Wolbachia Strain wAlbB Enhances Infection by the Rodent Malaria Parasite Plasmodium berghei in Anopheles gambiae Mosquitoes

Abstract

Wolbachia, a common bacterial endosymbiont of insects, has been shown to protect its hosts against a wide range of pathogens. However, not all strains exert a protective effect on their host. Here we assess the effects of two divergent Wolbachia strains, wAlbB from Aedes albopictus and wMelPop from Drosophila melanogaster, on the vector competence of Anopheles gambiae challenged with Plasmodium berghei. We show that the wAlbB strain significantly increases P. berghei oocyst levels in the mosquito midgut while wMelPop modestly suppresses oocyst levels. The wAlbB strain is avirulent to mosquitoes while wMelPop is moderately virulent to mosquitoes pre-blood meal and highly virulent after mosquitoes have fed on mice. These various effects on P. berghei levels suggest that Wolbachia strains differ in their interactions with the host and/or pathogen, and these differences could be used to dissect the molecular mechanisms that cause interference of pathogen development in mosquitoes.

INTRODUCTION

Wolbachia species are common intracellular bacteria that infect approximately 70% of arthropod species (17). The high percentage of infected arthropods has been attributed to the ability of Wolbachia to manipulate host reproduction to enhance its own transmission. In many cases, Wolbachia can directly or indirectly enhance the fitness of its host (4, 11, 12, 36, 39), which may explain the existence of strains which lack obvious reproductive phenotypes and yet are still prevalent in populations. Recently, it has emerged that Wolbachia-mediated inhibition of pathogens is another mechanism whereby Wolbachia can influence host fitness (1, 15, 16, 18, 21, 22, 27, 35).

Wolbachia-mediated pathogen interference occurs in both naturally infected (15, 16, 35) and artificially transinfected (21, 27) insect species. In naturally infected Drosophila species, only some strains confer protection against RNA viruses (29). Mosquitoes somatically infected with Wolbachia by adult microinjection also exhibit this phenotype (18, 21). Although the mechanism behind pathogen interference is unknown, there is evidence for induction of basal immunity in the insect by Wolbachia in some systems (1, 21, 22, 27). Priming of the immune system before exposure to pathogens can decrease the susceptibility of the host to infection (3, 9, 30, 32, 33). In cases where Wolbachia induces host immunity, the associations were artificially generated (1, 21, 22, 27, 41), whereas no change in immune gene expression was observed in the naturally infected Drosophila and Aedes albopictus associations (2). RNA interference (RNAi) knockdown of TEP1 in wMelPop-injected Anopheles mosquitoes restored Plasmodium berghei levels similar to controls, suggesting that the phenotype may be in part immune mediated (21).

Alternatively, other mechanisms may be the cause of Wolbachia-mediated pathogen inhibition, such as metabolic competition for resources between bacteria and the pathogen. Adding weight to this theory, dengue virus was not seen to colocalize in cells infected with wMelPop in Aedes aegypti mosquitoes, suggesting that Wolbachia is excluding the virus at the cellular level (27). Somatic Wolbachia infection in Anopheles appears atypical, where host immunity changes dynamically in response to Wolbachia, with initial induction of immunity followed by suppression of immune genes (18). It is unknown if this is a trait specific to Anopheles species, which are naturally uninfected, or is due to the artificial nature of somatic infection. Despite wAlbB and wMelPop suppressing immunity in older Anopheles gambiae, both these strains reduced levels of the human malaria parasite Plasmodium falciparum within the mosquito (18). Taken together, this suggests that mechanisms other than induction of basal immunity may be involved in interference of Plasmodium development.

Rodent malaria parasites are often used as models for human disease systems; however, the relevance of these models is not always clear. Here we assessed the pathogen interference phenotype of two strains of Wolbachia, wMelPop from Drosophila melanogaster and wAlbB from Aedes albopictus, against the rodent malaria parasite model P. berghei in Anopheles gambiae using adult somatic infection. Interestingly, we observed that in contrast to the human parasite P. falciparum (18), wAlbB enhances rather than suppresses P. berghei oocyst levels.

MATERIALS AND METHODS

Wolbachia culture and mosquito infection.

Both Wolbachia strains were cultured in Anopheles gambiae cells: wAlbB was cultured in Sua5B cells while wMelPop was cultured in Mos55 cells (25, 31). Wolbachia was extracted from cells as previously described (31). Female A. gambiae (Keele strain) adults (2 days postemergence) were anesthetized on ice and injected with Wolbachia or uninfected Mos55 cell lysate (control) according to previously established methods (16). Postinjection, mosquitoes were incubated in cups (n = 60 mosquitoes per cup) with 3 replicate cups per treatment at 19°C for 2 days for recovery and then maintained at 28°C. Mortality of mosquitoes was assessed daily. The entire experiment was replicated twice. Differences in life spans between treatments were statistically assessed by analysis of variance (with treatment and replicate included as factors).

Mouse maintenance and Plasmodium berghei infection.

Random-bred ND4 Swiss Webster female mice (Harlan Laboratories, Indianapolis, IN), 6 to 8 weeks old, weighing 21 to 30 g at the time of infection, were used throughout the study. Mice were kept at 22°C and a 12-h light/12-h dark cycle. Studies involving laboratory animals were performed in accordance with the regulations of the U.S. Institutional Animal Care and Use Committee (IACUC). Seven days post-Wolbachia injection, mosquitoes were fed on mice infected with P. berghei ANKA 2.34 (according to Johns Hopkins School of Public Health [JHSPH] animal welfare assurance protocol A3272-01) with a male gametocyte exflagellation rate of 1 to 2 exflagellations per field. Mice were anesthetized, and mosquitoes were allowed to feed on the infected mouse for a period of 15 min. Each treatment was fed on the same mouse. Unfed mosquitoes were removed from the experiment. After feeding, mosquitoes were kept at 21°C and 80% humidity. To determine the number of oocysts per mosquito, midguts were dissected at 10 days post-blood meal (pbm) and stained with 0.2% mercurochrome for 20 min at room temperature. Oocysts were counted under a light microscope (Olympus). The experiment was repeated three times. Data were analyzed by analysis of variance with treatment and replicate included as factors.

qPCR analysis of Wolbachia from mosquito carcass.

To determine the relationship between the density of Wolbachia in the mosquito and the level of parasite infection, quantitative PCR (qPCR) was performed on the carcasses of a subset of randomly chosen individual mosquitoes that survived 10 days pbm (replicate 1: wAlbB injected, n = 37; wMelPop injected, n = 34; replicate 2: wAlbB injected, n = 37; wMelPop injected, n = 46) according to the work of Hughes et al. (18). Briefly, DNA was extracted from the carcass of the mosquito using the Blood and Tissue DNA extraction kit (Qiagen). A fragment of the single-copy gene was amplified from wMelPop (WD_0550) and wAlbB (wsp) and normalized to a host reference gene (S7) to ascertain the relative abundance of Wolbachia in each carcass (18). Analysis of qPCR data was conducted using qGENE (20).

RESULTS

A. gambiae mosquitoes infected with diverse Wolbachia strains vary in their parasite load when challenged with P. berghei (Fig. 1). Pairwise contrasts showed that infection with wAlbB significantly increased oocyst numbers in A. gambiae midguts (P < 0.0001). Infection with wMelPop consistently decreased oocyst numbers in all replicates (as suggested by previous studies [21]), although in our experiment, these decreases were not statistically significant (P = 0.15). Replicate was significant in the model (P < 0.0001), but there was no significant treatment × replicate interaction (P = 0.98) (i.e., the trends were similar between replicates). Oocyst prevalence (percentage of mosquitoes with one or more oocysts) was not statistically affected by either Wolbachia strain (P > 0.05).

Fig 1.

Plasmodium berghei oocyst counts from midguts of Wolbachia-infected mosquitoes. Each dot represents an individual mosquito, and black horizontal lines represent median values. Mosquitoes were injected with either purified Wolbachia (wAlbB or wMelPop) or cell lysate from uninfected Mos55 cells. wAlbB-infected mosquitoes are significantly different from Mos55-injected control mosquitoes.

Recently, we reported that wMelPop dramatically reduces the survival of mosquitoes after feeding on human blood through a membrane feeder (18). Therefore, survival trajectories of Wolbachia-injected or control mosquitoes before and after feeding on P. berghei-infected mice were monitored. In pre-blood-fed mosquitoes, replicate was not significant in the model (P = 0.50) and there was no significant treatment × replicate interaction (P = 0.31), so replicate data were pooled for analysis. Wolbachia infection significantly affected pre-blood-fed mosquito survival (P < 0.0001). Pairwise contrasts indicated that mosquitoes injected with wAlbB had survival similar to control mosquitoes (P = 0.21) but mosquitoes injected with wMelPop had reduced survival compared to control mosquitoes (P < 0.0001) or wAlbB-injected mosquitoes (P = 0.0009) (see Fig. 3A). When we analyzed the survival of mosquitoes post-blood meal, replicate was significant in the model (P < 0.0001) but there was no significant treatment by replicate interaction (P = 0.21). Wolbachia infection significantly affected mosquito survival after feeding on mice (P < 0.0001). Pairwise contrasts showed that results were similar to those observed pre-blood meal: wAlbB-infected mosquitoes had similar mortality trajectories as control mosquitoes (P = 0.52) while wMelPop-infected mosquitoes had lower survival than either control or wAlbB-infected mosquitoes (P < 0.0001) (see Fig. 3B). While significantly reduced both pre- and post-blood meal, wMelPop-induced mortality was much more severe after blood feeding (see Fig. 3A and B).

Fig 3.

Survival trajectories of Wolbachia-infected mosquitoes. Life history of mosquitoes after Wolbachia injection and fed on sugar (A) and then given a blood meal on a P. berghei-infected mouse (B). The virulence of wMelPop is subtle when fed on sugar but becomes more dramatic post-blood meal, inducing approximately 50% mortality 3 days after feeding. There is no statistical difference between Mos55 and wAlbB fed on either diet. Different letters indicate statistical significance.

To determine if the density of Wolbachia was correlated with either the parasite load or the blood meal mortality phenotype, the titer of Wolbachia in the mosquito carcass of individuals that survived 10 days pbm was assessed. The density of wAlbB in mosquitoes that survived 10 days pbm was significantly higher than the density of wMelPop-infected mosquitoes (Fig. 2C). For both strains, in both replicates tested, there was no relationship between Wolbachia density and P. berghei levels (Fig. 2A and B). This was consistent with previous studies where neither wAlbB or wMelPop density was correlated with P. falciparum parasite load (18).

Fig 2.

qPCR analysis of the density of Wolbachia in the carcasses of mosquitoes compared to oocyst load for wAlbB (A)- and wMelPop (B)-injected mosquitoes. qPCR was completed on a subset of mosquito carcasses from replicates 1 and 2 that survived 10 days pbm (C). Wolbachia density is represented as Wolbachia genomes/host genomes.

DISCUSSION

Here we show that A. gambiae mosquitoes infected with diverse Wolbachia strains vary in their parasite load when challenged with P. berghei. In contrast to other studies that show pathogen inhibition due to Wolbachia infection, we show that wAlbB increases parasite load in somatically infected mosquitoes. The variation in response of P. berghei to wAlbB and wMelPop infection may be related to the influence of these strains on the immunity of the mosquito. Previously, we reported suppression of Anopheles immune genes by wAlbB in both cell culture and mosquitoes (18, 19), which may explain elevated parasite levels. Paradoxically, downregulation of immune transcripts was also observed in wMelPop-infected mosquitoes (18), yet parasite abundance was reduced by this Wolbachia strain (reference 21 and this study). If immunity affects P. berghei load in the mosquito, Wolbachia strain-specific variation in mosquito gene expression may explain these differences.

The wAlbB strain enhances P. berghei oocyst number in A. gambiae and yet suppresses P. falciparum (18). This differential response of the two Plasmodium parasites to wAlbB infection in mosquitoes is not surprising. It has been shown that Anopheles mosquitoes clear different parasites via specific immune pathways. The Toll pathway primarily regulates P. berghei, while the IMD pathway primarily regulates P. falciparum (14, 26). Dong et al. (7) demonstrated that P. berghei ookinete invasion of the midgut is far more complex and remarkably divergent from P. falciparum ookinete invasion and that P. berghei elicits a far greater immune response in the carcass than does P. falciparum. If pathogen interference is occurring in an immune-mediated manner, wAlbB may not modulate immune genes critical for clearance of P. berghei infection and yet affect genes related to P. falciparum resistance. P. berghei development also occurs at lower temperatures than does that of P. falciparum, which may influence Wolbachia infection dynamics and subsequent interactions within the mosquito.

Competition for cellular nutrients between Wolbachia and pathogens has been proposed as an alternative pathogen interference hypothesis (27). Although the metabolic requirements of Wolbachia and Plasmodium are poorly understood, in general both these organisms are amino acid heterotrophs (13, 40). In the blood stage of P. falciparum, glucose, amino acids, phosphate, and lipids have been identified as critical nutritional components (23, 34, 38). Supplementation experiments may elucidate key components of metabolic pathways critical for both the parasite and Wolbachia. Genomic evaluation of both the Wolbachia strains and the parasites may shed light on possible metabolites, either consumed or excreted, which may influence P. berghei oocyst intensity.

Previous studies suggest that there is a relationship between Wolbachia density and the strength of the antiviral phenotype in cell culture (10) and Drosophila (29). Here, we observed no correlation between the density of Wolbachia in the carcass after midgut dissection and oocyst number, similar to previous observations (18). However, Plasmodium parasites may modulate Wolbachia densities, leading to a more complex interaction. In Aedes albopictus, which is naturally infected with two Wolbachia strains, wAlbA and wAlbB, Chikungunya virus infection modulates bacterium levels, lowering the density of both strains (28, 37).

Our data suggest that wMelPop density influences mortality in injected Anopheles mosquitoes. The wMelPop-injected mosquitoes that survived 10 days pbm had significantly fewer Wolbachia organisms than did their wAlbB counterparts, suggesting that mosquitoes with high wMelPop titers did not survive this time period (Fig. 3). Previously, we saw that feeding wMelPop-infected A. gambiae on human blood elicits a similar phenotype, while Plasmodium infection had no observable effects on longevity (18). The mortality phenotype on human blood is more severe, perhaps due to adaptation of the Anopheles colony by continuous rearing on mouse blood or, alternatively, the different compositions of mouse and human blood. The source of blood influenced reproductive fitness in Aedes mosquitoes transinfected with wMelPop (24). The avirulence of wAlbB-injected mosquitoes after a blood meal suggests that this strain may be more suitable for creation of a stable line using adult microinjection.

Similarly to recently published work (21), we observed a consistent reduction of P. berghei oocysts in wMelPop-somatically infected mosquitoes compared to controls; however, our results were not statistically significant. These differences could be explained by differences in the mosquito genotypes or microflora between the two studies (5, 6, 8). In addition, the controls differed between the studies; our experiments used uninfected cell lysate while Kambris et al. (21) used cell culture medium.

This study confirms the utility of somatic infections to assay Wolbachia-mediated pathogen interference in insect species that are naturally uninfected with the symbiont. Somatic infections provide an amenable system to allow for the rapid assessment of pathogen interference phenotypes, enabling a comparison of different Wolbachia strains, host backgrounds, and pathogens. The variation in responses of P. berghei to divergent Wolbachia strains underpins the importance of studying relevant human disease systems rather than model malaria species before developing a stable Wolbachia-infected Anopheles line for disease control applications. The different responses of P. falciparum and P. berghei to wAlbB suggest that even closely related pathogens may elicit varied responses. Lastly, the disparity between different Wolbachia strains and parasites may be exploited to tease apart the molecular mechanism(s) causing pathogen interference against Plasmodium in Anopheles mosquitoes.