Wolbachia wAlbB Research Articles

Infectious Diseases: Antiviral Wolbachia Limits Dengue in Malaysia

Vector-borne viral diseases pose an urgent public health challenge, particularly in the tropics. Field releases of mosquitoes carrying bacterial symbionts that reduce vector competence are ongoing in Kuala Lumpur, Malaysia. Early results show that wAlbB Wolbachia can persist in mosquitoes in urban settings and decrease dengue incidence in humans.

Effect of Wolbachia wAlbB on a positive-sense RNA negev-like virus: a novel virus persistently infecting Aedes albopictus mosquitoes and cells.

The Aedes aegypti mosquito is the primary vector of several medically important arboviruses. The endosymbiotic bacterium, Wolbachia pipientis, has emerged as a means of blocking transmission of arboviruses such as dengue and Zika viruses. One Wolbachia strain that has shown potential in field trials is wAlbB, a naturally occurring Wolbachia strain of the Asian tiger mosquito Aedes albopictus. When transinfected into Ae. aegypti, wAlbB exhibits strong virus inhibition. In addition to modulating arboviruses, Wolbachia also modulates some insect-specific viruses. Here, we explored the effect of Wolbachia on the virome of the Ae. albopictus cell line Aa23 naturally infected with wAlbB and also a stably transinfected recipient Ae. aegypti cell line (Aag2.wAlbB). RNA sequencing and bioinformatic analysis on both cell lines revealed an 11 kb genome of a single-stranded positive-sense RNA negev-like virus related to the recently proposed negevirus taxon. We denoted this novel virus as Aedes albopictus negev-like virus (AalNLV). Tetracycline clearance of Wolbachia from Aa23 cells did not significantly affect AalNLV levels, while in Aag2.wAlbB cells, a significant increase in virus genome RNA copies was observed. We further investigated the inhibitory effect of wAlbB on AalNLV and another positive-sense RNA virus, cell fusing agent virus, which is present in Aag2 cells and known to be suppressed by Wolbachia. wAlbB suppressed both viruses, with the effect on AalNLV being more striking. The findings from this study further supplement our understanding of the complex interaction between Wolbachia, host and virome.

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions.

With Wolbachia-based arbovirus control programs being scaled and operationalised around the world, cost effective and reliable detection of Wolbachia in field samples and laboratory stocks is essential for quality control. Here we validate a modified loop-mediated isothermal amplification (LAMP) assay for routine scoring of Wolbachia in mosquitoes from laboratory cultures and the field, applicable to any setting. We show that this assay is a rapid and robust method for highly sensitive and specific detection of wAlbB Wolbachia infection within Aedes aegypti under a variety of conditions. We test the quantitative nature of the assay by evaluating pooled mixtures of Wolbachia-infected and uninfected mosquitoes and show that it is capable of estimating infection frequencies, potentially circumventing the need to perform large-scale individual analysis for wAlbB infection status in the course of field monitoring. These results indicate that LAMP assays are useful for routine screening particularly under field conditions away from laboratory facilities.

<p class="Body">Transfection and colonization of Tetranychus truncatus Wolbachia strain wTtru in cell lines of the mosquito Aedes albopictus

Wolbachia are endosymbiotic bacteria that commonly infect arthropods and cause reproductive manipulations in hosts. Artificial transfection is one of the best methods for the study of Wolbachia-induced reproductive manipulations and its application in production practice. However, the low success rate of artificial transfection laid burden for investigation. The success rate of artificial transfection of Wolbachia between closely related hosts is usually higher than distant hosts, but the mechanism of transfection is unknown. In our study, the wTtru strain (supergroup B) infecting the Arachnida Tetranychus truncatus was transfected into the Aedes albopictus cell line (native-infected with wAlB strain from supergroup B). The wTtru strain was quantified and localized in the cell line to observe dynamic changes after transfection. A phylogenetic analysis of the relationship between the wTtru strain and the wAlB strain showed that the wTtru (ST=278) strain belonged to supergroup B and was closely related to the mosquitoes native Wolbachia wAlbB (ST=464), so we speculate that successful transfection was related to the Wolbachia strain, and furthermore, Wolbachia strain may be more important than host relationship. Our results provide a factual basis for studying artificial transfection of Wolbachia, and show that Wolbachia transfection between distant hosts can be achieved.

Effects of Alternative Blood Sources on Wolbachia Infected Aedes aegypti Females within and across Generations.

Wolbachia bacteria have been identified as a tool for reducing the transmission of arboviruses transmitted by Aedes aegypti. Research groups around the world are now mass rearing Wolbachia-infected Ae. aegypti for deliberate release. We investigated the fitness impact of a crucial element of mass rearing: the blood meal required by female Ae. aegypti to lay eggs. Although Ae. aegypti almost exclusively feed on human blood, it is often difficult to use human blood in disease-endemic settings. When females were fed on sheep or pig blood rather than human blood, egg hatch rates decreased in all three lines tested (uninfected, or infected by wMel, or wAlbB Wolbachia). This finding was particularly pronounced when fed on sheep blood, although fecundity was not affected. Some of these effects persisted after an additional generation on human blood. Attempts to keep populations on sheep and pig blood sources only partly succeeded, suggesting that strong adaptation is required to develop a stably infected line on an alternative blood source. There was a decrease in Wolbachia density when Ae. aegypti were fed on non-human blood sources. Density increased in lines kept for multiple generations on the alternate sources but was still reduced relative to lines kept on human blood. These findings suggest that sheep and pig blood will entail a cost when used for maintaining Wolbachia-infected Ae. aegypti. These costs should be taken into account when planning mass release programs.

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37°C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions.

Wolbachia pipientis grows in Saccharomyces cerevisiae evoking early death of the host and deregulation of mitochondrial metabolism.

Wolbachia sp. has colonized over 70% of insect species, successfully manipulating host fertility, protein expression, lifespan, and metabolism. Understanding and engineering the biochemistry and physiology of Wolbachia holds great promise for insect vector‐borne disease eradication. Wolbachia is cultured in cell lines, which have long duplication times and are difficult to manipulate and study. The yeast strain Saccharomyces cerevisiae W303 was used successfully as an artificial host for Wolbachia wAlbB. As compared to controls, infected yeast lost viability early, probably as a result of an abnormally high mitochondrial oxidative phosphorylation activity observed at late stages of growth. No respiratory chain proteins from Wolbachia were detected, while several Wolbachia F1F0‐ATPase subunits were revealed. After 5 days outside the cell, Wolbachia remained fully infective against insect cells.

Comprehensive proteome profiling in Aedes albopictus to decipher Wolbachia-arbovirus interference phenomenon

BackgroundAedes albopictus is a vector of arboviruses that cause severe diseases in humans such as Chikungunya, Dengue and Zika fevers. The vector competence of Ae. albopictus varies depending on the mosquito population involved and the virus transmitted. Wolbachia infection status in believed to be among key elements that determine viral transmission efficiency. Little is known about the cellular functions mobilized in Ae. albopictus during co-infection by Wolbachia and a given arbovirus. To decipher this tripartite interaction at the molecular level, we performed a proteome analysis in Ae. albopictus C6/36 cells mono-infected by Wolbachia wAlbB strain or Chikungunya virus (CHIKV), and bi-infected.ResultsWe first confirmed significant inhibition of CHIKV by Wolbachia. Using two-dimensional gel electrophoresis followed by nano liquid chromatography coupled with tandem mass spectrometry, we identified 600 unique differentially expressed proteins mostly related to glycolysis, translation and protein metabolism. Wolbachia infection had greater impact on cellular functions than CHIKV infection, inducing either up or down-regulation of proteins associated with metabolic processes such as glycolysis and ATP metabolism, or structural glycoproteins and capsid proteins in the case of bi-infection with CHIKV. CHIKV infection inhibited expression of proteins linked with the processes of transcription, translation, lipid storage and miRNA pathways.ConclusionsThe results of our proteome profiling have provided new insights into the molecular pathways involved in tripartite Ae. albopictus-Wolbachia-CHIKV interaction and may help defining targets for the better implementation of Wolbachia-based strategies for disease transmission control.

The Maternally Inheritable Wolbachia wAlbB Induces Refractoriness to Plasmodium berghei in Anopheles stephensi.

The endosymbiont Wolbachia wAlbB induces refractoriness to Plasmodium falciparum in Anopheles stephensi, the primary mosquito vector of human malaria in the Middle East and South Asia. However, it remains unknown whether such refractoriness can be extended to other malaria species. In particular, it was reported that under very specific conditions, wAlbB can enhance Plasmodium infection in some hosts. Here, we measured the impact of wAlbB on the rodent malaria parasite Plasmodium berghei in A. stephensi by comparing the load of oocysts and sporozoites in midguts and salivary glands, respectively, between wAlbB-infected and -uninfected mosquitoes. To investigate whether wAlbB modulated mosquito immune defense against parasites, we compared the expression of the immune genes, which were previously reported to involve in antimalarial response, in both midguts and the remaining carcass tissues of mosquitoes. The stable association of wAlbB with A. stephensi resulted in reduction of parasites by more than half at the oocyst stage, and up to 91.8% at the sporzoite stage. The anti-plasmodium immune genes, including TEP1, LRIM1, Toll pathway gene Rel1 and the effector Defensin 1, were induced by wAlbB in different mosquito body tissues. These findings suggest that immune priming is a potential cause of wAlbB-mediated antimalarial response in A. stephensi. More importantly, no evidence was found for any enhancement of Plasmodium infection in A. stephensi stably infected with wAlbB. We discuss these findings with possible implementations of Wolbachia for malaria control in disease endemic areas.

Wolbachia strain wAlbB confers both fitness costs and benefit on Anopheles stephensi.

BackgroundWolbachia is a maternally transmitted intracellular bacterium that is estimated to infect up to 65% of insect species, but it is not naturally present in Anopheles malaria vectors. Wolbachia-based strategies for malaria vector control can be developed either through population replacement to reduce vectorial capacity or through population suppression to reduce the mosquito population. We have previously generated An. stephensi mosquitoes carrying a stable wAlbB Wolbachia infection and have demonstrated their ability to invade wild-type laboratory populations and confer resistance to Plasmodium on these populations.MethodsWe assessed wAlbB-associated fitness by comparing the female fecundity, immature development and survivorship, body size, male mating competiveness, and adult longevity of the infected An. stephensi to that of wild-type mosquitoes.ResultsWe found that wAlbB reduced female fecundity and caused a minor decrease in male mating competiveness. We also observed that wAlbB increased the life span of both male and female mosquitoes when they were maintained solely on sugar meals; however, there was no impact on the life span of blood-fed females. In addition, wAlbB did not influence either immature development and survivorship or adult body sizes.ConclusionsThese results provide significant support for developing Wolbachia-based strategies for malaria vector control.

Wolbachia strain wPip yields a pattern of cytoplasmic incompatibility enhancing a Wolbachia-based suppression strategy against the disease vector Aedes albopictus.

BackgroundCytoplasmic incompatibility (CI) is induced in nature by Wolbachia bacteria, resulting in conditional male sterility. Previous research demonstrated that the two Wolbachia strains (wAlbA and wAlbB) that naturally co-infect the disease vector mosquito Aedes albopictus (Asian tiger mosquito) can be replaced with the wPip Wolbachia strain from Culex pipiens. Since Wolbachia-based vector control strategies depend upon the strength and consistency of CI, a greater understanding is needed on the CI relationships between wPip, wAlbA and wAlbB Wolbachia in Ae. albopictus.MethodsThis work consisted of a collaborative series of crosses carried out in Italy and in US to study the CI relationships between the “wPip” infected Ae. albopictus strain (ARwP) and the superinfected SR strain. The Ae. albopictus strains used in Italian tests are the wPip infected ARwP strain (ARwPIT), the superinfected SR strain and the aposymbiotic AR strain. To understand the observed pattern of CI, crossing experiments carried out in USA focused on the study of the CI relationships between ARwP (ARwPUS) and artificially-generated single infected lines, in specific HTA and HTB, harbouring only wAlbA and wAlbB Wolbachia respectively.ResultsThe paper reports an unusual pattern of CI observed in crossing experiments between ARwP and SR lines. Specifically, ARwP males are able to induce full sterility in wild type females throughout most of their lifetime, while crosses between SR males and ARwP females become partially fertile with male aging. We demonstrated that the observed decrease in CI penetrance with SR male age, is related to the previously described decrease in Wolbachia density, in particular of the wAlbA strain, occurring in aged superinfected males.ConclusionsThe results here reported support the use of the ARwP Ae. albopictus line as source of “ready-made sterile males”, as an alternative to gamma radiation sterilized males, for autocidal suppression strategies against the Asian tiger mosquito. In addition, the age dependent CI weakening observed in the crosses between SR males and ARwP females simplifies the downstream efforts to preserve the genetic variability within the laboratory ARwP colonies, to date based on the antibiotic treatment of wild captured superinfected mosquitoes, also reducing the costs.

Bacterial diversity of field-caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar

Symbiotic bacteria are known to play important roles in the biology of insects, but the current knowledge of bacterial communities associated with mosquitoes is very limited and consequently their contribution to host behaviors is mostly unknown. In this study, we explored the composition and diversity of mosquito-associated bacteria in relation with mosquitoes' habitats. Wild Aedes albopictus and Aedes aegypti were collected in three different geographic regions of Madagascar. Culturing methods and denaturing gradient gel electrophoresis (DGGE) and sequencing of the rrs amplicons revealed that Proteobacteria and Firmicutes were the major phyla. Isolated bacterial genera were dominated by Bacillus, followed by Acinetobacter, Agrobacterium and Enterobacter. Common DGGE bands belonged to Acinetobacter, Asaia, Delftia, Pseudomonas, Enterobacteriaceae and an uncultured Gammaproteobacterium. Double infection by maternally inherited Wolbachia pipientis prevailed in 98% of males (n=272) and 99% of females (n=413); few individuals were found to be monoinfected with Wolbachia wAlbB strain. Bacterial diversity (Shannon-Weaver and Simpson indices) differed significantly per habitat whereas evenness (Pielou index) was similar. Overall, the bacterial composition and diversity were influenced both by the sex of individuals and by the environment inhabited by the mosquitoes; the latter might be related to both the vegetation and the animal host populations that Aedes used as food sources.

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.