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, differenti....

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 ov....

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