Wolbachia strain wMel induces cytoplasmic incompatibility and blocks dengue transmission in Aedes albopictus

Abstract

Wolbachia inherited bacteria are able to invade insect populations using cytoplasmic incompatibility and provide new strategies for controlling mosquito-borne tropical diseases, such as dengue. The overreplicating wMelPop strain was recently shown to strongly inhibit the replication of dengue virus when introduced into Aedes aegypti mosquitoes, as well as to stimulate chronic immune up-regulation. Here we show that stable introduction of the wMel strain of Drosophila melanogaster into Aedes albopictus, a vector of dengue and other arboviruses, abolished the transmission capacity of dengue virus-challenged mosquitoes. Immune up-regulation was observed in the transinfected line, but at a much lower level than that previously found for transinfected Ae. aegypti. Transient infection experiments suggest that this difference is related to Ae. albopictus immunotolerance of Wolbachia, rather than to the Wolbachia strain used. This study provides an example of strong pathogen inhibition in a naturally Wolbachia-infected mosquito species, demonstrating that this inhibition is not limited to naturally naïve species, and suggests that the Wolbachia strain is more important than host background for viral inhibition. Complete bidirectional cytoplasmic incompatibility was observed with WT strains infected with the naturally occurring Ae. albopictus Wolbachia, and this provides a mechanism for introducing wMel into natural populations of this species.

Fig. 1.

Uju.wMel crossing type. Experiments to characterize the crossing type of the Uju.wMel line at G₆ were performed using UjuT and Ascoli strains. Error bars represent the SEM of hatch rates between females; 15 adult males and 15 females were used for each mass cross. A second round of crossing experiments was performed two generations later (G₈) using Uju.wMel × Uju.wMel, Ascoli × Uju.wMel, Uju.wMel × Ascoli, and Uju.wMel × UjuT following to the same procedure as in the previous experiment. No statistically significant difference was found between G₆ and G₈ of the same cross using Wilcoxon tests. The number of eggs counted for each generation is shown under the x-axis.

Fig. 2.

Transmission capacity (A) and titer of dengue virus in mosquito saliva (B). Three Ae. albopictus strains (Uju.wAlbA/wAlbB, UjuT, and Uju.wMel) were orally infected with dengue 2 virus using glass feeders covered with a chicken skin membrane containing the infectious blood meal at a titer of 10⁷ FFU/mL. After blood feeding, mosquitoes were transferred into cardboard containers and maintained in BSL3 insectaries at 28 °C. After 14 d, surviving mosquitoes were tested for the presence of viral particles in saliva collected using the forced salivation technique. The number of fluorescent foci in the saliva of each mosquito was estimated on C6/36 Aedes albopictus cell culture. The transmission capacity representing the percentage of mosquitoes with infectious saliva among tested mosquitoes was also calculated. The number of fed and assayed females was 44 for Uju.wAlbA/wAlbB, 26 for UjuT, and 44 for Uju.wMel.

Fig. 3.

Immune gene expression in Wolbachia-infected and uninfected mosquitoes. (A) RNA was extracted from adult females of G₅ Uju.wMel, UjuT, and Ascoli, and separately from G₁₀ Uju.wMel, UjuT, and Uju.wAlbA/wAlbB, at 11 d posteclosion. The expression of four Ae. albopictus orthologs for Ae. aegypti immune genes was analyzed by qRT-PCR: a peptidoglycan recognition protein (PGRPS1), cecropin D (CECD), CLIP-domain serine protease (CLIPB37), and a thioester-containing protein (TEP20). Expression was normalized to that of the UjuT adult females. Error bars show the SEM of three biological replicates, each containing four adult females for the G₅ experiment and six adults for the G₁₀ experiment (a total of 12 and 18 mosquitoes per condition). *P < 0.05 compared with UjuT, Wilcoxon test. (B) Adult females were transiently infected with Wolbachia using intrathoracic injections with either wMelPop or wAlbB, controls of either heat-killed E. coli, or the buffer alone, approximately 3 d after eclosion. RNA was extracted from half of the females at 5 d postinjection and from the other half at 9 d postinjection. The expression of four Ae. aegypti immune genes was analyzed by qRT-PCR: a peptidoglycan recognition protein (PGRPS1) cecropin D (CECD), CLIP-domain serine protease (CLIPB37), and a thioester-containing protein (TEP20). Orthologs for these genes in Ae. albopictus were also analyzed by qRT-PCR. Expression was normalized to noninjected adult females of the same age from the same colony. Error bars show the SEM of three biological replicates, each containing five adult females (a total of 30 mosquitoes per condition). *P < 0.05 compared with noninjected control, Wilcoxon test.

Fig. 4.

Concentration of Wolbachia in Ascoli and Uju.wMel lines. DNA was extracted from adult females of Uju.wMel and Ascoli at 11 d after eclosion. The ratio between Wolbachia wsp DNA and host S17 DNA was used to estimate the concentration of Wolbachia. The concentration of wAlbA/wAlbB in the Ascoli strain was arbitrarily designated “1,” and the concentration of wMel in Uju.wMel was plotted relative to this. Error bars show the SEM of three biological replicates, each containing four adult females (a total of 12 mosquitoes per condition). *P < 0.05, Wilcoxon test.