Detection of Wolbachia endobacteria in Culex quinquefasciatus by Gimenez staining and confirmation by PCR

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, includingthe induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm-eggincompatibility. Since they can also move horizontally across species boundaries, Wolbachia is gaining importancein recent days as it could be used as a biological control agent to control vector mosquitoes or for paratransgenicapproaches. However, the study of Wolbachia requires sophisticated techniques such as PCR and cell culturefacilities which cannot be affordable for many laboratories where the diseases transmitted by arthropod vectorsare common. Hence, it would be beneficial to develop a simple method to detect the presence of Wolbachia inarthropods.Method: In this study, we described a method of staining Wolbachia endobacteria, present in the reproductivetissues 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 wildcaught and Wolbachia (+) Cx. quinquefasciatus revealed the presence of pink coloured pleomorphic cells ofWolbachia 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 otherbacteria, the bacterial cells in the smears from the ovaries of wild caught mosquitoes did not take the stain properlyand the cells were not clearly visible. The PCR amplified product from the pooled remains of wild caught andWolbachia (+) Cx. quinquefasciatus showed clear banding, whereas, no banding was observed for the negativecontrol (distilled water) and Wolbachia (–) Cx. quinquefasciatus.Interpretation & conclusion: The Gimenez staining technique applied, could be used to detect the members of theendobacteria Wolbachia easily, even in a simple laboratory without any special facilities or even in the fieldcondition and for handling large number of samples in a shorter duration