In order to obtain a Wolbachia free line, a Cx. quinquefasciatus wild type colony was treated with tetracycline as previously described31,46. A tetracycline solution (0.1 mg/mL) was used for exposure of first larvae during development. Pupae were transferred to clean water without antibiotic and reared to adulthood, and adults allowed to feed ad libitum with sucrose 10% containing 0.05 mg/mL of tetracycline hydrochloride (final concentration, Sigma, St Louis, MO; Cat. No. T33 83) for three consecutive generations. To confirm Wolbachia in each mosquito generation, ten individual females and ten larvae were screened using the wsp-specific amplification as described above. The line wild type naturally infected with Wolbachia (w+) and Wolbachia free treated with tetracycline (w) were kept under same environmental conditions previously described. The antibiotic was removed in the fourth generation and the experimental work was initiated after at least three generations permitting capture of environmental microbiota, and as well as recovery from the side effects of the treatment with antibiotic. The absence of Wolbachia in mosquito adults cured with tetracycline was confirmed by PCR amplification of the wsp gene.

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