chapter fourteen

Wolbachia and Anopheles mosquitoes Jason L. Rasgon

Contents The problem of malaria..................................................................................................... 321 Wolbachia........................................................................................................................... 321 Wolbachia in anopheles....................................................................................................... 323 Has Wolbachia ever been associated with anopheles mosquitoes?................................ 324 In vitro studies of Wolbachia–anopheles interactions....................................................... 324 In vivo anopheles infections with Wolbachia...................................................................... 325 Conclusions......................................................................................................................... 325 References............................................................................................................................ 326

The problem of malaria Human malaria, caused exclusively by Plasmodium parasites, infects up to 500 million people and results in almost 3 million deaths per year (Hay et al., 2004). The malaria parasites are dependent on Anopheles mosquitoes for transmission between human hosts (Collins and Paskewitz, 1995). Control of the disease is currently limited to antiparasitic drugs and mosquito control (Beaty, 2000) and is hampered by the evolution of drug and insecticide resistance (Talisuna et al., 2004; Hemingway and Ranson, 2000). Thus, there has been a recent concerted effort to develop genetically modified Anopheles mosquitoes that are unable to transmit Plasmodium (Ito et al., 2002; Marrelli et al., 2007). Before deployment of genetically modied mosquitoes for malaria control can be implemented, three critical milestones must be met. These include engineering gene effectors that block pathogen transmission in the mosquito, integration and expression of effectors in the mosquito genome, and spread of the transgene into natural mosquito populations (Rasgon and Scott, 2003; Rasgon and Gould, 2005; James, 2005). Although there has been significant progress toward the first two items (Ito et al., 2002; Marrelli et al., 2007), there is no available drive mechanism to spread transgenes into natural Anopheles populations.

Wolbachia One such potential transgene driver is the endosymbiont Wolbachia (Rasgon and Scott, 2003; James, 2005). In mosquitoes, the maternally inherited symbionts are generally associated with cytoplasmic incompatibility (CI)—reduced egg hatch in matings between uninfected females and infected males. Matings between infected females and infected or uninfected males are fertile. Thus, infected females have a reproductive advantage, 321

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32 Insect symbiosis, volume three which, coupled with maternal inheritance, can cause Wolbachia to spread rapidly through host populations. Model predictions of Wolbachia dynamics have been validated in cage experiments (Curtis, 1976; Xi et al., 2005) and by observations of the dynamics of Wolbachia infections in natural insect populations (Turelli et al., 1992; Turelli and Hoffmann, 1995; Rasgon and Scott, 2003). If a transgene is inserted into the Wolbachia genome, or placed on a separate maternally inherited construct, the transgene will “hitchhike” with the symbiont into the population (Figure 14.1), replacing the natural population with one that is refractory to parasite transmission (Turelli and Hoffmann, 1999). An alternative Wolbachia-based malaria control strategy is to reduce mosquito population levels by releasing Wolbachia-infected (i.e., incompatible) males into uninfected natural populations. In this scenario, released males are reproductively incompatible with the wild females, resulting in sterility. This strategy is functionally equivalent to the sterile insect technique (SIT), but with the advantage that males do not need to be exposed to damaging radiation or chemosterilants that might lower their mating competitiveness (Arunachalam and Curtis, 1985; Shahid and Curtis, 1987; Dobson et al., 2002a; Zabalou et al., 2004; Brelsfoard et al., 2008). The third strategy is to release mosquitoes infected with virulent Wolbachia strains that shorten mosquito lifespan. After feeding on an infected host, a mosquito must survive for a period of up to 2 weeks before it is able to transmit the parasites. Thus, the daily probability of survival is the most sensitive component of a vector’s role in pathogen transmission (Garrett-Jones, 1964; Rasgon et al., 2003). Control strategies that reduce mosquito lifespan are theoretically more efficient in reducing disease than other strategies because

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Generation Figure 14.1  Wolbachia can drive maternally inherited transgenes into mosquito populations. Solid line = Wolbachia. Dotted line = maternally inherited transgene. Wolbachia maternal transmission efficiency is 95%, causes 100% CI and has no fitness cost. Transgene is neutral and is maternally inherited by 100% of progeny. Transgene frequency increases from an initial level of 5% to fixation in approximately 40 generations.

Chapter fourteen: Wolbachia and Anopheles mosquitoes 323 Table 14.1  Species of Anopheles That Have Been Assayed for Wolbachia Infection, and Found Negative (Kittayapong et al. 2000, Ricci et al. 2002, Rasgon and Scott 2004) Anopheles gambiae s.s.s.

Anopheles annularis s.l.l.

Anopheles funestusus

Anopheles culicifacieses

Anopheles arabiensisis

Anopheles dirus species A & B B

Anopheles nilili

Anopheles dravidicusus

Anopheles pharoensisis

Anopheles jamesiiii

Anopheles mouchetiti

Anopheles kochihi

Anopheles maculipenisis

Anopheles maculatusus

Anopheles atroparousus

Anopheles minimusus

Anopheles sacharovivi

Anopheles nivipeses

Anopheles superpictusus

Anopheles pseudowillmoriri

Anopheles plumbeusus

Anopheles sawadwongpornini

Anopheles freebornini

Anopheles spendidusus

Anopheles barbirostrisis

Anopheles subpictusus

Anopheles peditaeniatusus

Anopheles tessellatusus

Anopheles hyrcanusus

Anopheles vagusus

Anopheles aconitisis

Anopheles varunana

small changes in the daily survival rate result in large changes in the number of new host infections. A pathogenic Wolbachia strain (denoted popcorn or wMelPop) has been shown to kill adult Drosophila melanogaster by over-replication in the central nervous system of the fly. Adult life span of infected flies is approximately one-half that of uninfected flies (Min and Benzer, 1997). Similar results were seen when wMelPop was artificially transferred to D. simulans (McGraw et al., 2002). If a virulent popcorn-like Wolbachia strain were transferred into Anopheles, it might be possible to use CI to counteract the fitness disadvantages conferred by increased mortality and spread pathogenic symbionts through the population, reducing pathogen transmission and malaria incidence by shortening vector lifespan (Rasgon et al., 2003).

Wolbachia in Anopheles Wolbachia symbionts have been identified in many mosquito species (Kittayapong et al., 2000; Ricci et al., 2002; Rasgon and Scott, 2004a), the processes that govern symbiont spread in natural mosquitoes populations have been examined in detail empirically (Rasgon and Scott, 2003, 2004a) and theoretically (Dobson et al., 2002a; Rasgon et al., 2003; Rasgon and Scott, 2004b), and several different Wolbachia-based control strategies have been discussed (Dobson et al., 2002a; Rasgon et al., 2003; Rasgon and Scott, 2003; Sinkins and Godfray, 2004), but no Wolbachia infections have ever been identified in any species of Anopheles (Kittayapong et al., 2000; Ricci et al., 2002; Rasgon and Scott, 2004a). Over 30 species of Anopheles from 4 continents have been assayed for Wolbachia infections with negative results (Table 14.1). Because preexisting natural infections can interact with and alter the behavior of introduced infections (Hoffmann and Turelli, 1997), the naive infection status of natural Anopheles gambiae populations offer a clean slate for Wolbachia-based malaria control strategies.

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Has Wolbachia ever been associated with Anopheles mosquitoes? In a study examining the An. gambiae salivary gland transcriptome, researchers identified a region on chromosome 3R encoding for several transcripts coding for putatively “Wolbachia-like” proteins (e.g., ENSANGP00000026834) associated with cell membrane biogenesis (Arca et al., 2005). This region was flanked by transposons, suggesting a possible transposable element-mediated horizontal transfer event from a past Wolbachia infection into the Anopheles genome. Horizontal movement of Wolbachia DNA into the host nuclear chromosomes is now an established phenomenon (Kondo et al., 2002; Hotopp et al., 2007) and these data, taken at face value, suggest that sometime in the past Anopheles may have been infected with Wolbachia. The researchers also suggested that the presence of these Wolbachia-like transcripts may be responsible for the apparent resistance of Anopheles to current infection. However, homologues to these genes are also found in Aedes aegypti (e.g., Genbank #EAT45021), indicating that if a horizontal transfer from Wolbachia to mosquitoes occurred, it happened before the split of the genera Anopheles and Aedes. Thus, there is no genomic evidence that Anopheles mosquitoes have ever had Wolbachia infection.

In vitro studies of Wolbachia–Anopheles interactions Due to the absence of infection in Anopheline mosquitoes, some have suggested that Anopheles mosquitoes may be genetically incapable of sustaining Wolbachia infections (Sinkins, 2004; Arca et al., 2005). Our group hypothesized that if there was an intrinsic genetic block to Wolbachia infection in Anopheles gambiae, we would be unable to infect cultured Anopheles gambiae cells with the symbiont. Using the modified shell-vial technique (Dobson et al., 2002b), we successfully infected 2 phylogenetically distinct Wolbachia strains—wRi from Drosophila simulans (Figure 14.2) and wAlbB from Aedes albopictus Aa23 cells—into the immune-competent An. gambiae cell line Sua5B (Rasgon et al., 2006). Another An. gambiae

Figure 14.2  Wolbachia strain wRi in Sua5B cells. Cell nuclei and Wolbachia are stained with DAPI and visualized by epifluorescent microscopy.

Chapter fourteen: Wolbachia and Anopheles mosquitoes 325 cell line (Moss55) has been infected with the virulent Wolbachia strain wMelPop (gift of Dr. SL O’Neill, University of Queensland). Some infections have reached very high levels in the cell cultures, where almost 100% of cells are infected at high levels (wAlbB in Sua5B and wMelPop in Moss55). wRi, however, never reached high levels in Sua5B cells (maximum 30% of cells infected) (Rasgon et al., 2006) and was eventually eliminated from the cell line after approximately 150 passages (Rasgon, unpublished). Although in vitro data do not always translate to results in vivo, the cell line data indicate that there is no genetic block to some strains of Wolbachia in Anopheles gambiae cells and thus there is no a priori reason to suggest that Anopheles mosquitoes are refractory to Wolbachia infection in general, although certain strains of Wolbachia may be more able to colonize Anopheles than others. Therefore, with proper technique, and selection of an appropriate Wolbachia strain, establishment of in vivo Anopheles infections may well be feasible.

In vivo anopheles infections with Wolbachia Artificial cross-taxa Wolbachia transfections by embryonic microinjection are routine in Drosophila (Poinsot et al., 1998; Rousset et al., 1999; McGraw et al., 2002; Riegler et al., 2004; Veneti et al., 2004) and have succeeded in several other insect taxa (Chang and Wade, 1996; Van Meer and Stouthamer, 1999; Sasaki and Ishikawa, 2000; Zabalou et al., 2004). Recently, embryonic microinjection protocols for Wolbachia transfection have been developed for the mosquitoes Aedes albopictus (Xi et al., 2006) and Aedes aegypti (Xi et al., 2005), as well as Wolbachia transfer protocols based on injection of symbionts directly into adult Drosophila and Aedes mosquitoes (Frydman et al., 2006; Ruang-Areerate and Kittayapong, 2006). We are currently using both embryonic and adult injection protocols to transfer Wolbachia into Anopheles gambiae, and experiments are ongoing.

Conclusions Although there has been much recent progress toward the goal of developing transgenic Anopheles mosquitoes that are refractory to transmission of malaria parasites, there has been little corresponding research toward the development of drive mechanisms. Without a drive mechanism, most transgenic control strategies are doomed to failure. Wolbachia has shown considerable promise in both manipulated and natural systems as a viable method for driving genes into populations for disease control. In vitro data suggests that the Anopheles genetic background is competent to harbor some Wolbachia strains and there is thus no a priori reason to suspect that the mosquitoes are refractory to infection in vivo. Wolbachia transfer technologies have been developed for a variety of vector and nonvector insects, and we have every reason to believe that similar techniques can be adapted to Anopheles mosquitoes. The successful transfer of Wolbachia into Anopheles mosquitoes will lay the foundation for the successful deployment of genetically modified Anopheles mosquitoes for malaria control.

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