Abstract:
BACKGROUND: The maternal transformation of Wolbachia (Wb) to Aedes mosquitoes is an approach that has been widely used in Aedes mosquito population control strategies in the integrated dengue vector control efforts. This study details usage of such approach in the maternal transformation of Aedes aegypti using the Wb isolated from infected mosquito hosts using micro-injection based procedure for the first time in Sri Lanka. METHODS: Twenty five ovaries of each adult female Aedes albopictus and Culex quinquifaciatus mosquitoes (14-18 days old) were dissected and Wb was isolated and suspended in 20 μl of 1X Phosphate Buffered Saline (PBS). The isolated samples were kept on ice to maximize the bacteria viability. The exudate was then used in the trans-infection of Ae. aegypti mosquitoes. Infection of Wb into the mosquito Ae. aegypti was carried out by maternal/adult microinjection. To create stable trans-infected lines, the regions near the 3rd leg emerged from the thorax of Ae. aegypti were microinjected to incorporate Wb into the developing germ line and then to facilitate the transmission of Wb to offspring. The volume used for injection based on the swelling of the abdomen, which was monitored throughout the experiment while maintaining the injection pressure, time and number of injecting times. Virgin female Aedes mosquitoes survived after microinjection was fed with 1 ml of 10% sugar solution containing 10,000 units Penicillin/Streptomycin per 100 ml. Survival rate was monitored at 1, 3 and 24 hour/s following microinjection procedure. Survived female mosquitoes resulted from injected line (generation 0 [G0]) was placed in vials with three uninfected male mosquitoes to establish iso-female lines. After egg laying 3 times, G0 females were sacrificed and total DNA was extracted, and PCR was carried out (wsp primers 81F and 691R) using DNA extracted from Ae. aegypti to detect the presence of Wb DNA and its density in the latter mosquito using dilution series of extracted DNA. To select for a stable infection, only offspring from females that came positive for Wb by PCR screening was used as parental stocks. For the samples positive by wsp PCR, strain identification was carried out by performing PCR with 4 different primer sets; wPip, wAlbA, wAlbB and wsp specific for Wb. Females that tested negative for Wb were discarded along with their progeny. The resulting triple infected line will be monitored periodically by PCR to confirm infection status. RESULTS: Wolbachia-triple infected line of Ae. aegypti mosquito was created by using isolated wAlbA+wAlbB+wPip Wolbachia strains. Infection status of newly developed line was monitored by PCR up to G2 and will be continued to G8. Experimental work will be started from G5 to evaluate the use of newly develop Ae. aegypti mosquito line for Wb-based approach under laboratory condition. CONCLUSION: Direct maternal micro-injection technique was used to create Wb infected mosquito line of Ae aegypti, which could be applied in Wb-based dengue vector control activities in Sri Lanka once the line is established.