Wolbachia Induces Male-Specific Mortality in the Mosquito Culex pipiens (LIN Strain)

Background: Wolbachia are maternally inherited endosymbionts that infect a diverse range of invertebrates, including insects, arachnids, crustaceans and filarial nematodes. Wolbachia are responsible for causing diverse reproductive alterations in their invertebrate hosts that maximize their transmission to the next generation. Evolutionary theory suggests that due to maternal inheritance, Wolbachia should evolve toward mutualism in infected females, but strict maternal inheritance means there is no corresponding force to select for Wolbachia strains that are mutualistic in males. Methodology/Principal findings: Using cohort life-table analysis, we demonstrate that in the mosquito Culex pipiens (LIN strain), Wolbachia-infected females show no fitness costs due to infection. However, Wolbachia induces up to a 30% reduction in male lifespan. Conclusions/significance: These results indicate that the Wolbachia infection of the Culex pipiens LIN strain is virulent in a sex-specific manner. Under laboratory situations where mosquitoes generally mate at young ages, Wolbachia strains that reduce male survival could evolve by drift because increased mortality in older males is not a significant selective force

Viral Paratransgenesis in the Malaria Vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae

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

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

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

Wolbachia in the flesh: symbiont intensities in germ-line and somatic tissues challenge the conventional view of Wolbachia transmission routes

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought

Wolbachia infections that reduce immature insect survival: Predicted impacts on population replacement

<p>Abstract</p> <p>Background</p> <p>The evolutionary success of <it>Wolbachia </it>bacteria, infections of which are widespread in invertebrates, is largely attributed to an ability to manipulate host reproduction without imposing substantial fitness costs. Here, we describe a stage-structured model with deterministic immature lifestages and a stochastic adult female lifestage. Simulations were conducted to better understand <it>Wolbachia </it>invasions into uninfected host populations. The model includes conventional <it>Wolbachia </it>parameters (the level of cytoplasmic incompatibility, maternal inheritance, the relative fecundity of infected females, and the initial <it>Wolbachia </it>infection frequency) and a new parameter termed relative larval viability (<it>RLV</it>), which is the survival of infected larvae relative to uninfected larvae.</p> <p>Results</p> <p>The results predict the <it>RLV </it>parameter to be the most important determinant for <it>Wolbachia </it>invasion and establishment. Specifically, the fitness of infected immature hosts must be close to equal to that of uninfected hosts before population replacement can occur. Furthermore, minute decreases in <it>RLV </it>inhibit the invasion of <it>Wolbachia </it>despite high levels of cytoplasmic incompatibility, maternal inheritance, and low adult fitness costs.</p> <p>Conclusions</p> <p>The model described here takes a novel approach to understanding the spread of <it>Wolbachia </it>through a population with explicit dynamics. By combining a stochastic female adult lifestage and deterministic immature/adult male lifestages, the model predicts that even those <it>Wolbachia </it>infections that cause minor decreases in immature survival are unlikely to invade and spread within the host population. The results are discussed in relation to recent theoretical and empirical studies of natural population replacement events and proposed applied research, which would use <it>Wolbachia </it>as a tool to manipulate insect populations.</p

Wolbachia Infections in Anopheles gambiae Cells: Transcriptomic Characterization of a Novel Host-Symbiont Interaction

The endosymbiotic bacterium Wolbachia is being investigated as a potential control agent in several important vector insect species. Recent studies have shown that Wolbachia can protect the insect host against a wide variety of pathogens, resulting in reduced transmission of parasites and viruses. It has been proposed that compromised vector competence of Wolbachia-infected insects is due to up-regulation of the host innate immune system or metabolic competition. Anopheles mosquitoes, which transmit human malaria parasites, have never been found to harbor Wolbachia in nature. While transient somatic infections can be established in Anopheles, no stable artificially-transinfected Anopheles line has been developed despite numerous attempts. However, cultured Anopheles cells can be stably infected with multiple Wolbachia strains such as wAlbB from Aedes albopictus, wRi from Drosophila simulans and wMelPop from Drosophila melanogaster. Infected cell lines provide an amenable system to investigate Wolbachia-Anopheles interactions in the absence of an infected mosquito strain. We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes. Wolbachia infection had a dramatic strain-specific effect on gene expression in this cell line, with almost 700 genes in total regulated representing a diverse array of functional classes. Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts. This is in stark contrast to the induction of immune genes observed in other insect hosts. We also identified genes that may be potentially involved in Wolbachia-induced reproductive and pathogenic phenotypes. Somatically-infected mosquitoes had similar responses to cultured cells. The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria

African tropical rainforest net carbon dioxide fluxes in the twentieth century

The African humid tropical biome constitutes the second largest rainforest region, significantly impacts global carbon cycling and climate, and has undergone major changes in functioning owing to climate and land-use change over the past century. We assess changes and trends in CO2 fluxes from 1901 to 2010 using nine land surface models forced with common driving data, and depict the inter-model variability as the uncertainty in fluxes. The biome is estimated to be a natural (no disturbance) net carbon sink (−0.02 kg C m−2 yr−1 or −0.04 Pg C yr−1, p < 0.05) with increasing strength fourfold in the second half of the century. The models were in close agreement on net CO2 flux at the beginning of the century (σ1901 = 0.02 kg C m−2 yr−1), but diverged exponentially throughout the century (σ2010 = 0.03 kg C m−2 yr−1). The increasing uncertainty is due to differences in sensitivity to increasing atmospheric CO2, but not increasing water stress, despite a decrease in precipitation and increase in air temperature. However, the largest uncertainties were associated with the most extreme drought events of the century. These results highlight the need to constrain modelled CO2 fluxes with increasing atmospheric CO2 concentrations and extreme climatic events, as the uncertainties will only amplify in the next century

Cytoplasmic Incompatibility as a Means of Controlling Culex pipiens quinquefasciatus Mosquito in the Islands of the South-Western Indian Ocean

The use of the bacterium Wolbachia is an attractive alternative method to control vector populations. In mosquitoes, as in members of the Culex pipiens complex, Wolbachia induces a form of embryonic lethality called cytoplasmic incompatibility, a sperm-egg incompatibility occurring when infected males mate either with uninfected females or with females infected with incompatible Wolbachia strain(s). Here we explore the feasibility of the Incompatible Insect Technique (IIT), a species-specific control approach in which field females are sterilized by inundative releases of incompatible males. We show that the Wolbachia wPip(Is) strain, naturally infecting Cx. p. pipiens mosquitoes from Turkey, is a good candidate to control Cx. p. quinquefasciatus populations on four islands of the south-western Indian Ocean (La Réunion, Mauritius, Grande Glorieuse and Mayotte). The wPip(Is) strain was introduced into the nuclear background of Cx. p. quinquefasciatus mosquitoes from La Réunion, leading to the LR[wPip(Is)] line. Total embryonic lethality was observed in crosses between LR[wPip(Is)] males and all tested field females from the four islands. Interestingly, most crosses involving LR[wPip(Is)] females and field males were also incompatible, which is expected to reduce the impact of any accidental release of LR[wPip(Is)] females. Cage experiments demonstrate that LR[wPip(Is)] males are equally competitive with La Réunion males resulting in demographic crash when LR[wPip(Is)] males were introduced into La Réunion laboratory cages. These results, together with the geographic isolation of the four south-western Indian Ocean islands and their limited land area, support the feasibility of an IIT program using LR[wPip(Is)] males and stimulate the implementation of field tests for a Cx. p. quinquefasciatus control strategy on these islands