male and female offspring, is a cornerstone of behavioral ecology^1,2,3. 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 whiteflies^{4,5,6,7,8,9,10} 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 pests^12,13. 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 ("MAC1," collected from the Maricopa Agricultural Center), we test whether Rickettsia-infected (R⁺) females fertilize more eggs than uninfected (R^-) females.

Protocol

NOTE: Ensure that all work is performed at room temperature in a well-ventilated area or under a fume hood. All ‘drops’ in this protocol are defined as 5–20 µL, depending on the operator’s preference.

1. Initial Setup

1. 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. 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.
2. 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.

2. Dechorionation

1. 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 2–3 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.
2. Remove the adult whiteflies from the leaf.
3. 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 pin at 45° or at a comfortable working angle into a melted pipette tip (Figure 2).
4. 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: 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.
5. 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.

3. Fixation

NOTE: These steps are taken from a Hymenopteran protocol¹⁷.

1. 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.
2. Remove the glacial acetic acid with a glass Pasteur pipette and add drops of Clarke’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).
3. 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).

4. Staining

1. 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.
2. Remove the 1x PBS with a glass Pasteur pipette, and add drops of 0.1 μg/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.

5. Washing

1. Remove the DAPI with a glass Pasteur pipette.
2. 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.

6. Mounting

1. 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 µL 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.
2. For long-term storage, seal the cover slide with clear nail polish and, then, either store the slide in the dark at 2 °C or immediately view it under a fluorescent microscope.

Results

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 °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 µL strip tubes. When adults emerged, ...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x PBS	Any
1x TBST	Any		5x solution made from 30 g Tris, 43.8 g NaCl, 5 mL Tween-20 and 1.0 g NaN3 pH 7.5, and brought to 1 L with PCR grade water
Bleach	Clorox		Any household bleach will work as long as it can be diluted to 0.83% Sodium hypochlorite
Clear nail polish	Any
DAPI dilactate	Santa Cruz Biotechnology	sc300415
Ethanol	Any		Dilute to 70% EtOH
Fluorescent microscope	Nikon		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
Glacial acetic acid	Mallinckrodt	UN2789
Glycerol	Any
Microscope	Wild		A Wild M5A microscope was used for this experiment, but any microscope where the operator can clearly see the whitefly eggs can be used
Microscope slide covers	Any		Methods are for 18 mm x 18 mm sized slide covers. More mounting media will need to be added for larger slide covers.
Microscope slides	Any
Minuten nadel pins	BioQuip	1208SA	Minuten nadel pins are optional for fashioning as probes with pipette tips
NaCl	Any
NaN3	Any
n-propyl-gallate	Sigma/Santa Cruz Biotechnology	P3130/sc-250794
Parafilm	Bemis
Pasteur pipettes	Fisher Scientific	13-678-20A	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
PCR grade water	Any
Pipette tips	Any		Pipette tips are optional for fashioning as probes with minuten nadel pins
Small dropper bulb	Any		Must fit on Pasteur pipette
Tris	Any
Tween-20	Any