Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae

Endosymbiotic Wolbachia bacteria are potent modulators of pathogen infection and transmission in multiple naturally and artificially infected insect species, including important vectors of human pathogens. Anopheles mosquitoes are naturally uninfected with Wolbachia, and stable artificial infections have not yet succeeded in this genus. Recent techniques have enabled establishment of somatic Wolbachia infections in Anopheles. Here, we characterize somatic infections of two diverse Wolbachia strains (wMelPop and wAlbB) in Anopheles gambiae, the major vector of human malaria. After infection, wMelPop disseminates widely in the mosquito, infecting the fat body, head, sensory organs and other tissues but is notably absent from the midgut and ovaries. Wolbachia initially induces the mosquito immune system, coincident with initial clearing of the infection, but then suppresses expression of immune genes, coincident with Wolbachia replication in the mosquito. Both wMelPop and wAlbB significantly inhibit Plasmodium falciparum oocyst levels in the mosquito midgut. Although not virulent in non-bloodfed mosquitoes, wMelPop exhibits a novel phenotype and is extremely virulent for approximately 12-24 hours post-bloodmeal, after which surviving mosquitoes exhibit similar mortality trajectories to control mosquitoes. The data suggest that if stable transinfections act in a similar manner to somatic infections, Wolbachia could potentially be used as part of a strategy to control the Anopheles mosquitoes that transmit malaria.     

Wolbachia strain wAlbB enhances infection by the rodent malaria parasite Plasmodium berghei in Anopheles gambiae mosquitoes

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.     

Can Anopheles gambiae be infected with Wolbachia pipientis? Insights from an in vitro system

Wolbachia pipientis are maternally inherited endosymbionts associated with cytoplasmic incompatibility, a potential mechanism to drive transgenic traits into Anopheles populations for malaria control. W. pipientis infections are common in many mosquito genera but have never been observed in any Anopheles species, leading to the hypothesis that Anopheles mosquitoes are incapable of harboring infection. We used an in vitro system to evaluate the ability of Anopheles gambiae cells to harbor diverse W. pipientis infections. We successfully established W. pipientis infections (strains wRi and wAlbB) in the immunocompetent Anopheles gambiae cell line Sua5B. Infection was confirmed by PCR, antibiotic curing, DNA sequencing, and direct observation using fluorescence in situ hybridization. The infections were maintained at high passage rates for >30 passages. Our results indicate that there is no intrinsic genetic block to W. pipientis infection in A. gambiae cells, suggesting that establishment of in vivo W. pipientis infections in Anopheles mosquitoes may be feasible.     

Genetic and functional characterization of the type IV secretion system in Wolbachia

A type IV secretion system (T4SS) is used by many symbiotic and pathogenic intracellular bacteria for the successful infection of and survival, proliferation, and persistence within hosts. In this study, the presence and function of the T4SS in Wolbachia strains were investigated by a combination of genetic screening and immunofluorescence microscopy. Two operons of virB-virD4 loci were found in the genome of Wolbachia pipientis strain wAtab3, from the Hymenoptera Asobara tabida, and strain wRi, infecting Drosophila simulans. One operon consisted of five vir genes (virB8, virB9, virB10, virB11, and virD4) and the downstream wspB locus. The other operon was composed of three genes (virB3, virB4, and virB6) and included four additional open reading frames (orf1 to orf4) orientated in the same direction. In cell culture and insect hosts infected with different Wolbachia strains, the bona fide vir genes were polycistronically transcribed, together with the downstream adjacent loci, notably, as virB8 to virD4 and wspB and as virB3, virB4, virB6, and orf1 to orf4. Two peptides encompassing conserved C and N termini of the Wolbachia VirB6 protein were used for the production of polyclonal antibodies. Anti-VirB6 antibodies could detect the corresponding recombinant protein by chemifluorescence on Western blots of total proteins from Escherichia coli transformants and Wolbachia strains cultured in cell lines. Using immunofluorescence microscopy, we further demonstrated that the VirB6 protein was produced by Wolbachia strains in ovaries of insects harboring wAtab3 or wRi and cell lines infected with wAlbB or wMelPop. As VirB6 is known to associate with other VirB proteins to form a membrane-spanning structure, this finding suggests that a T4SS may function in Wolbachia.     

Interspecific transfer of Wolbachia into the mosquito disease vector Aedes albopictus

Intracellular Wolbachia bacteria are obligate, maternally inherited endosymbionts found frequently in insects and other invertebrates. The evolutionary success of Wolbachia is due in part to an ability to manipulate reproduction. In mosquitoes and many other insects, Wolbachia causes a form of sterility known as cytoplasmic incompatibility (CI). Wolbachia-induced CI has attracted interest as a potential agent for affecting medically important disease vectors. However, application of the approach has been restricted by an absence of appropriate, naturally occurring Wolbachia infections. Here, we report the interspecific transfer of Wolbachia infection into a medically important mosquito. Using embryonic microinjection, Wolbachia is transferred from Drosophila simulans into the invasive pest and disease vector: Aedes albopictus (Asian tiger mosquito). The resulting infection is stably maintained and displays a unique pattern of bidirectional CI in crosses with naturally infected mosquitoes. Laboratory population cage experiments examine a strategy in which releases of Wolbachia-infected males are used to suppress mosquito egg hatch. We discuss the results in relation to developing appropriate Wolbachia-infected mosquito strains for population replacement and population suppression strategies.     

Sperm chromatin remodelling and Wolbachia-induced cytoplasmic incompatibility in Drosophila.

Wolbachia pipientis is an obligate bacterial endosymbiont, which has successfully invaded approximately 20% of all insect species by manipulating their normal developmental patterns. Wolbachia-induced phenotypes include parthenogenesis, male killing, and, most notably, cytoplasmic incompatibility. In the future these phenotypes might be useful in controlling or modifying insect populations but this will depend on our understanding of the basic molecular processes underlying insect fertilization and development. Wolbachia-infected Drosophila simulans express high levels of cytoplasmic incompatibility in which the sperm nucleus is modified and does not form a normal male pronucleus when fertilizing eggs from uninfected females. The sperm modification is somehow rescued in eggs infected with the same strain of Wolbachia. Thus, D. simulans has become an excellent model organism for investigating the manner in which endosymbionts can alter reproductive programs in insect hosts. This paper reviews the current knowledge of Drosophila early development and particularly sperm function. Developmental mutations in Drosophila that are known to affect sperm function will also be discussed.incompatibility.     

Invasion of Wolbachia into Anopheles and other insect germlines in an ex vivo organ culture system

The common bacterial endosymbiont Wolbachia manipulates its host's reproduction to promote its own maternal transmission, and can interfere with pathogen development in many insects making it an attractive agent for the control of arthropod-borne disease. However, many important species, including Anopheles mosquitoes, are uninfected. Wolbachia can be artificially transferred between insects in the laboratory but this can be a laborious and sometimes fruitless process. We used a simple ex vivo culturing technique to assess the suitability of Wolbachia-host germline associations. Wolbachia infects the dissected germline tissue of multiple insect species when the host tissue and bacteria are cultured together. Ovary and testis infection occurs in a density-dependent manner. Wolbachia strains are more capable of invading the germline of their native or closely related rather than divergent hosts. The ability of Wolbachia to associate with the germline of novel hosts is crucial for the development of stably-transinfected insect lines. Rapid assessment of the suitability of a strain-host combination prior to transinfection may dictate use of a particular Wolbachia strain. Furthermore, the cultured germline tissues of two major Anopheline vectors of Plasmodium parasites are susceptible to Wolbachia infection. This finding further enhances the prospect of using Wolbachia for the biological control of malaria.     

Generation of a novel Wolbachia infection in Aedes albopictus (Asian tiger mosquito) via embryonic microinjection

Genetic strategies that reduce or block pathogen transmission by mosquitoes are being investigated as a means to augment current control measures. Strategies of vector suppression and replacement are based upon intracellular Wolbachia bacteria, which occur naturally in many insect populations. Maternally inherited Wolbachia have evolved diverse mechanisms to manipulate host insect reproduction and promote infection invasion. One mechanism is cytoplasmic incompatibility (CI) through which Wolbachia promotes infection spread by effectively sterilizing uninfected females. In a prior field test, releases of Wolbachia-infected males were used to suppress a field population of Culex pipiens. An additional strategy would employ Wolbachia as a vehicle to drive desired transgenes into vector populations (population replacement). Wolbachia-based population suppression and population replacement strategies require an ability to generate artificial Wolbachia associations in mosquitoes. Here, we demonstrate a technique for transferring Wolbachia (transfection) in a medically important mosquito species: Aedes albopictus (Asian tiger mosquito). Microinjection was used to transfer embryo cytoplasm from a double-infected Ae. albopictus line into an aposymbiotic line. The resulting mosquito line is single-infected with the wAlbB Wolbachia type. The artificially generated infection type is not known to occur naturally and displays a new CI crossing type and the first known example of bidirectional CI in Aedes mosquitoes. We discuss the results in relation to applied mosquito control strategies and the evolution of Wolbachia infections in Ae. albopictus.     

Immune responses in Anopheles gambiae

Transmission of human malaria requires a successful development of Plasmodium parasites in anopheline mosquitoes. Insects have developed efficient immune responses to oppose microbial and eukaryotic invaders. The completion of the sequencing of the Anopheles genome provides a wealth of information on putative immune genes that are homologous to components of the Drosophila and mammalian immune systems. In this review, we will summarize our present knowledge of immune responses in the mosquito Anopheles gambiae and attempt a comparative analysis of insect immune systems.     

Can Wolbachia be used to control malaria?

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.     

Wolbachia stimulates immune gene expression and inhibits plasmodium development in Anopheles gambiae

The over-replicating wMelPop strain of the endosymbiont Wolbachia pipientis has recently been shown to be capable of inducing immune upregulation and inhibition of pathogen transmission in Aedes aegypti mosquitoes. In order to examine whether comparable effects would be seen in the malaria vector Anopheles gambiae, transient somatic infections of wMelPop were created by intrathoracic inoculation. Upregulation of six selected immune genes was observed compared to controls, at least two of which (LRIM1 and TEP1) influence the development of malaria parasites. A stably infected An. gambiae cell line also showed increased expression of malaria-related immune genes. Highly significant reductions in Plasmodium infection intensity were observed in the wMelPop-infected cohort, and using gene knockdown, evidence for the role of TEP1 in this phenotype was obtained. Comparing the levels of upregulation in somatic and stably inherited wMelPop infections in Ae. aegypti revealed that levels of upregulation were lower in the somatic infections than in the stably transinfected line; inhibition of development of Brugia filarial nematodes was nevertheless observed in the somatic wMelPop infected females. Thus we consider that the effects observed in An. gambiae are also likely to be more pronounced if stably inherited wMelPop transinfections can be created, and that somatic infections of Wolbachia provide a useful model for examining effects on pathogen development or dissemination. The data are discussed with respect to the comparative effects on malaria vectorial capacity of life shortening and direct inhibition of Plasmodium development that can be produced by Wolbachia.     

The Plasmodium parasite--a 'new' challenge for insect innate immunity

Though lacking adaptive immunity, insects possess a powerful innate immune system, a genome-encoded defence machinery used to confront infections. Studies in the fruit fly Drosophila melanogaster revealed a remarkable capacity of the innate immune system to differentiate between and subsequently respond to different bacteria and fungi. However, hematophagous compared to non-hematophagous insects encounter additional blood-borne infectious agents, such as parasites and viruses, during their lifetime. Anopheles mosquitoes become infected with the malaria parasite Plasmodium during feeding on infected human hosts and may then transmit the parasite to new hosts during subsequent bites. Whether Anopheles has developed mechanisms to confront these infections is the subject of this review. Initially, we review our current understanding of innate immune reactions and give an overview of the Anopheles immune system as revealed through comparative genomic analyses. Then, we examine and discuss the capacity of mosquitoes to recognize and respond to infections, especially to Plasmodium, and finally, we explore approaches to investigate and potentially utilize the vector immune competence to prevent pathogen transmission. Such approaches constitute a new challenge for insect immunity research, a challenge for global health.     

Expression of cytoplasmic incompatibility in Drosophila simulans and its impact on infection frequencies and distribution of Wolbachia pipientis

The aim of this study is to examine the expression of cytoplasmic incompatibility and investigate the distribution and population frequencies of Wolbachia pipientis strains in Drosophila simulans. Nucleotide sequence data from 16S rDNA and a Wolbachia surface protein coding sequence and cytoplasmic incompatibility assays identify four distinct Wolbachia strains: wHa, wRi, wMa, and wAu. The levels of cytoplasmic incompatibility between six lines carrying these strains of bacteria and three control lines without bacteria are characterized. Flies infected with wHa and wRi are bidirectionally incompatible, and males that carry either strain can only successfully produce normal numbers of offspring with females carrying the same bacterial strain. Males infected with wAu do not express incompatibility. Males infected with the wMa strain express intermediate incompatibility when mated to females with no bacteria and no incompatibility with females with any other Wolbachia strain. We conduct polymerase chain reaction/restriction fragment length polymorphism assays to distinguish the strain of Wolbachia and the mitochondrial haplotype to survey populations for each type and associations between them. Drosophila simulans is known to have three major mitochondrial haplotypes (siI, sill, and siIII) and two subtypes (siIIA and siIIB). All infected lines of the sil haplotype carry wHa, wNo, or both; wMa and wNo are closely related and it is not clear whether they are distinct strains or variants of the same strain. Infected lines with the silIA haplotype harbor wRi and the siIIB haplotype carries wAu. The wMa infection is found in siIII haplotype lines. The phenotypic expression of cytoplasmic incompatibility and its relation to between-population differences in frequencies of Wolbachia infection are discussed.     

No effect of Wolbachia on resistance to intracellular infection by pathogenic bacteria in Drosophila melanogaster

Multiple studies have shown that infection with the endosymbiotic bacterium Wolbachia pipientis confers Drosophila melanogaster and other insects with resistance to infection by RNA viruses. Studies investigating whether Wolbachia infection induces the immune system or confers protection against secondary bacterial infection have not shown any effect. These studies, however, have emphasized resistance against extracellular pathogens. Since Wolbachia lives inside the host cell, we hypothesized that Wolbachia might confer resistance to pathogens that establish infection by invading host cells. We therefore tested whether Wolbachia-infected D. melanogaster are protected against infection by the intracellular pathogenic bacteria Listeria monocytogenes and Salmonella typhimurium, as well as the extracellular pathogenic bacterium Providencia rettgeri. We evaluated the ability of flies infected with Wolbachia to suppress secondary infection by pathogenic bacteria relative to genetically matched controls that had been cured of Wolbachia by treatment with tetracycline. We found no evidence that Wolbachia alters host ability to suppress proliferation of any of the three pathogenic bacteria. Our results indicate that Wolbachia-induced antiviral protection does not result from a generalized response to intracellular pathogens.     

Natural Wolbachia infections in the Drosophila yakuba species complex do not induce cytoplasmic incompatibility but fully rescue the wRi modification

In this study, we report data about the presence of Wolbachia in Drosophila yakuba, D. teissieri, and D. santomea. Wolbachia strains were characterized using their wsp gene sequence and cytoplasmic incompatibility assays. All three species were found infected with Wolbachia bacteria closely related to the wAu strain, found so far in D. simulans natural populations, and were unable to induce cytoplasmic incompatibility. We injected wRi, a CI-inducing strain naturally infecting D. simulans, into the three species and the established transinfected lines exhibited high levels of CI, suggesting that absence of CI expression is a property of the Wolbachia strain naturally present or that CI is specifically repressed by the host. We also tested the relationship between the natural infection and wRi and found that it fully rescues the wRi modification. This result was unexpected, considering the significant evolutionary divergence between the two Wolbachia strains.     

Insect immunity and its implication in mosquito-malaria interactions

Insects' resistance to infectious agents is essential for their own survival and also for the health of the plant, animal and human populations with which they closely interact. Several of the major human diseases are spread by insects and are rapidly expanding as a result of the development of insecticide resistance in vectors and drug resistance in parasites. A vector insects' permissiveness to a pathogen, and hence the spread of the disease, will largely depend on the compatibility of the molecular interactions between the two species and the capability of the insect immune system to recognize and kill the pathogen. The innate immune system comprises a variety of components and mechanisms that can discriminate between different microorganisms and mount specific responses to control pathogenic infections. An impressive body of knowledge on the insects' innate immunity has been generated from studies in the model organism Drosophila. These studies are now guiding the exploration of the immune system in the vector mosquito of human malaria, Anopheles, and its implication in the elimination of parasites. Anopheles immune responses have been linked to parasite losses and some refractory mosquitoes can kill all parasites through specific defence mechanisms. The recently sequenced Drosophila and Anopheles genomes provide a detailed and comparative view on their immune gene repertoires that in combination with post-genomic analyses is used to further dissect the complex mechanisms of Plasmodium killing in the mosquito.     

Wolbachia-mediated antibacterial protection and immune gene regulation in Drosophila

The outcome of microbial infection of insects is dependent not only on interactions between the host and pathogen, but also on the interactions between microbes that co-infect the host. Recently the maternally inherited endosymbiotic bacteria Wolbachia has been shown to protect insects from a range of microbial and eukaryotic pathogens. Mosquitoes experimentally infected with Wolbachia have upregulated immune responses and are protected from a number of pathogens including viruses, bacteria, Plasmodium and filarial nematodes. It has been hypothesised that immune upregulation underpins Wolbachia-mediated protection. Drosophila is a strong model for understanding host-Wolbachia-pathogen interactions. Wolbachia-mediated antiviral protection in Drosophila has been demonstrated for a number of different Wolbachia strains. In this study we investigate whether Wolbachia-infected flies are also protected against pathogenic bacteria. Drosophila simulans lines infected with five different Wolbachia strains were challenged with the pathogenic bacteria Pseudomonas aeruginosa PA01, Serratia marcescens and Erwinia carotovora and mortality compared to paired lines without Wolbachia. No difference in mortality was observed in the flies with or without Wolbachia. Similarly no antibacterial protection was observed for D. melanogaster infected with Wolbachia. Interestingly, D. melanogaster Oregon RC flies which are naturally infected with Wolbachia showed no upregulation of the antibacterial immune genes TepIV, Defensin, Diptericin B, PGRP-SD, Cecropin A1 and Attacin D compared to paired flies without Wolbachia. Taken together these results indicate that Wolbachia-mediated antibacterial protection is not ubiquitous in insects and furthermore that the mechanisms of antibacterial and antiviral protection are independent. We suggest that the immune priming and antibacterial protection observed in Wolbachia-infected mosquitoes may be a consequence of the recent artificial introduction of the symbiont into insects that normally do not carry Wolbachia and that antibacterial protection is unlikely to be found in insects carrying long-term Wolbachia infections.     

Impact of population age structure on Wolbachia transgene driver efficacy: ecologically complex factors and release of genetically modified mosquitoes

Wolbachia symbionts hold theoretical promise as a way to drive transgenes into insect vector populations for disease prevention. For simplicity, current models of Wolbachia dynamics and spread ignore ecologically complex factors such as the age structure of vector populations and overlapping vector generations. We developed a model including these factors to assess their impact on the process of Wolbachia spread into populations of three mosquito species (Anopheles gambiae, Aedes aegypti and Culex pipiens). Depending on the mosquito species, Wolbachia parameters, released mosquito life stage and initial age structure of the target population, the number of Wolbachia-infected mosquitoes that we predict would need to be released ranged from less than the threshold calculated by the simple model to a 10-30-fold increase. Transgenic releases into age-structured populations, which is an expectation for wild mosquitoes, will be difficult and depending on the circumstances may not be economically or logistically feasible due to the large number of infected mosquitoes that must be released. Our results support the perspective that understanding ecological factors is critical for designing transgenic vector-borne disease control strategies.     

Wolbachia infection alters olfactory-cued locomotion in Drosophila spp

Wolbachia pipientis is an endosymbiotic bacterium present in diverse insect species. Although it is well studied for its dramatic effects on host reproductive biology, little is known about its effects on other aspects of host biology, despite its presence in a wide array of host tissues. This study examined the effects of three Wolbachia strains on two different Drosophila species, using a laboratory performance assay for insect locomotion in response to olfactory cues. The results demonstrate that Wolbachia infection can have significant effects on host responsiveness that vary with respect to the Wolbachia strain-host species combination. The wRi strain, native to Drosophila simulans, increases the basal activity level of the host insect as well as its responsiveness to food cues. In contrast, the wMel strain and the virulent wMelPop strain, native to Drosophila melanogaster, cause slight decreases in responsiveness to food cues but do not alter basal activity levels in the host. Surprisingly, the virulent wMelPop strain has very little impact on host responsiveness in D. simulans. This novel strain-host relationship was artificially created previously by transinfection. These findings have implications for understanding the evolution and spread of Wolbachia infections in wild populations and for Wolbachia-based vector-borne disease control strategies currently being developed.     

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections.