Detection of Wolbachia endobacteria in Culex quinquefasciatus by Gimenez staining and confirmation by PCR.
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Date
2012-12
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Abstract
Background & objectives: Wolbachia are common intracellular bacteria that are found in arthropods and nematodes.
These endosymbionts are transmitted vertically through host eggs and alter host biology in diverse ways, including
the induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm-egg
incompatibility. Since they can also move horizontally across species boundaries, Wolbachia is gaining importance
in recent days as it could be used as a biological control agent to control vector mosquitoes or for paratransgenic
approaches. However, the study of Wolbachia requires sophisticated techniques such as PCR and cell culture
facilities which cannot be affordable for many laboratories where the diseases transmitted by arthropod vectors
are common. Hence, it would be beneficial to develop a simple method to detect the presence of Wolbachia in
arthropods.
Method: In this study, we described a method of staining Wolbachia endobacteria, present in the reproductive
tissues of mosquitoes. The reliability of this method was compared with Gram staining and PCR based detection.
Results: The microscopic observation of the Gimenez stained smear prepared from the teased ovary of wild
caught and Wolbachia (+) Cx. quinquefasciatus revealed the presence of pink coloured pleomorphic cells of
Wolbachia ranging from cocci, comma shaped cells to bacillus and chain forms. The ovaries of Wolbachia (–)
cured mosquito did not show any cell. Although Gram’s staining is a reliable differential staining for the other
bacteria, the bacterial cells in the smears from the ovaries of wild caught mosquitoes did not take the stain properly
and the cells were not clearly visible. The PCR amplified product from the pooled remains of wild caught and
Wolbachia (+) Cx. quinquefasciatus showed clear banding, whereas, no banding was observed for the negative
control (distilled water) and Wolbachia (–) Cx. quinquefasciatus.
Interpretation & conclusion: The Gimenez staining technique applied, could be used to detect the members of the
endobacteria Wolbachia easily, even in a simple laboratory without any special facilities or even in the field
condition and for handling large number of samples in a shorter duration.
Description
Keywords
Culex quinquefasciatus, Gimenez staining, PCR, Wolbachia endobacteria
Citation
Muniaraj M, Paramasivan R, Sunish I P, Arunachalam N, Mariappan T, Leo S Victor Jerald, Dhananjeyan K J. Detection of Wolbachia endobacteria in Culex quinquefasciatus by Gimenez staining and confirmation by PCR. Journal of Vector Borne Diseases. 2012 Dec; 49(4): 258-261.