Genome sequencing and comparative analysis of Wolbachia strain wAlbA reveals Wolbachia-associated plasmids are common.

Wolbachia are widespread maternally-transmitted bacteria of arthropods that often spread by manipulating their host's reproduction through cytoplasmic incompatibility (CI). Their invasive potential is currently being harnessed in field trials aiming to control mosquito-borne diseases. Wolbachia genomes commonly harbour prophage regions encoding the cif genes which confer their ability to induce CI. Recently, a plasmid-like element was discovered in wPip, a Wolbachia strain infecting Culex mosquitoes; however, it is unclear how common such extra-chromosomal elements are in Wolbachia. Here we sequenced the complete genome of wAlbA, a strain of the symbiont found in Aedes albopictus, after eliminating the co-infecting and higher density wAlbB strain that previously made sequencing of wAlbA challenging. We show that wAlbA is associated with two new plasmids and identified additional Wolbachia plasmids and related chromosomal islands in over 20% of publicly available Wolbachia genome datasets. These plasmids encode a variety of accessory genes, including several phage-like DNA packaging genes as well as genes potentially contributing to host-symbiont interactions. In particular, we recovered divergent homologues of the cif genes in both Wolbachia- and Rickettsia-associated plasmids. Our results indicate that plasmids are common in Wolbachia and raise fundamental questions around their role in symbiosis. In addition, our comparative analysis provides useful information for the future development of genetic tools to manipulate and study Wolbachia symbionts.

Wolbachia strain wAlbB shows favourable characteristics for dengue control use in Aedes aegypti from Burkina Faso.

Dengue represents an increasing public health burden worldwide. In Africa, underreporting and misdiagnosis often mask its true epidemiology, and dengue is likely to be both more widespread than reported data suggest and increasing in incidence and distribution. Wolbachia-based dengue control is underway in Asia and the Americas but has not to date been deployed in Africa. Due to the genetic heterogeneity of African Aedes aegypti populations and the complexity of the host-symbiont interactions, characterization of key parameters of Wolbachia-carrying mosquitoes is paramount for determining the potential of the system as a control tool for dengue in Africa. The wAlbB Wolbachia strain was stably introduced into an African Ae. aegypti population by introgression, and showed high intracellular density in whole bodies and different mosquito tissues; high intracellular density was also maintained following larval rearing at high temperatures. No effect on the adult lifespan induced by Wolbachia presence was detected. Moreover, the ability of this strain to strongly inhibit DENV-2 dissemination and transmission in the host was also demonstrated in the African background. Our findings suggest the potential of harnessing Wolbachia for dengue control for African populations of Ae. aegypti.

Genomic and Phenotypic Comparisons Reveal Distinct Variants of Wolbachia Strain wAlbB.

The intracellular bacterium Wolbachia inhibits virus replication and is being harnessed around the world to fight mosquito-borne diseases through releases of mosquitoes carrying the symbiont. Wolbachia strains vary in their ability to invade mosquito populations and suppress viruses in part due to differences in their density within the insect and associated fitness costs. Using whole-genome sequencing, we demonstrate the existence of two variants in wAlbB, a Wolbachia strain being released in natural populations of Aedes aegypti mosquitoes. The two variants display striking differences in genome architecture and gene content. Differences in the presence/absence of 52 genes between variants include genes located in prophage regions and others potentially involved in controlling the symbiont's density. Importantly, we show that these genetic differences correlate with variation in wAlbB density and its tolerance to heat stress, suggesting that different wAlbB variants may be better suited for field deployment depending on local environmental conditions. Finally, we found that the wAlbB genome remained stable following its introduction in a Malaysian mosquito population. Our results highlight the need for further genomic and phenotypic characterization of Wolbachia strains in order to inform ongoing Wolbachia-based programs and improve the selection of optimal strains in future field interventions. IMPORTANCE Dengue is a viral disease transmitted by Aedes mosquitoes that threatens around half of the world population. Recent advances in dengue control involve the introduction of Wolbachia bacterial symbionts with antiviral properties into mosquito populations, which can lead to dramatic decreases in the incidence of the disease. In light of these promising results, there is a crucial need to better understand the factors affecting the success of such strategies, in particular the choice of Wolbachia strain for field releases and the potential for evolutionary changes. Here, we characterized two variants of a Wolbachia strain used for dengue control that differ at the genomic level and in their ability to replicate within the mosquito. We also found no evidence for the evolution of the symbiont within the 2 years following its deployment in Malaysia. Our results have implications for current and future Wolbachia-based health interventions.

TRIM69 Inhibits Vesicular Stomatitis Indiana Virus.

Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.IMPORTANCE Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.

The Wolbachia strain wAu provides highly efficient virus transmission blocking in Aedes aegypti.

Introduced transinfections of the inherited bacteria Wolbachia can inhibit transmission of viruses by Aedes mosquitoes, and in Ae. aegypti are now being deployed for dengue control in a number of countries. Only three Wolbachia strains from the large number that exist in nature have to date been introduced and characterized in this species. Here novel Ae. aegypti transinfections were generated using the wAlbA and wAu strains. In its native Ae. albopictus, wAlbA is maintained at lower density than the co-infecting wAlbB, but following transfer to Ae. aegypti the relative strain density was reversed, illustrating the strain-specific nature of Wolbachia-host co-adaptation in determining density. The wAu strain also reached high densities in Ae. aegypti, and provided highly efficient transmission blocking of dengue and Zika viruses. Both wAu and wAlbA were less susceptible than wMel to density reduction/incomplete maternal transmission resulting from elevated larval rearing temperatures. Although wAu does not induce cytoplasmic incompatibility (CI), it was stably combined with a CI-inducing strain as a superinfection, and this would facilitate its spread into wild populations. Wolbachia wAu provides a very promising new option for arbovirus control, particularly for deployment in hot tropical climates.

Wolbachia Modulates Lipid Metabolism in Aedes albopictus Mosquito Cells.

UNLABELLED: Certain strains of the intracellular endosymbiont Wolbachia can strongly inhibit or block the transmission of viruses such as dengue virus (DENV) by Aedes mosquitoes, and the mechanisms responsible are still not well understood. Direct infusion and liquid chromatography-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry-based lipidomics analyses were conducted using Aedes albopictus Aa23 cells that were infected with the wMel and wMelPop strains of Wolbachia in comparison to uninfected Aa23-T cells. Substantial shifts in the cellular lipid profile were apparent in the presence of Wolbachia Most significantly, almost all sphingolipid classes were depleted, and some reductions in diacylglycerols and phosphatidylcholines were also observed. These lipid classes have previously been shown to be selectively enriched in DENV-infected mosquito cells, suggesting that Wolbachia may produce a cellular lipid environment that is antagonistic to viral replication. The data improve our understanding of the intracellular interactions between Wolbachia and mosquitoes. IMPORTANCE: Mosquitoes transmit a variety of important viruses to humans, such as dengue virus and Zika virus. Certain strains of the intracellular bacterial genus called Wolbachia found in or introduced into mosquitoes can block the transmission of viruses, including dengue virus, but the mechanisms responsible are not well understood. We found substantial shifts in the cellular lipid profiles in the presence of these bacteria. Some lipid classes previously shown to be enriched in dengue virus-infected mosquito cells were depleted in the presence of Wolbachia, suggesting that Wolbachia may produce a cellular lipid environment that inhibits mosquito-borne viruses.

Transcriptional regulation of Culex pipiens mosquitoes by Wolbachia influences cytoplasmic incompatibility.

Cytoplasmic incompatibility (CI) induced by the endosymbiont Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility.

Wolbachia strain wMel induces cytoplasmic incompatibility and blocks dengue transmission in Aedes albopictus.

Wolbachia inherited bacteria are able to invade insect populations using cytoplasmic incompatibility and provide new strategies for controlling mosquito-borne tropical diseases, such as dengue. The overreplicating wMelPop strain was recently shown to strongly inhibit the replication of dengue virus when introduced into Aedes aegypti mosquitoes, as well as to stimulate chronic immune up-regulation. Here we show that stable introduction of the wMel strain of Drosophila melanogaster into Aedes albopictus, a vector of dengue and other arboviruses, abolished the transmission capacity of dengue virus-challenged mosquitoes. Immune up-regulation was observed in the transinfected line, but at a much lower level than that previously found for transinfected Ae. aegypti. Transient infection experiments suggest that this difference is related to Ae. albopictus immunotolerance of Wolbachia, rather than to the Wolbachia strain used. This study provides an example of strong pathogen inhibition in a naturally Wolbachia-infected mosquito species, demonstrating that this inhibition is not limited to naturally naïve species, and suggests that the Wolbachia strain is more important than host background for viral inhibition. Complete bidirectional cytoplasmic incompatibility was observed with WT strains infected with the naturally occurring Ae. albopictus Wolbachia, and this provides a mechanism for introducing wMel into natural populations of this species.

Comparative genome analysis of Wolbachia strain wAu.

BACKGROUND: Wolbachia intracellular bacteria can manipulate the reproduction of their arthropod hosts, including inducing sterility between populations known as cytoplasmic incompatibility (CI). Certain strains have been identified that are unable to induce or rescue CI, including wAu from Drosophila. Genome sequencing and comparison with CI-inducing related strain wMel was undertaken in order to better understand the molecular basis of the phenotype. RESULTS: Although the genomes were broadly similar, several rearrangements were identified, particularly in the prophage regions. Many orthologous genes contained single nucleotide polymorphisms (SNPs) between the two strains, but a subset containing major differences that would likely cause inactivation in wAu were identified, including the absence of the wMel ortholog of a gene recently identified as a CI candidate in a proteomic study. The comparative analyses also focused on a family of transcriptional regulator genes implicated in CI in previous work, and revealed numerous differences between the strains, including those that would have major effects on predicted function. CONCLUSIONS: The study provides support for existing candidates and novel genes that may be involved in CI, and provides a basis for further functional studies to examine the molecular basis of the phenotype.

Invertebrate post-segregation distorters: a new embryo-killing gene.

Cytoplasmic incompatibility induced by inherited intracellular bacteria of arthropods, and Medea elements found in flour beetles, are both forms of postsegregation distortion involving the killing of embryos in order to increase the ratio of progeny that inherit them. The recently described peel-zeel element of Caenorhabditis elegans also uses this mechanism; like Medea the genes responsible are in the nuclear genome but it shares a paternal mode of action with the bacteria. The peel-1 gene has now been shown to encode a potent toxin that is delivered by sperm, and rescued by zygotic transcription of the linked zeel-1. The predominance of self-fertilization in C. elegans has produced an unusual distribution pattern for a selfish genetic element; further population and functional studies will shed light on its evolution. The element might also have potential for use in disease control.

Evidence of Differences in Cellular Regulation of Wolbachia-Mediated Viral Inhibition between Alphaviruses and Flaviviruses.

The intracellular bacterium Wolbachia is increasingly being utilised in control programs to limit the spread of arboviruses by Aedes mosquitoes. Achieving a better understanding of how Wolbachia strains can reduce viral replication/spread could be important for the long-term success of such programs. Previous studies have indicated that for some strains of Wolbachia, perturbations in lipid metabolism and cholesterol storage are vital in Wolbachia-mediated antiviral activity against the flaviviruses dengue and Zika; however, it has not yet been examined whether arboviruses in the alphavirus group are affected in the same way. Here, using the reporters for the alphavirus Semliki Forest virus (SFV) in Aedes albopictus cells, we found that Wolbachia strains wMel, wAu and wAlbB blocked viral replication/translation early in infection and that storage of cholesterol in lipid droplets is not key to this inhibition. Another alphavirus, o'nyong nyong virus (ONNV), was tested in both Aedes albopictus cells and in vivo in stable, transinfected Aedes aegypti mosquito lines. The strains wMel, wAu and wAlbB show strong antiviral activity against ONNV both in vitro and in vivo. Again, 2-hydroxypropyl-ß-cyclodextrin (2HPCD) was not able to rescue ONNV replication in cell lines, suggesting that the release of stored cholesterol caused by wMel is not able to rescue blockage of ONNV. Taken together, this study shows that alphaviruses appear to be inhibited early in replication/translation and that there may be differences in how alphaviruses are inhibited by Wolbachia in comparison to flaviviruses.

Transgenic expression of cif genes from Wolbachia strain wAlbB recapitulates cytoplasmic incompatibility in Aedes aegypti.

The endosymbiotic bacteria Wolbachia can invade insect populations by modifying host reproduction through cytoplasmic incompatibility (CI), an effect that results in embryonic lethality when Wolbachia-carrying males mate with Wolbachia-free females. Here we describe a transgenic system for recreating CI in the major arbovirus vector Aedes aegypti using CI factor (cif) genes from wAlbB, a Wolbachia strain currently being deployed to reduce dengue transmission. CI-like sterility is induced when cifA and cifB are co-expressed in testes; this sterility is rescued by maternal cifA expression, thereby reproducing the pattern of Wolbachia-induced CI. Expression of cifB alone is associated with extensive DNA damage and disrupted spermatogenesis. The strength of rescue by maternal cifA expression is dependent on the comparative levels of cifA/cifB expression in males. These findings are consistent with CifB acting as a toxin and CifA as an antitoxin, with CifA attenuating CifB toxicity in both the male germline and in developing embryos. These findings provide important insights into the interactions between cif genes and their mechanism of activity and provide a foundation for the building of a cif gene-based drive system in Ae. aegypti.

Introduction of Aedes aegypti mosquitoes carrying wAlbB Wolbachia sharply decreases dengue incidence in disease hotspots.

Partial replacement of resident Aedes aegypti mosquitoes with introduced mosquitoes carrying certain strains of inherited Wolbachia symbionts can result in transmission blocking of dengue and other viruses of public health importance. Wolbachia strain wAlbB is an effective transmission blocker and stable at high temperatures, making it particularly suitable for hot tropical climates. Following trial field releases in Malaysia, releases using wAlbB Ae. aegypti have become operationalized by the Malaysian health authorities. We report here on an average reduction in dengue fever of 62.4% (confidence intervals 50-71%) in 20 releases sites when compared to 76 control sites in high-rise residential areas. Importantly the level of dengue reduction increased with Wolbachia frequency, with 75.8% reduction (61-87%) estimated at 100% Wolbachia frequency. These findings indicate large impacts of wAlbB Wolbachia invasions on dengue fever incidence in an operational setting, with incidence expected to further decrease as wider areas are invaded.

Population studies of the filarial vector Aedes polynesiensis (Diptera: Culicidae) in two island settings of French Polynesia.

A mark-release-recapture study was conducted to estimate the adult population size, migration, and dispersal patterns of male and female Aedes (Stegomyia) polynesiensis (Marks) in a valley of Moorea, a volcanic island, and a motu (islet) on the atoll of Tetiaroa, two settings typical of the Society Islands. Aedes polynesiensis recapture rate was high for females and low for males. The distribution of Aedes species in the valley was heterogeneous. Marked individuals dispersed to most parts of the motu and over great distances in the valley for some females. The study provides insights into the field dynamics of Ae. polynesiensis populations and confirms that more efficient sampling methods are warranted. There was no evidence of active migration between motus on the atoll, suggesting that Tetiaroa is a suitable site for small-scale initial open releases of Wolbachia incompatible insect technique and other sterile insect technique-like suppression or replacement strategies.

Field evaluation of selected traps and lures for monitoring the filarial and arbovirus vector, Aedes polynesiensis (Diptera: Culicidae), in French Polynesia.

The efficacy of the BG-Sentinel (BGS) and the BG-Mosquitito (BGM) mosquito traps for sampling populations of the important filariasis and dengue vector Aedes (Stegomyia) polynesiensis (Marks) was evaluated in French Polynesia against human bait collections (HBC) using a modified Centers for Disease Control and Prevention backpack aspirator. Traps were baited with BG-Lure (a combination of lactic acid, ammonia, and caproic acid) or carbon dioxide plus octenol (1-octen-3-ol) known as attractants to aedine mosquitoes. Mosquito sampling was conducted on two typical islands of French Polynesia: the high, volcanic island of Moorea, and the low, coral island (atoll) of Tetiaroa Sampling efficacy was measured in a randomized Latin Square design. Production of carbon dioxide from yeast-sugar fermentation was used as an alternative source of CO2 because supply via dry ice, gas cylinders, or propane combustion in remote tropical islands is costly and challenging. Although the BGS trap captured the greatest number ofAe. polynesiensis in both island settings, catch rates of BGS or BGM baited with either lure were not significantly different from that of HBC. On Moorea, the number of collected aedes species in the BGS trap baited with either lure was significantly greater than the BGM with BG-lure. On Tetiaroa, BGM trapping was severely hampered by damage from rats, and the traps were removed from the study. Our study confirms the efficiency, comparability, and convenience of the BGS trap, a robust and safe alternative to HBC for sampling Aedes mosquitoes in research and surveillance efforts against filariasis and arboviruses in the South Pacific.

Wolbachia-Virus interactions and arbovirus control through population replacement in mosquitoes.

Following transfer into the primary arbovirus vector Aedes aegypti, several strains of the intracellular bacterium Wolbachia have been shown to inhibit the transmission of dengue, Zika, and chikungunya viruses, important human pathogens that cause significant morbidity and mortality worldwide. In addition to pathogen inhibition, many Wolbachia strains manipulate host reproduction, resulting in an invasive capacity of the bacterium in insect populations. This has led to the deployment of Wolbachia as a dengue control tool, and trials have reported significant reductions in transmission in release areas. Here, we discuss the possible mechanisms of Wolbachia-virus inhibition and the implications for long-term success of dengue control. We also consider the evidence presented in several reports that Wolbachia may cause an enhancement of replication of certain viruses under particular conditions, and conclude that these should not cause any concerns with respect to the application of Wolbachia to arbovirus control.

Evaluation of an Engineered Zika Virus-Like Particle Vaccine Candidate in a Mosquito-Mouse Transmission Model.

The primary route of Zika virus (ZIKV) transmission is through the bite of an infected Aedes mosquito, when it probes the skin of a vertebrate host during a blood meal. Viral particles are injected into the bite site together with mosquito saliva and a complex mixture of other components. Some of them are known to play a key role in the augmentation of the arbovirus infection in the host, with increased viremia and/or morbidity. This vector-derived contribution to the infection is not usually considered when vaccine candidates are tested in preclinical animal models. In this study, we performed a preclinical validation of a promising ZIKV vaccine candidate in a mosquito-mouse transmission model using both Asian and African ZIKV lineages. Mice were immunized with engineered ZIKV virus-like particles and subsequently infected through the bite of ZIKV-infected Aedes aegypti mosquitoes. Despite a mild increase in viremia in mosquito-infected mice compared to those infected through traditional needle injection, the vaccine protected the animals from developing the disease and strongly reduced viremia. In addition, during peak viremia, naive mosquitoes were allowed to feed on infected vaccinated and nonvaccinated mice. Our analysis of viral titers in mosquitos showed that the vaccine was able to inhibit virus transmission from the host to the vector. IMPORTANCE Zika is a mosquito-borne viral disease, causing acute debilitating symptoms and complications in infected individuals and irreversible neuronal abnormalities in newborn children. The primary vectors of ZIKV are Aedes aegypti mosquitoes. Despite representing a significant public health burden with a widespread transmission in many regions of the world, Zika remains a neglected disease with no effective antiviral therapies or approved vaccines. It is known that components of the mosquito bite lead to an enhancement of viral infection and spread, but this aspect is often overlooked when vaccine candidates undergo preclinical validation. In this study, we included mosquitoes as viral vectors, demonstrating the ability of a promising vaccine candidate to protect animals against ZIKV infections after the bite of an infected mosquito and to also prevent its further transmission. These findings represent an additional crucial step for the development of an effective prevention tool for clinical use.

Differences in proteome perturbations caused by the Wolbachia strain wAu suggest multiple mechanisms of Wolbachia-mediated antiviral activity.

Some strains of the inherited bacterium Wolbachia have been shown to be effective at reducing the transmission of dengue virus (DENV) and other RNA viruses by Aedes aegypti in both laboratory and field settings and are being deployed for DENV control. The degree of virus inhibition varies between Wolbachia strains. Density and tissue tropism can contribute to these differences but there are also indications that this is not the only factor involved: for example, strains wAu and wAlbA are maintained at similar intracellular densities but only wAu produces strong DENV inhibition. We previously reported perturbations in lipid transport dynamics, including sequestration of cholesterol in lipid droplets, with strains wMel/wMelPop in Ae. aegypti. To further investigate the cellular basis underlying these differences, proteomic analysis of midguts was carried out on Ae. aegypti lines carrying strains wAu and wAlbA: with the hypothesis that differences in perturbations may underline Wolbachia-mediated antiviral activity. Surprisingly, wAu-carrying midguts not only showed distinct proteome perturbations when compared to non-Wolbachia carrying and wAlbA-carrying midguts but also wMel-carrying midguts. There are changes in RNA processing pathways and upregulation of a specific set of RNA-binding proteins in the wAu-carrying line, including genes with known antiviral activity. Lipid transport and metabolism proteome changes also differ between strains, and we show that strain wAu does not produce the same cholesterol sequestration phenotype as wMel. Moreover, in contrast to wMel, wAu antiviral activity was not rescued by cyclodextrin treatment. Together these results suggest that wAu could show unique features in its inhibition of arboviruses compared to previously characterized Wolbachia strains.

Spatial Distribution and Long-Term Persistence of Wolbachia-Infected Aedes aegypti in the Mentari Court, Malaysia.

Dengue is endemic in Malaysia, and vector control strategies are vital to reduce dengue transmission. The Wolbachia strain wAlbB carried by both sexes of Ae. aegypti was released in Mentari Court, a high-rise residential site, in October 2017 and stopped after 20 weeks. Wolbachia frequencies are still being monitored at multiple traps across this site, providing an opportunity to examine the spatiotemporal distribution of Wolbachia and mosquito density with respect to year, residential block, and floor, using spatial interpolation in ArcGIS, GLMs, and contingency analyses. In just 12 weeks, Wolbachia-infected mosquitoes were established right across the Mentari Court site with an overall infection frequency of >90%. To date, the Wolbachia frequency of Ae. aegypti has remained high in all areas across the site despite releases finishing four years ago. Nevertheless, the Wolbachia invaded more rapidly in some residential blocks than others, and also showed a relatively higher frequency on the eighth floor. The Ae. aegypti index tended to differ somewhat between residential blocks, whilst the Ae. albopictus index was relatively higher at the top and bottom floors of buildings. In Mentari Court, only a short release period was required to infiltrate Wolbachia completely and stably into the natural population. The results inform future releases in comparable sites in a dengue control programme.

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.