Wolbachia Invades Anopheles stephensi Populations and Induces Refractoriness to Plasmodium Infection

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Science  10 May 2013:
Vol. 340, Issue 6133, pp. 748-751
DOI: 10.1126/science.1236192

Infections Against Infection

In the same way that infection with the bacteria Wolbachia spp. can make Aedes mosquitoes resistant to dengue virus, there have been hints that these bacteria can interfere with the reproduction of malaria parasites. Bian et al. (p. 748) established a heritable Wolbachia infection in anopheline mosquitoes, which simultaneously suppressed the reproduction of malaria parasites within the adult female mosquitoes. The results hold promise for developing the model into a biocontrol agent to assist malaria control.


Wolbachia is a maternally transmitted symbiotic bacterium of insects that has been proposed as a potential agent for the control of insect-transmitted diseases. One of the major limitations preventing the development of Wolbachia for malaria control has been the inability to establish inherited infections of Wolbachia in anopheline mosquitoes. Here, we report the establishment of a stable Wolbachia infection in an important malaria vector, Anopheles stephensi. In A. stephensi, Wolbachia strain wAlbB displays both perfect maternal transmission and the ability to induce high levels of cytoplasmic incompatibility. Seeding of naturally uninfected A. stephensi populations with infected females repeatedly resulted in Wolbachia invasion of laboratory mosquito populations. Furthermore, wAlbB conferred resistance in the mosquito to the human malaria parasite Plasmodium falciparum.

The ability of Wolbachia to spread through cytoplasmic incompatibility (CI) (1, 2) and render mosquitoes resistant to a variety of human pathogens (36) has instigated the development of Wolbachia-based strategies for both suppression and replacement of disease vector populations (2, 7). Given their medical importance, there have been considerable efforts to extend this approach to anopheline malaria vector mosquitoes, which are not naturally infected by Wolbachia spp. (8). Over the past two decades, various attempts to artificially generate stably infected Anopheles spp. have failed, raising concern that the Anopheles germ line is inhospitable to Wolbachia or that Wolbachia infection might cause reproductive ablation in Anopheles mosquitoes (6). Studies based on a transient somatic infection have recently indicated that Wolbachia can inhibit the development of the malaria parasite in the Anopheles mosquito, possibly by stimulating a mosquito antiparasitic immune response (5, 6). These results reinforced the potential of a Wolbachia-based intervention for malaria vector control, but only if the bacterium could be made to form a stable association with this mosquito.

Anopheles stephensi is the major vector of human malaria in the Middle East and South Asia. We infected A. stephensi [Liston strain (LIS)] by embryonic microinjection of the wAlbB Wolbachia strain derived from Aedes albopictus (Houston strain) (9). Cytoplasm was withdrawn from A. albopictus embryos and directly injected into the posterior of A. stephensi early embryos (1). After oviposition, we used polymerase chain reaction (PCR) to test females (G0) developed from surviving embryos for Wolbachia infection. We observed a stable wAlbB infection in one isofemale line (designated LB1) at G1 with a 100% infection frequency maintained through G34 (the last generation assayed thus far). At G9, G10, and G11, we randomly selected 20 individuals (10 males and 10 females) from the LB1 cage population and tested them by diagnostic PCR (10). All individuals (n = 60) were infected with wAlbB (fig. S1).

The 100% maternal transmission efficiency was also confirmed by fluorescence in situ hybridization (FISH) of LB1 mosquito ovaries showing heavy wAlbB infection of all ovarian egg chambers. In the ovaries of the 5-day-old non–blood-fed females, wAlbB was mainly found in the oocytes of the egg chambers, with a low-level presence in nurse cells (Fig. 1A). This observation is consistent with a previous model showing Wolbachia migration from nurse cells to the oocytes through the ring canals during oogenesis (11). As in A. albopictus and the transinfected Aedes aegypti WB1 line (1, 12), Wolbachia was concentrated in the anterior and posterior part of LB1 mosquito oocytes 3 days after a blood meal (fig. S2), indicating that the wAlbB distribution pattern in the ovaries is conserved between mosquito species.

Fig. 1 Establishment and invasion of Wolbachia wAlbB in A. stephensi populations.

(A) wAlbB distribution in the ovarian egg chambers of 5-day-old non–blood-fed LB1 females with LIS females as controls. Wolbachia, cytoplasm, and nuclear DNA were stained with 16S ribosomal DNA Wolbachia probes (green), propidium iodide (red), and 4′,6-diamidino-2-phenylindole (blue), respectively. White arrows indicate Wolbachia. (B) wAlbB induces nearly complete CI in A. stephensi when infected males are crossed with uninfected females. Error bars indicate SE. The number of replicates for each of the four cross types is shown in parentheses. (C) wAlbB invades the A. stephensi laboratory populations. Female infection frequency was measured by PCR after a single release of LB1 females into LIS populations and continued inundative release of LB1 males at a rate of twice the male population size for each generation.

Of 8087 eggs resulting from crosses between LB1 males and the naturally uninfected LIS females, only 1.2% (95% confidence interval = 0.15 to 2.16) hatched (Fig. 1B), indicating a typical CI pattern. We observed a >50% egg-hatch rate in the other cross types. The egg hatches resulting from LB1 self-crosses (52.4%) were significantly lower than those observed in compatible crosses of wild-type individuals (91.0%; P < 0.01, χ2 = 2016.4). Outcrossing of the LB1 females with LIS males for four generations did not improve the egg-hatch rate, but tetracycline treatment of the outcrossed line increased the rate to 85.9 ± 5.3% (fig. S3), supporting the hypothesis that the wAlbB infection is responsible for the reduced hatch rate.

To assess the ability of the wAlbB infection to invade a natural uninfected population, we seeded LB1 females at ratios of 5, 10, and 20% into uninfected LIS cage populations composed of 50 females and 50 males. To promote population replacement, we also released 100 LB1 males at every generation to suppress the effective mating of LIS females. In all populations, wAlbB increased to 100% infection frequency within eight generations and remained fixed in subsequent generations (Fig. 1C). These results support the potential for Wolbachia to mediate population replacement in a public health intervention strategy. Specifically, the wAlbB infection was able to invade and replace the naturally uninfected cytotype within eight generations after an introduction rate as low as 5% and continued inundative release of males at a rate of two times the male population size each generation. These results also raise the challenge in application that large-scale programs for breeding and releasing male infected mosquitoes might be necessary, perhaps in conjunction with short-term intensive mosquito abatement.

A transient Wolbachia infection in Anopheles gambiae mosquitoes is known to inhibit Plasmodium falciparum development (5, 6). To assess the possible anti–P. falciparum activity of wAlbB in the transinfected LB1 mosquitoes, we fed them on a gametocyte culture, along with LIS and the aposymbiotic line LBT mosquitoes (generated by tetracycline treatment of the LB1 strain to remove wAlbB) as controls. Although the presence of wAlbB had no impact on the ookinete stage parasites before midgut invasion (Fig. 2A and table S1), it did result in a significantly reduced prevalence and mean intensity of the oocyst stage parasite on the basal side of the midgut, as assayed at 7 days postinfection (dpi). Specifically, the LB1 strain displayed significantly lower infection prevalence and intensity than the LIS strain (Mann-Whitney U test, P < 0.0001) and the aposymbiotic LBT strain (Mann-Whitney U test, P < 0.01), whereas no difference was observed between the LIS and LBT strains (Fig. 2B and table S1). We also investigated the impact of wAlbB on the salivary gland sporozoite stage infection at 14 dpi and observed a greater inhibition than at the oocyst stage (Fig. 2C and table S1). wAlbB infection resulted in a 3.4- and 3.7-fold reduction in the sporozoite loads in salivary glands of LB1 mosquitoes when compared with LIS and LBT mosquitoes, respectively (Mann-Whitney U test, P < 0.001) (Fig. 2C and table S1). These data suggest that wAlbB inhibit P. falciparum development between the preinvasion lumenal ookinete and oocyst stages and between the oocyst and salivary gland sporozoite stages.

Fig. 2 Wolbachia wAlbB-mediated inhibition of Plasmodium development.

P. falciparum ookinete (A), oocyst (B), and sporozoite (C) loads in midgut lumens, midguts, and salivary glands, respectively, of A. stephensi LIS, LB1, and LBT strains. Points represent the number of parasites from an individual mosquito; horizontal lines indicate the median number of parasites per tissue. Different letters above each column signify distinct statistical groups [(B) P < 0.0001 for LB1 versus LIS and P < 0.01 for LB1 versus LBT; (C) P < 0.001 for both LB1 versus LIS and LB1 versus LBT; Mann-Whitney test].

A local distribution of wAlbB and wMelPop strains in mosquito somatic tissues, especially those in which pathogens replicate, develop, and travel, is important for Wolbachia to induce pathogen interference (4, 13). We examined the wAlbB density in midguts, salivary glands, and fat bodies from 7-day-old LB1 non–blood-fed females by real-time PCR. We detected Wolbachia wAlbB in all tissues, with a marked 5.9-fold higher density in the fat bodies than in the ovaries (Fig. 3A). Salivary glands and ovaries contained similar levels of wAlbB, whereas midguts had lower infection than ovaries, a distribution confirmed by FISH assay (Fig. 3B). This result is similar to observations made in the transiently infected A. gambiae, in that Wolbachia resided primarily within cells of the fat bodies and had a low affinity for midgut cells (6).

Fig. 3 Wolbachia wAlbB distribution in somatic tissues of LB1 mosquitoes (G27).

(A) The genome copy of Wolbachia surface protein (WSP) was measured by real-time PCR and normalized by A. stephensi ribosomal protein S6 (RPS6). Different letters above each column signify distinct statistical groups (P < 0.05 for comparison between a, b, and c; Student’s t test). Error bars indicate SEM of at least 10 biological replicates. (B) Wolbachia wAlbB distribution in fat body, midgut, and salivary gland of an LB1 mosquito, assayed by FISH as described in Fig. 1A. White arrows indicate Wolbachia.

We have previously shown that wAlbB induces the production of reactive oxygen species (ROS) in Aedes (14), and other work has shown that ROS can inhibit Plasmodium infection in Anopheles (15, 16). To explore whether wAlbB-induced ROS could play a role in the mosquitoes’ resistance to Plasmodium, we compared the levels of H2O2 in midguts, fat bodies, and whole bodies of LB1 and LIS mosquitoes. The levels of H2O2 were significantly higher in tissues of LB1 mosquitoes than in those of LIS mosquitoes (Student’s t test, P < 0.01) (Fig. 4) and nearly twofold higher in whole LB1 than in LIS mosquitoes.

Fig. 4 Wolbachia-induced ROS production in LB1 mosquitoes.

This figure shows a comparison of H2O2 levels in the fat body, midgut, and whole mosquito in 7-day-old LB1 and LIS females before a blood meal. The data shown are means of 6 (fat body and midgut) or 10 (whole-mosquito) replicates. Different letters above each column signify distinct statistical groups (P < 0.01 for each pair of comparison between a, b, c, d, e, and f; Student’s t test). Error bars indicate SEM.

In conclusion, we show that the Wolbachia wAlbB strain can form a stable symbiosis with A. stephensi, invade laboratory mosquito populations through CI, and confer elevated resistance to Plasmodium infection, potentially through ROS generation. Previous failures in establishing a stable Wolbachia infection in Anopheles mosquitoes may be due to the Wolbachia strains used. To form a symbiosis, the Wolbachia strain should be sufficiently invasive to establish an infection in germ tissues but without being lethal to the host. The success of wAlbB may be attributed to its ability to confer a fitness advantage to its host (10) and its high infectivity to Anopheles germ tissues (17). We used a previously described embryo microinjection technique (1) but observed a lower survivor rate, possibly due to the greater sensitivity of Anopheles eggs to desiccation. The low egg-hatch rate associated with the wAlbB infection in A. stephensi, which is not observed in wAlbB-infected A. aegypti, may be related to the use of mouse (an unnatural host) blood in this study. A previous study has reported suppression of egg hatch after a long-distance transfer of wMelPop into A. aegypti feeding on nonhuman blood sources, but only mild decreases when the mosquitoes fed on human blood (18).

The recent success of a field trial has demonstrated that Wolbachia can be deployed as a practical dengue intervention strategy, with the potential for area-wide implementation (2). The design of Wolbachia-based malaria control strategies would have to accommodate the fact that Plasmodium is vectored by multiple and frequently sympatric Anopheles species in different parts of the world (19, 20). However, this complication can be resolved by integrating a Wolbachia-based approach with other vector control strategies and by targeting the dominant malaria vectors that are the most difficult to control. For example, Wolbachia could be used to target outdoor-biting and -resting species that can evade current vector control methods, such as insecticide-treated nets and residual insecticide sprays (21). In our studies, we used a laboratory P. falciparum infection model that results in unnaturally high infection intensities, reaching a median of 20 oocysts per midgut, whereas infection levels in nature rarely exceed 2 to 3 oocysts (22). As we have shown in other studies comparing natural and laboratory infection intensities (23), it is quite likely that a stable wAlbB infection would confer complete refractoriness under natural field conditions. Our success in rendering A. stephensi resistant to P. falciparum by stable introduction of wAlbB offers a potential approach to permanently reduce the vectorial capacities of dominant malaria vectors in sub-Saharan Africa, one of the most challenging goals in current malaria vector control (21). However, it is still unknown whether Plasmodium will develop resistance to ROS or other Wolbachia-mediated inhibitory mechanisms in mosquitoes.

Supplementary Materials

Materials and Methods

Figs. S1 to S3

Table S1

References (2430)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: This work was supported by NIH grants R01AI080597, R21AI082141, and R01AI061576 and a grant from the Foundation for the NIH through the Grand Challenges in Global Health Initiative of the Bill and Melinda Gates Foundation. We are grateful to the Johns Hopkins Malaria Research Institute Parasitology and Insectary Core Facilities and thank S. O’Neill, A. A. Hoffmann, and S. L. Dobson for their comments and suggestions and D. McClellan and S. Thiem for editorial assistance. Z.X. is also affiliated with Guangzhou WolbaKi Biotech Co., LTD. Data for this report are archived as supplementary materials on Science Online.
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