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.
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.
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 tissue2,3. 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 antibiotics (Aa23-T). Given that Aa23 retains only wAlbB, it is a useful model for the study of host-endosymbiont interactions4,5,6.
Wolbachia is maternally transmitted and infects an estimated 65% of insect species8,9 and 28% of mosquito species10. It infects a variety of tissues and forms an intimate symbiotic relationship with the host, usually inducing cytoplasmic incompatibility (CI)11 and population replacement by manipulating the host reproductive system12,13. These host responses have been observed in natural populations of Drosophila simulans14 and in A. aegypti in a laboratory cage and field trial15. An important nonreproductive manipulation elicited by Wolbachia is induced host resistance to a variety of pathogens, including DENV, Chikungunya virus (CHIKV), and West Nile virus (WNV)16,17, that may be mediated by an improved innate immune system of the symbiont18,19, competition between Wolbachia and viruses for essential host resources20, and manipulation of host viral defense pathways21.
This protocol has been developed for studying these underlying mechanisms of Wolbachia-induced host antiviral responses. It uses four methods of detection of intracellular Wolbachia infection of Aa23 cells. These methods provide a strong theoretical basis for studies of intracellular Wolbachia infection of other host species. The first method, PCR-a powerful technique allowing the enzymatic amplification of specific regions of DNA without utilizing conventional cloning procedures-was used to detect Wolbachia DNA and determine the presence/absence of Wolbachia infection22. The second method measures Wolbachia DNA copy density using quantitative PCR (qPCR) for reliable detection and measurement of products generated during each PCR cycle that is directly proportionate to the amount of template before PCR23. The third method detects the presence of intracellular Wolbachia proteins, using western blot-one of the most powerful tools for detecting specific proteins in complex mixtures by combining the high separation power of electrophoresis, the specificity of antibodies, and the sensitivity of chromogenic enzymatic reactions. The final method is an immunofluorescence assay (IFA) that combines immunology, biochemistry, and microscopy to detect the Wolbachia surface protein (wsp) through an antigen-antibody reaction to confirm the cellular uptake of Wolbachia and determine its cellular localization.
This paper describes the four methods listed above to verify the existence of Wolbachia in the cells, which can be used to detect whether the exogenous Wolbachia was successfully transfected and the Wolbachia in the cell was cleared. After determining whether Wolbachia is present in the cells or not, a variety of different analyses can be performed, including genomics, proteomics, or metabolomics. This protocol demonstrates the detection of Wolbachia through Aa23 cells but can also be used in other cells.
1. Materials and reagents
2. Cell culture
3. DNA extraction
4. Detection of Wolbachia nucleic acid
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 diagnostic primers 81F/691R was possible in Aa23 cells (Lanes 1 and 2) but not in Aa23-T cells (Lanes 3 and 4) (Figure 2A). The analysis of Wolbachia density in the cell lines using qPCR showed a wsp/rps6 ratio of 2.4 in Aa23 cells but no Wolbachia in Aa23-T cells (Figure 2B). Western blot analysis of protein extracts showed a strong wsp signal for Aa23 and no signal for Aa23-T (Figure 3A), while the indirect immunofluorescence assay detected the wsp protein (Figure 3B) in Aa23 cells (green), while only DNA stained with DAPI (blue) was detected in Aa23-T cells.
Figure 1: Light microscopy of unstained Aa23 and Aa23-T cells. Heterogeneous morphology indicates that more than one cell type is present in both cell lines, as indicated by the arrows. Scale bar = 20 µm. Please click here to view a larger version of this figure.
Figure 2: Detection of Wolbachia infection in cells at the nucleic acid level. (A) PCR analysis of Aa23 and Aa23-T cells resolved on a 1% agarose gel. Positive PCR amplification of the Wolbachiawsp gene using the diagnostic primers, 81F/691R, observed in Aa23 cells (Lanes 1 and 2) but not in Aa23-T cells (Lanes 3 and 4). (B) Wolbachia wsp copy density of Aa23 and Aa23-T cells. Density was greater in Aa23 cells and undetectable in Aa23-T cells. Copy number was normalized using Aedes albopictus rps6; data are mean ± SEM, n = 6. Please click here to view a larger version of this figure.
Figure 3: Detection of Wolbachia infection in cells at the protein level. (A) Immunoblotting with wsp antibody in Aa23 cells (Lane 1), with an obvious band near 25 kDa; Aa23-T (Lane 2), which was treated with tetracycline. (B) Immunofluorescence labeling of cells showing the localization of Wolbachia (green); cells were probed with polyclonal anti-wsp antibody (Wolbachia), followed by goat anti-mouse (green) Alexa Fluor 488-conjugated antibodies, and nuclei (blue) are stained with DAPI. Mouse serum was used as a negative control because the anti-wsp antibody was not purified, and the polyclonal antibody was mouse-derived. Aa23 cells (Aa23-anti-wsp) infected with Wolbachia are indicated with a white arrow. Scale bars = 10 µm. Abbreviation: DAPI = 4',6-diamidino-2-phenylindole; M = marker. Please click here to view a larger version of this figure.
Supplemental File: Plasmid generation and anti-wsp antibody preparation. Please click here to download this File.
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 presence of Wolbachia infection of cells at the nucleic acid level. However, as it does not quantify levels of infection28,29, qPCR was used to quantify DNA copy density28,30. As the accuracy of PCR methods may be affected by low amplification efficiency and false positives, it is important to ensure good quality of DNA, especially for qPCR detection, where reagents and templates must be placed on ice to prevent denaturation, and operations should be carried out according to standard methods. Low amplification efficiency tends to be caused by reagent degradation in the reaction system, insufficient template amounts, and/or excessive length of extended primer fragments28. False positives are usually caused by contamination of reagents and templates, high levels of primer concentration, and/or dimer and hairpin primer structures. Therefore, it is important to ensure that reagents are of high quality, primers are optimized based on the literature, and appropriate software used for primer design.
Two methods described above detect intracellular Wolbachia infection at the nucleic acid level; the other two detect intracellular Wolbachia infection at the protein level. Western blotting was used to determine intracellular Wolbachia infection of Aa23 and Aa23-T cells at the protein level. Problems associated with this method are related to sample preparation, antibody incubation, and detection31. Critical steps in the western blotting detection process include using fresh and fully lysed samples and correct secondary antibodies, which must be validated and diluted according to the instructions at a dilution of 1:12,000. According to the experimental requirements, this method can also be combined with other immunization methods. Therefore, Immunofluorescence was used to localize intracellular infection by Wolbachia31 , because this is not directly detected using PCR, qPCR, or western blot analyses. There are some critical steps to note about immunofluorescence. First, ensure the optimal quality of cell samples and avoid contamination. Contamination of cells by black glue bug, which can also be stained, will seriously affect the interpretation of the results. Second, cell density in the laser confocal dishes must be ~60-70%. Finally, washing with PBS after sample incubation is critical to get clear fluorescence images.
Using relative and absolute quantitative and visual methods to detect intracellular Wolbachia infection at the nucleic acid and protein levels, this protocol is more comprehensive and accurate than the use of any single method. In addition, constructing a double standard curve (for wsp and RPS6) results in less stringent requirements for the PCR system and amplification efficiency and partial elimination of the experimental error in the final data processing, which greatly reduces the experimental work. However, there were some limitations to this protocol. First, the anti-wsp antibody is prepared in-house and has not yet been commercialized. Second, although there were no extra bands due to the unpurified polyclonal anti-wsp antibody in western blotting, indicating antibody purity, the negative control group using mouse serum also had a green fluorescence background, which was not conducive to the interpretation of the results. While the accuracy of the western blot and immunofluorescence methods may be improved by ensuring the use of purified antibodies or accurate fluorescent probes, further study and analysis of empirical data are required. In summary, a more comprehensive and accurate protocol is reported for the detection of Wolbachia infection in Aa23 cells using four methods used in previous studies of Wolbachia. The protocol described here is applicable to the detection of Wolbachia in other types of cells and tissues.
The authors have nothing to disclose.
We thank Dr. Xin-Ru Wang from the University of Minnesota for insightful suggestions and guidance. This work was supported by a grant from the National Natural Science Foundation of China (No.81760374).
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 |