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Wolbachia Establishment and Invasion in an Aedes aegypti Laboratory Population

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Science  14 Oct 2005:
Vol. 310, Issue 5746, pp. 326-328
DOI: 10.1126/science.1117607

Abstract

A proposed strategy to aid in controlling the growing burden of vector-borne disease is population replacement, in which a natural vector population is replaced by a population with a reduced capacity for disease transmission. An important component of such a strategy is the drive system, which serves to spread a desired genotype into the targeted field population. Endosymbiotic Wolbachia bacteria are potential transgene drivers, but infections do not naturally occur in some important mosquito vectors, notably Aedes aegypti. In this work, stable infections of wAlbB Wolbachia were established in A. aegypti and caused high rates of cytoplasmic incompatibility (that is, elimination of egg hatch). Laboratory cage tests demonstrated the ability of wAlbB to spread into an A. aegypti population after seeding of an uninfected population with infected females, reaching infection fixation within seven generations.

Aedes aegypti (yellow fever mosquito) is the principle vector of dengue viruses throughout the tropical world. Without a registered vaccine or other prophylactic measures, efforts to reduce cases of dengue fever and dengue hemorrhagic fever are limited to vector control. Unfortunately, traditional mosquito control measures are not succeeding. With an estimated 100 million human cases of dengue fever every year (1), substantial effort is being devoted to the development of new strategies to complement existing vector control methods. One such method is population replacement, in which natural A. aegypti populations would be replaced with modified populations that are refractory to dengue transmission. Recent advances toward the production of refractory A. aegypti strains include the development of methods for stable genetic transformation of A. aegypti, RNA interference technology, and genomic sequencing efforts (24). In contrast, there has been relatively little progress toward the development of a vehicle that will serve to drive the refractory genotype into the field population.

A gene-drive vehicle is an important component of vector population replacement strategies, providing a mechanism for the autonomous spread of desired transgenes into the targeted population. Compared with strategies that rely on inundative releases and Mendelian inheritance, gene-drive strategies would require relatively small “seedings” of transgenic individuals into a field population. Perhaps more important than increased cost efficacy, gene-drive strategies can facilitate population replacement with transgenic individuals that have a lower fitness relative to the natural population.

Cytoplasmic incompatibility (CI), induced by naturally occurring intracellular Wolbachia bacteria, has attracted scientific attention as a potential vehicle for gene drive. CI occurs when a Wolbachia-infected male mates with an uninfected female, resulting in karyogamy failure and early developmental arrest of the mosquito embryo (5). Although CI and other forms of reproductive parasitism have made Wolbachia an evolutionary success, with an estimate that infections occur in ∼20% of insect species (6), Wolbachia infections do not naturally occur in A. aegypti, raising the question of whether A. aegypti can support a Wolbachia infection. Although Wolbachia infections have been introduced into Drosophila and other insects (7), Wolbachia infection may not cause CI in A. aegypti and may not invade an uninfected population. Key parameters in Wolbachia infection dynamics include the intensity of CI (number of hatching eggs resulting from an incompatible cross), the maternal inheritance rates (number of uninfected progeny produced by an infected female), and mosquito fitness costs associated with the infection (8). These parameters also determine the infection frequency after a population replacement event, an important consideration because the goal of population replacement is for the entire mosquito population to carry the desired genotype.

A. aegypti were infected by embryonic microinjection with the wAlbB Wolbachia infection from A. albopictus (7). In brief, cytoplasm from A. albopictus eggs (Hou strain superinfected with wAlbA and wAlbB) was injected into A. aegypti eggs (Waco strain). Wolbachia were detected by polymerase chain reaction (PCR) as previously described (9) in each of the five females (G0) that survived from injection to adult. Only three females successfully produced progeny (G1). PCR tests of G1 individuals demonstrated the offspring of one female to be uninfected. The lines established from the remaining two females were only infected with the wAlbB, and one line (designated WB1) was selected for additional tests. In previous work on A. albopictus, wAlbB infections were obtained and not wAlbA (7). This may reflect the lower infection rate of wAlbA relative to wAlbB (10).

PCR assays of WB1 individuals in subsequent generations (≤G12) consistently identified Wolbachia infection. As a specific test of the maternal inheritance rate, progeny were collected from isolated WB1 females (G12). After PCR confirmation of Wolbachia infection in 10 G12 females, the progeny (10 daughters and 10 sons for each G12 female) were assayed with PCR. All of the G13 progeny (n = 200) were infected by Wolbachia (95% binomial confidence interval between 0.9851 and 1.0). Fluorescence in situ hybridization (FISH) confirms high Wolbachia infection rates in WB1 oocytes (Fig. 1). The infection appears highest in the anterior, posterior, and cortical regions of oocytes, similar to the pattern observed in naturally infected A. albopictus (7).

Fig. 1.

(Left) Oocytes of uninfected Waco and wAlbB-infected WB1 stained with a Wolbachia-specific FISH probe. FISH staining methods were as previously described (7). Fig. 2. (Right) Wolbachia infection frequency (A) and egg hatch rates (B) after a single release of WB1 females into Waco populations. (Inset) Graph displays model predictions (8) of Wolbachia infection dynamics assuming complete CI, 100% maternal transmission, and a 15% fecundity cost associated with Wolbachia infection.

Crosses were conducted to determine whether CI occurs as a result of the Wolbachia infection in the WB1 strain. The design of the cross experiment was as previously described (9). As shown in Table 1, the pattern of egg hatch resulting from crosses is consistent with strong CI, similar to that observed in A. albopictus, from which the wAlbB infection was derived (7). No egg hatch resulted from >3800 eggs examined from crosses of uninfected Waco females and infected WB1 males.

Table 1.

CI pattern resulting from crosses of the naturally uninfected Waco and the wAlbB-transfected WB1 A. aegypti strains. Percent egg hatch ± standard deviation and number of cross replicates are shown for each of the four cross types. Crosses were conducted as previously described (7).

Male Waco Male WB1
Female 92.5 ± 3.7% 0.0 ± 0.0%
    Waco (n = 9) (n = 15)
Female 69.1 ± 11.7% 50.6 ± 12.9%
    WB1 (n = 9) (n = 15)

Among the compatible crosses, egg hatches resulting from WB1 crosses [51% and 69% (Table 1)] were significantly lower [Kruskal-Wallis, df (degrees of freedom) = 1, P < 0.01] than the egg hatch observed in compatible crosses of Waco individuals (92%). Because the progeny of the WB1 G0 female were sibling-mated during production of the WB1 isofemale line, the low egg hatch may reflect an inbreeding effect. Therefore, virgin WB1 females (G3) were mated with uninfected Waco males. After the repeat of this introgression for six generations, the egg hatch increased to an average of 89% (G9).

Strong CI and high maternal transmission rates suggest that wAlbB infection will invade an uninfected population. To test this prediction, we released WB1 females at different ratios into replicate Waco laboratory populations (Fig. 2A). The population cage experimental design was as previously described (9). In the 20% initial release cage, the wAlbB infection frequency was observed to increase to 100% infection frequency within seven generations. Additional sampling in the eighth and ninth generations demonstrated that the infection frequency remained fixed at 100% (Fig. 2A). Consistent with model predictions (11), a transient drop in egg hatch was observed during the cytotype replacement (circa generation four) (Fig. 2B). The latter is expected owing to the frequent occurrence of CI crosses; however, once the infection becomes fixed within the population, CI crosses no longer occur, and the egg hatch rates recover.

In cages established with an initial infection frequency of ≤10%, the infection was detected for up to four generations before its disappearance from the population (Fig. 2A). Infection could not be detected in populations in cages initially infected at a rate of 2% release of WB1 females. Hence, the loss of Wolbachia infection from a population is predicted if the initial infection frequency is below a required threshold determined by CI level, fidelity of maternal transmission, and fitness costs associated with Wolbachia infection (8). Complete CI and no evidence of maternal transmission failure were observed in this study. If no fitness costs were associated with the infection, we would predict Wolbachia invasion in all cages in which WB1 females were released. However, we estimated a threshold infection frequency of ∼20%, suggesting there is a substantial fitness cost associated with wAlbB infection in A. aegypti. By using a previously developed model (8), we predicted an approximate 15% fecundity cost to be associated with the wAlbB infection on the basis of the observed population replacement events (Fig. 2A inset).

An analysis of fecundity costs associated with the wAlbB infection neither revealed any differences in egg number (P > 0.3, t test) in comparisons of WB1 females (57.8 ± 17.6 eggs per female, n = 12) with Waco females (52.4 ± 8.5 eggs per female, n = 14) nor revealed any differences in egg hatch rate between Waco and WB1 strains (χ2 test, df = 1, P > 0.05). Thus, future experiments need to investigate additional types of fitness costs (e.g., reduced mating competitiveness or immature survivorship). Furthermore, the laboratory population cage tests conducted here represent an initial proof of principle, but future field cage tests will provide a more accurate prediction of infection dynamics in natural populations (12).

Nevertheless, important obstacles currently prevent implementation of genetic modification strategies using Wolbachia as a vehicle to drive transgenes into A. aegypti populations. Notably absent is a method for linking transgenes to Wolbachia. However, the ability to artificially infect A. aegypti with wAlbB into a major disease vector is an important step toward proposed population replacement strategies. The observed high CI rates, high maternal inheritance, and ability of wAlbB to invade an uninfected laboratory population to infection fixation represent desired characteristics for population replacement strategies. Additional study is needed to define the fitness of wAlbB-infected A. aegypti relative to uninfected individuals, and population replacement experiments need to be performed under conditions that resemble the natural environment. Transfection success with A. aegypti suggests that the transfection approach may be successful with other medically important disease vectors, including Anopheles species.

Supporting Online Material

www.sciencemag.org/cgi/content/full/310/5746/326/DC1

Materials and Methods

Fig. S1

Tables S1 and S2

References and Notes

References and Notes

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