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.
ABSTRACT
Understanding Wolbachia mosquito interactions have been recognized as an important concept to develop novel vector control strategies. The prevalence of Wolbachia endobacteria in a natural population of the filariasis vector Culex quinquefasciatus was determined by the polymerase chain reaction method. Earlier workers had estimated the infection rates of Wolbachia with only one or very few individuals per species. In our study large number of specimens were assayed, and a total of 750 adult Culex quinquefasciatus mosquitoes were collected from three south Indian villages of Tirukoilur and Mugaiyur blocks, monthly for a period of five months (December 2006 to April 2007) and screened for the presence of Wolbachia. The percentage prevalence in adult males ranged from 88% to 96%; while in females from 84% to 100%. An overall prevalence of 91.2% was observed. There was no significant difference observed in the proportion of mosquitoes positive for Wolbachia between males and females, and also between different months of the survey; except during the month of February ‘07. The wsp gene sequence of the Wolbachia strain of Cx. quinquefasciatus detected was BLAST analysed and showed 99% sequence similarity with Wolbachia sp. of Culex pipiens isolated from different geographical regions. Phylogenetic analysis based on wsp gene fragments showed that the present Wolbachia isolate was closely related with Wolbachia from Culex pipens pipiens, Niphotettix virescens (Order: Hemiptera) and Cnaphalocrosis medinalis (Order: Lepidoptera).