Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Summary

Four methods were used to detect intracellular Wolbachia, which complemented each other and improved the detection accuracy of Wolbachia infection of Aedes albopictus-derived Aa23 and Aa23-T cured of native Wolbachia infection using antibiotics.

Abstract

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Introduction

The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae), which is a key vector of dengue virus (DENV) in Asia and other parts of the world¹, is a natural host of two types of the intracellular bacteria, Wolbachia (wAlbA and wAlbB), which are distributed throughout the germ line and somatic tissue^2,3. The Aa23 cell line derived from A. albopictus embryos consists of at least two morphological cell types, both of which support infection⁴ and may be cured of native Wolbachia infection using an....

Protocol

1. Materials and reagents

1. Use pyrogen-free solutions and media for cell culture (see the Table of Materials).
2. Use ultrapure water to prepare all solutions.
3. Be careful in selecting fetal bovine serum (FBS) for cell culture, following a lot-check process.
   NOTE: As FBS lots are subject to regular change, it is impossible to state the relevant catalog and lot numbers in this protocol.
4. Select Aa23 cell strains derived from A. albopictus eggs, corresponding to Wolbachia-free Aa23-T.

2. Cell culture

1. Prepare 25 cm

Results

Before Wolbachia was detected, Aa23 and Aa23-T cells were observed under a light microscope to determine any morphological differences between the two cell lines. Aa23 and Aa23-T cells have at least two cell morphologies but no obvious morphological difference between the two cell types (Figure 1). Here, Aa23 cells were used as a model system to detect Wolbachia infection using four methods. Positive amplification of the Wolbachiawsp gene using the.......

Discussion

Detection of intracellular Wolbachia infection is essential for the study of Wolbachia-host interactions and the confirmation of successful transfection of cells with novel strains. In this protocol, four methods were used to successfully detect intracellular Wolbachia infection at the nucleic acid and protein levels. These four experimental methods corroborated and improved the detection accuracy of Wolbachia infection of cells.

PCR was used to detect the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microscope	Zeiss		SteREO Discovery V8
Petri dish	Fisher Scietific	FB0875713
Pipette	Pipetman	F167380	P10
inSituX platform
Analysis software	In-house developed
Cerium doped yttrium aluminum garnet	MSE Supplies		Ce:Y3Al5O12, YAG single crystal substrates
Chip holder	In-house developed
Control software	In-house developed
Immersion oil	Cargille Laboratories	16482	Type A low viscosity 150 cSt
inSituX platform	In-house developed
IR light source	Thorlabs Incorporated	LED1085L	LED with a Glass Lens, 1085 nm, 5 mW, TO-18
Outer ring	In-house developed
Pump lasers	Thorlabs Incorporated	LD785-SE400	785 nm, 400 mW, Ø9 mm, E Pin Code, Laser Diode
Raspberry Pi	Raspberry Pi Fundation
Retaining ring	Thorlabs Incorporated	SM1RR	SM1 retaining ring for Ø1" lens tubes and mounts
Seedless quartz crystal	University Wafers, Inc.	U01-W2-L-190514	25.4 mm diameter Z-cut 0.05 mm thickness double side polish 8 mm on -X
Shim	In-house developed
X-ray beam stop	In-house developed