A metapopulation approach to identify targets for Wolbachia-based dengue control.

Over the last decade, the release of Wolbachia-infected Aedes aegypti into the natural habitat of this mosquito species has become the most sustainable and long-lasting technique to prevent and control vector-borne diseases, such as dengue, zika, or chikungunya. However, the limited resources to generate such mosquitoes and their effective distribution in large areas dominated by the Aedes aegypti vector represent a challenge for policymakers. Here, we introduce a mathematical framework for the spread of dengue in which competition between wild and Wolbachia-infected mosquitoes, the cross-contagion patterns between humans and vectors, the heterogeneous distribution of the human population in different areas, and the mobility flows between them are combined. Our framework allows us to identify the most effective areas for the release of Wolbachia-infected mosquitoes to achieve a large decrease in the global dengue prevalence.

Two Newly Introduced Wolbachia Endosymbionts Induce Cell Host Differences in Competitiveness and Metabolic Responses.

Wolbachia endosymbionts can induce multiple reproductive manipulations in their hosts, with cytoplasmic incompatibility (CI) being one of the most common manipulations. Two important agricultural pests, the white-backed planthopper (Sogatella furcifera) and the brown planthopper (Nilaparvata lugens), are usually infected with CI-inducing Wolbachia strain wFur and non-CI-inducing Wolbachia strain wLug, respectively. The biological effects of these infections when present in a host cell are unknown. Here, we introduced the two Wolbachia strains into an Aedes albopictus cell line to stably establish a wFur-infected cell line (WFI) and a wLug-infected cell line (WLI). In a mixed culture, WFI cells were completely replaced by WLI cells, pointing to a stronger competitiveness of the WLI cell line. We found that infection by both Wolbachia strains reduced cell growth rates, but WLI had a higher cell growth rate than WFI, and this difference in cell growth rate combined with possible Wolbachia differences in diffusivity may have affected cell competitiveness. By examining gene expression and metabolites in the two lines, we found that some genes and key metabolites responded to differences in cell competitiveness. These results point to potential mechanisms that could contribute to the relative performance of hosts infected by these strains and also highlight the substantial impact of a non-CI Wolbachia on metabolism, which may in turn influence the fitness of its native host. IMPORTANCEWolbachia transinfection in insects can be used to suppress pests and block virus transmission. We stably introduced two Wolbachia strains from rice planthoppers into cell lines of an important arbovirus mosquito vector, Aedes albopictus. The levels of competitiveness of host cells from the lines infected by the two Wolbachia strains were different, as were metabolic responses of the cell lines. These results suggest potential metabolic effects of Wolbachia on native hosts that could be exploited when they are transinfected into novel hosts for pest control.

Forward genetics in Wolbachia: Regulation of Wolbachia proliferation by the amplification and deletion of an addictive genomic island.

Wolbachia is one of the most prevalent bacterial endosymbionts, infecting approximately 40% of terrestrial arthropod species. Wolbachia is often a reproductive parasite but can also provide fitness benefits to its host, as, for example, protection against viral pathogens. This protective effect is currently being applied to fight arboviruses transmission by releasing Wolbachia-transinfected mosquitoes. Titre regulation is a crucial aspect of Wolbachia biology. Higher titres can lead to stronger phenotypes and fidelity of transmission but can have a higher cost to the host. Since Wolbachia is maternally transmitted, its fitness depends on host fitness, and, therefore, its cost to the host may be under selection. Understanding how Wolbachia titres are regulated and other aspects of Wolbachia biology has been hampered by the lack of genetic tools. Here we developed a forward genetic screen to identify new Wolbachia over-proliferative mutant variants. We characterized in detail two new mutants, wMelPop2 and wMelOctoless, and show that the amplification or loss of the Octomom genomic region lead to over-proliferation. These results confirm previous data and expand on the complex role of this genomic region in the control of Wolbachia proliferation. Both new mutants shorten the host lifespan and increase antiviral protection. Moreover, we show that Wolbachia proliferation rate in Drosophila melanogaster depends on the interaction between Octomom copy number, the host developmental stage, and temperature. Our analysis also suggests that the life shortening and antiviral protection phenotypes of Wolbachia are dependent on different, but related, properties of the endosymbiont; the rate of proliferation and the titres near the time of infection, respectively. We also demonstrate the feasibility of a novel and unbiased experimental approach to study Wolbachia biology, which could be further adapted to characterize other genetically intractable bacterial endosymbionts.

In silico drug repurposing for filarial infection predicts nilotinib and paritaprevir as potential inhibitors of the Wolbachia 5'-aminolevulinic acid synthase.

Filarial infections affect millions of individuals and are responsible for some notorious disabilities. Current treatment options involve repeated mass drug administrations, which have been met with several challenges despite some successes. Administration of doxycycline, an anti-Wolbachia agent, has shown clinical effectiveness but has several limitations, including long treatment durations and contraindications. We describe the use of an in silico drug repurposing approach to screening a library of over 3200 FDA-approved medications against the filarial endosymbiont, Wolbachia. We target the enzyme which catalyzes the first step of heme biosynthesis in the Wolbachia. This presents an opportunity to inhibit heme synthesis, which leads to depriving the filarial worm of heme, resulting in a subsequent macrofilaricidal effect. High throughput virtual screening, molecular docking and molecular simulations with binding energy calculations led to the identification of paritaprevir and nilotinib as potential anti-Wolbachia agents. Having higher binding affinities to the catalytic pocket than the natural substrate, these drugs have the structural potential to bind and engage active site residues of the wolbachia 5'-Aminolevulinic Acid Synthase. We hereby propose paritaprevir and nilotinib for experimental validations as anti-Wolbachia agents.

Parasite-bacteria interrelationship.

Parasites and bacteria have co-evolved with humankind, and they interact all the time in a myriad of ways. For example, some bacterial infections result from parasite-dwelling bacteria as in the case of Salmonella infection during schistosomiasis. Other bacteria synergize with parasites in the evolution of human disease as in the case of the interplay between Wolbachia endosymbiont bacteria and filarial nematodes as well as the interaction between Gram-negative bacteria and Schistosoma haematobium in the pathogenesis of urinary bladder cancer. Moreover, secondary bacterial infections may complicate several parasitic diseases such as visceral leishmaniasis and malaria, due to immunosuppression of the host during parasitic infections. Also, bacteria may colonize the parasitic lesions; for example, hydatid cysts and skin lesions of ectoparasites. Remarkably, some parasitic helminths and arthropods exhibit antibacterial activity usually by the release of specific antimicrobial products. Lastly, some parasite-bacteria interactions are induced as when using probiotic bacteria to modulate the outcome of a variety of parasitic infections. In sum, parasite-bacteria interactions involve intricate processes that never cease to intrigue the researchers. However, understanding and exploiting these interactions could have prophylactic and curative potential for infections by both types of pathogens.

Wolbachia strain wAu efficiently blocks arbovirus transmission in Aedes albopictus.

The global incidence of arboviral diseases transmitted by Aedes mosquitoes, including dengue, chikungunya, yellow fever, and Zika, has increased dramatically in recent decades. The release of Aedes aegypti carrying the maternally inherited symbiont Wolbachia as an intervention to control arboviruses is being trialled in several countries. However, these efforts are compromised in many endemic regions due to the co-localization of the secondary vector Aedes albopictus, the Asian tiger mosquito. Ae. albopictus has an expanding global distribution following incursions into a number of new territories. To date, only the wMel and wPip strains of Wolbachia have been reported to be transferred into and characterized in this vector. A Wolbachia strain naturally infecting Drosophila simulans, wAu, was selected for transfer into a Malaysian Ae. albopictus line to create a novel triple-strain infection. The newly generated line showed self-compatibility, moderate fitness cost and complete resistance to Zika and dengue infections.

Wolbachia-mediated antiviral protection is cell-autonomous.

Vector-borne viral diseases pose significant risks to human health. To control the transmission of these viruses, a number of approaches are required. The ability of the intracellular bacteria Wolbachia to limit viral accumulation and transmission in some arthropod hosts, highlights its potential as a biocontrol agent. Whilst Wolbachia can reduce the transmission of several epidemiologically important viruses, protection is not consistent amongst all insects, viruses and strains of Wolbachia, which confounds elucidation of the mechanisms that underly this protection. Evidence of different mechanisms has emerged, but is not always consistent, suggesting the tripartite interaction may be complex. Here we provide evidence that Wolbachia-mediated antiviral protection is dependent on the presence of Wolbachia in individual cells, and cannot be conferred to surrounding cells. Our results suggest that protection is cell-autonomous, and this has several mechanistic implications, which can direct future research.

Lack of influence by endosymbiont Wolbachia on virus titer in the common bed bug, Cimex lectularius.

BACKGROUND: The common bed bug, Cimex lectularius, is an obligatory blood-feeding ectoparasite that requires a blood meal to molt and produce eggs. Their frequent biting to obtain blood meals and intimate association with humans increase the potential for disease transmission. However, despite more than 100 years of inquiry into bed bugs as potential disease vectors, they still have not been conclusively linked to any pathogen or disease. This ecological niche is extraordinarily rare, given that nearly every other blood-feeding arthropod is associated with some type of human or zoonotic disease. Bed bugs rely on the bacteria Wolbachia as an obligate endosymbiont to biosynthesize B vitamins, since they acquire a nutritionally deficient diet, but it is unknown if Wolbachia confers additional benefits to its bed bug host. In some insects, Wolbachia induces resistance to viruses such as Dengue, Chikungunya, West Nile, Drosophila C and Zika, and primes the insect immune system in other blood-feeding insects. Wolbachia might have evolved a similar role in its mutualistic association with the bed bug. In this study, we evaluated the influence of Wolbachia on virus replication within C. lectularius. METHODS: We used feline calicivirus as a model pathogen. We fed 40 bed bugs from an established line of Wolbachia-cured and a line of Wolbachia-positive C. lectularius a virus-laden blood meal, and quantified the amount of virus over five time intervals post-feeding. The antibiotic rifampicin was used to cure bed bugs of Wolbachia. RESULTS: There was a significant effect of time post-feeding, as the amount of virus declined by ~90% over 10 days in both groups, but no significant difference in virus titer was observed between the Wolbachia-positive and Wolbachia-cured groups. CONCLUSIONS: These findings suggest that other mechanisms are involved in virus suppression within bed bugs, independent of the influence of Wolbachia, and our conclusions underscore the need for future research.

Quantitative methods for assessing local and bodywide contributions to Wolbachia titer in maternal germline cells of Drosophila.

BACKGROUND: Little is known about how bacterial endosymbionts colonize host tissues. Because many insect endosymbionts are maternally transmitted, egg colonization is critical for endosymbiont success. Wolbachia bacteria, carried by approximately half of all insect species, provide an excellent model for characterizing endosymbiont infection dynamics. To date, technical limitations have precluded stepwise analysis of germline colonization by Wolbachia. It is not clear to what extent titer-altering effects are primarily mediated by growth rates of Wolbachia within cell lineages or migration of Wolbachia between cells. RESULTS: The objective of this work is to inform mechanisms of germline colonization through use of optimized methodology. The approaches are framed in terms of nutritional impacts on Wolbachia. Yeast-rich diets in particular have been shown to suppress Wolbachia titer in the Drosophila melanogaster germline. To determine the extent of Wolbachia sensitivity to diet, we optimized 3-dimensional, multi-stage quantification of Wolbachia titer in maternal germline cells. Technical and statistical validation confirmed the identity of Wolbachia in vivo, the reproducibility of Wolbachia quantification and the statistical power to detect these effects. The data from adult feeding experiments demonstrated that germline Wolbachia titer is distinctly sensitive to yeast-rich host diets in late oogenesis. To investigate the physiological basis for these nutritional impacts, we optimized methodology for absolute Wolbachia quantification by real-time qPCR. We found that yeast-rich diets exerted no significant effect on bodywide Wolbachia titer, although ovarian titers were significantly reduced. This suggests that host diets affects Wolbachia distribution between the soma and late stage germline cells. Notably, relative qPCR methods distorted apparent wsp abundance, due to altered host DNA copy number in yeast-rich conditions. This highlights the importance of absolute quantification data for testing mechanistic hypotheses. CONCLUSIONS: We demonstrate that absolute quantification of Wolbachia, using well-controlled cytological and qPCR-based methods, creates new opportunities to determine how bacterial abundance within the germline relates to bacterial distribution within the body. This methodology can be applied to further test germline infection dynamics in response to chemical treatments, genetic conditions, new host/endosymbiont combinations, or potentially adapted to analyze other cell and tissue types.

Population Dynamics of Wolbachia in Laodelphax striatellus (Fallén) Under Successive Stress of Antibiotics.

Wolbachia are the most common symbionts in arthropods; antibiotic treatment for eliminating the symbionts from their host is necessary to investigate the functions. Tetracycline antibiotics are widely used to remove endosymbiont Wolbachia from insect hosts. However, very little has been known on the effects of tetracycline on population size of Wolbachia in small brown planthopper (SBPH), Laodelphax striatellus (Fallén), an important insect pest of rice in Asia. Here, we investigated the dynamics of Wolbachia population density in females and males of L. striatellus by real-time fluorescent quantitative PCR method. The Wolbachia density in females and males of L. striatellus all declined sharply after treatment with 2 mg/mL tetracycline for one generation, and continued to decrease to a level which could not be detected by both qPCR and diagnostic PCR after treated for another generation, then maintained at 0 in the following three generations with continuous antibiotic treatment. Wolbachia infection did not recover in L. striatellus after stopping tetracycline treatment for ten generations. This is the first report to precisely monitor the population dynamics of Wolbachia in L. striatellus during successive tetracycline treatment and after that. The results provide a useful method for evaluating the efficiency of artificial operation of endosymbionts.

A systems biology approach for studying Wolbachia metabolism reveals points of interaction with its host in the context of arboviral infection.

Wolbachia are alpha-proteobacteria known to infect arthropods, which are of interest for disease control since they have been associated with improved resistance to viral infection. Although several genomes for different strains have been sequenced, there is little knowledge regarding the relationship between this bacterium and their hosts, particularly on their dependency for survival. Motivated by the potential applications on disease control, we developed genome-scale models of four Wolbachia strains known to infect arthropods: wAlbB (Aedes albopictus), wVitA (Nasonia vitripennis), wMel and wMelPop (Drosophila melanogaster). The obtained metabolic reconstructions exhibit a metabolism relying mainly on amino acids for energy production and biomass synthesis. A gap analysis was performed to detect metabolic candidates which could explain the endosymbiotic nature of this bacterium, finding that amino acids, requirements for ubiquinone precursors and provisioning of metabolites such as riboflavin could play a crucial role in this relationship. This work provides a systems biology perspective for studying the relationship of Wolbachia with its host and the development of new approaches for control of the spread of arboviral diseases. This approach, where metabolic gaps are key objects of study instead of just additions to complete a model, could be applied to other endosymbiotic bacteria of interest.

Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictus.

Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that influence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We find both species share a limited, yet highly variable core microbiota, reflecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We find significant differences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we find little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama.

Infection of Aedes albopictus Mosquito C6/36 Cells with the wMelpop Strain of Wolbachia Modulates Dengue Virus-Induced Host Cellular Transcripts and Induces Critical Sequence Alterations in the Dengue Viral Genome.

Dengue virus (DENV) causes frequent epidemics infecting ∼390 million people annually in over 100 countries. There are no approved vaccines or antiviral drugs for treatment of infected patients. However, there is a novel approach to control DENV transmission by the mosquito vectors, Aedes aegypti and Aedes albopictus, using the Wolbachia symbiont. The wMelPop strain of Wolbachia suppresses DENV transmission and shortens the mosquito life span. However, the underlying mechanism is poorly understood. To clarify this mechanism, either naive A. albopictus (C6/36) or wMelPop-C6/36 cells were infected with DENV serotype 2 (DENV2). Analysis of host transcript profiles by transcriptome sequencing (RNAseq) revealed that the presence of wMelPop dramatically altered the mosquito host cell transcription in response to DENV2 infection. The viral RNA evolved from wMelPop-C6/36 cells contained low-frequency mutations (∼25%) within the coding region of transmembrane domain 1 (TMD1) of E protein. Mutations with >97% frequencies were distributed within other regions of E, the NS5 RNA-dependent RNA polymerase (NS5POL) domain, and the TMDs of NS2A, NS2B, and NS4B. Moreover, while DENV2-infected naive C6/36 cells showed syncytium formation, DENV2-infected wMelPop-C6/36 cells did not. The Wolbachia-induced mutant DENV2 can readily infect and replicate in naive C6/36 cells, whereas in mutant DENV2-infected BHK-21 or Vero cells, virus replication was delayed. In LLC-MK2 cells, the mutant failed to produce plaques. Additionally, in BHK-21 cells, many mutations in the viral genome reverted to the wild type (WT) and compensatory mutations in NS3 gene appeared. Our results indicate that wMelPop impacts significantly the interactions of DENV2 with mosquito and mammalian host cells.IMPORTANCE Mosquito-borne diseases are of global significance causing considerable morbidity and mortality throughout the world. Dengue virus (DENV; serotypes 1 to 4), a member of the Flavivirus genus of the Flaviviridae family, causes millions of infections annually. Development of a safe vaccine is hampered due to absence of cross-protection and increased risk in secondary infections due to antibody-mediated immune enhancement. Infection of vector mosquitoes with Wolbachia bacteria offers a novel countermeasure to suppress DENV transmission, but the mechanisms are poorly understood. In this study, the host transcription profiles and viral RNA sequences were analyzed in naive A. albopictus (C6/36) and wMelPop-C6/36 cells by RNAseq. Our results showed that the wMelPop symbiont caused profound changes in host transcription profiles and morphology of DENV2-infected C6/36 cells. Accumulation of several mutations throughout DENV2 RNA resulted in loss of infectivity of progeny virions. Our findings offer new insights into the mechanism of Wolbachia-mediated suppression of DENV transmission.

Loss of cytoplasmic incompatibility in Wolbachia-infected Aedes aegypti under field conditions.

Wolbachia bacteria are now being introduced into Aedes aegypti mosquito populations for dengue control. When Wolbachia infections are at a high frequency, they influence the local transmission of dengue by direct virus blocking as well as deleterious effects on vector mosquito populations. However, the effectiveness of this strategy could be influenced by environmental temperatures that decrease Wolbachia density, thereby reducing the ability of Wolbachia to invade and persist in the population and block viruses. We reared wMel-infected Ae. aegypti larvae in the field during the wet season in Cairns, North Queensland. Containers placed in the shade produced mosquitoes with a high Wolbachia density and little impact on cytoplasmic incompatibility. However, in 50% shade where temperatures reached 39°C during the day, wMel-infected males partially lost their ability to induce cytoplasmic incompatibility and females had greatly reduced egg hatch when crossed to infected males. In a second experiment under somewhat hotter conditions (>40°C in 50% shade), field-reared wMel-infected females had their egg hatch reduced to 25% when crossed to field-reared wMel-infected males. Wolbachia density was reduced in 50% shade for both sexes in both experiments, with some mosquitoes cleared of their Wolbachia infections entirely. To investigate the critical temperature range for the loss of Wolbachia infections, we held Ae. aegypti eggs in thermocyclers for one week at a range of cyclical temperatures. Adult wMel density declined when eggs were held at 26-36°C or above with complete loss at 30-40°C, while the density of wAlbB remained high until temperatures were lethal. These findings suggest that high temperature effects on Wolbachia are potentially substantial when breeding containers are exposed to partial sunlight but not shade. Heat stress could reduce the ability of Wolbachia infections to invade mosquito populations in some locations and may compromise the ability of Wolbachia to block virus transmission in the field. Temperature effects may also have an ecological impact on mosquito populations given that a proportion of the population becomes self-incompatible.

Wolbachia introduction into Lutzomyia longipalpis (Diptera: Psychodidae) cell lines and its effects on immune-related gene expression and interaction with Leishmania infantum.

BACKGROUND: The leishmaniases are important neglected diseases caused by Leishmania spp. which are transmitted by sand flies, Lutzomyia longipalpis being the main vector of visceral leishmaniasis in the Americas. The methodologies for leishmaniasis control are not efficient, causing 1.5 million reported cases annually worldwide, therefore showing the need for development of novel strategies and interventions to control transmission of the disease. The bacterium Wolbachia pipientis is being used to control viruses transmitted by mosquitoes, such as dengue and Zika, and its introduction in disease vectors has been effective against parasites such as Plasmodium. Here we show the first successful establishment of Wolbachia into two different embryonic cell lines from L. longipalpis, LL-5 and Lulo, and analysed its effects on the sand fly innate immune system, followed by in vitro Leishmania infantum interaction. RESULTS: Our results show that LL-5 cells respond to wMel and wMelPop-CLA strains within the first 72 h post-infection, through the expression of antimicrobial peptides and inducible nitric oxide synthase resulting in a decrease of Wolbachia detection in the early stages of infection. In subsequent passages, the wMel strain was not able to infect any of the sand fly cell lines while the wMelPop-CLA strain was able to stably infect Lulo cells and LL-5 at lower levels. In Wolbachia stably infected cells, the expression of immune-related genes involved with downregulation of the IMD, Toll and Jak-Stat innate immune pathways was significantly decreased, in comparison with the uninfected control, suggesting immune activation upon Wolbachia transinfection. Furthermore, Wolbachia transinfection did not promote a negative effect on parasite load in those cells. CONCLUSIONS: Initial strong immune responses of LL5 cells might explain the inefficiency of stable infections in these cells while we found that Lulo cells are more permissive to infection with Wolbachia causing an effect on the cell immune system, but not against in vitro L. infantum interaction. This establishes Lulo cells as a good system for the adaptation of Wolbachia in L. longipalpis.

Matching the genetics of released and local Aedes aegypti populations is critical to assure Wolbachia invasion.

BACKGROUND: Traditional vector control approaches such as source reduction and insecticide spraying have limited effect on reducing Aedes aegypti population. The endosymbiont Wolbachia is pointed as a promising tool to mitigate arbovirus transmission and has been deployed worldwide. Models predict a rapid increase on the frequency of Wolbachia-positive Ae. aegypti mosquitoes in local settings, supported by cytoplasmic incompatibility (CI) and high maternal transmission rate associated with the wMelBr strain. METHODOLOGY/PRINCIPLE FINDINGS: Wolbachia wMelBr strain was released for 20 consecutive weeks after receiving >87% approval of householders of the isolated community of Tubiacanga, Rio de Janeiro. wMelBr frequency plateued~40% during weeks 7-19, peaked 65% but dropped as releases stopped. A high (97.56%) maternal transmission was observed. Doubling releases and deploying mosquitoes with large wing length and low laboratory mortality produced no detectable effects on invasion trend. By investigating the lab colony maintenance procedures backwardly, pyrethroid resistant genotypes in wMelBr decreased from 68% to 3.5% after 17 generations. Therefore, we initially released susceptible mosquitoes in a local population highly resistant to pyrethroids which, associated with the over use of insecticides by householders, ended jeopardizing Wolbachia invasion. A new strain (wMelRio) was produced after backcrossing wMelBr females with males from field to introduce mostly pyrethroid resistance alleles. The new strain increased mosquito survival but produced relevant negative effects on Ae. aegypti fecundity traits, reducing egg clutche size and egg hatch. Despite the cost on fitness, wMelRio successful established where wMelBr failed, revealing that matching the local population genetics, especially insecticide resistance background, is critical to achieve invasion. CONCLUSIONS/SIGNIFICANCE: Local householders support was constantly high, reaching 90% backing on the second release (wMelRio strain). Notwithstanding the drought summer, the harsh temperature recorded (daily average above 30°C) did not seem to affect the expression of maternal transmission of wMel on a Brazilian background. Wolbachia deployment should match the insecticide resistance profile of the wild population to achieve invasion. Considering pyrethroid-resistance is a widely distributed phenotype in natural Ae. aegypti populations, future Wolbachia deployments must pay special attention in maintaining insecticide resistance in lab colonies for releases.

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 F1 F0 -ATPase subunits were revealed. After 5 days outside the cell, Wolbachia remained fully infective against insect cells.

Short-term impacts of anthropogenic stressors on Aedes albopictus mosquito vector microbiota.

Recent studies have highlighted the potential role of microbiota in the biology of the Aedes albopictus mosquito vector. This species is highly anthropogenic and exhibits marked ecological plasticity, with a resulting high potential to colonize a wide range of habitats-including anthropized areas-under various climatic conditions. We put forward the hypothesis that climate and anthropogenic activities, such as the use of antibiotics in agriculture and human medicine, might affect the mosquito-associated bacterial community. We thus studied the additive impact of a temperature decrease and antibiotic ingestion on the temporal dynamics of Ae. albopictus survival and its associated bacterial communities. The results showed no effects of disturbances on mosquito survival. However, short-term temperature impacts on bacterial diversity were observed, while both the community structure and bacterial diversity were affected by early antibiotic ingestion. The genera Elizabethkingia, Chryseobacterium and Wolbachia, as well as an unclassified member of the Bacteroidales order were particularly affected. Antibiotics negatively impacted Elizabethkingia abundance, while Chryseobacterium was completely eliminated following both disturbances, to the benefit of Wolbachia and the unclassified Bacteroidales species. These results generated fresh insight into the effects of climate and anthropogenic activities such as the use of antibiotics on mosquito microbiota.

Growth kinetics of endosymbiont Wolbachia in the common bed bug, Cimex lectularius.

The common bed bug, Cimex lectularius harbors the endosymbiotic microorganism, Wolbachia (wCle), in a gonad-associated bacteriome as an obligate nutritional mutualist. The obligatory nature of this association suggests that all individuals in C. lectularius populations would be infected with wCle. However, studies spanning the past several decades have reported variation in both infection frequency and relative abundance of wCle in field-collected samples of bed bugs. Since the growth kinetics of wCle is poorly understood, the objective of this study was to quantify wCle over the life cycle of two strains of C. lectularius. Our results highlight that wCle is dynamic during bed bug development, changing relative to life stage, intermolt stage, and blood-fed status. These results suggest new hypotheses about the coordination of Wolbachia growth and regression with its host's physiology and endocrine events. The observed quantitative modulation of wCle during the bed bug life cycle and during periods of starvation may explain the disparities in wCle infections reported in field-collected C. lectularius.

Wolbachia's role in mediating its flea's reproductive success differs according to flea origin.

Endosymbionts-microbes that live within and engage in prolonged and intimate associations with their hosts-are gaining recognition for their direct impact on plant and animal reproduction. Here we used the overlooked Wolbachia-flea system to explore the possibility that endosymbionts may also play a role as mediators in shaping the reproductive success of their hosts. We simultaneously quantified the Wolbachia density in field- and laboratory-originated fleas that fed and mated on rodents for either 5 or 10 days and assessed their body size and current reproductive success. By combining multigroup analysis and model selection approaches, we teased apart the contribution of the direct effects of the flea's physiological age and body size and the mediation effect of its Wolbachia endosymbionts on flea reproductive success, and we showed that the latter was stronger than the former. However, interestingly, the mediation effect was manifested only in laboratory-originated fleas, for which the increase in Wolbachia with age translated into lower reproductive success. These results suggest that some well-supported phenomena, such as aging effects, may be driven by endosymbionts and show once again that the role of endosymbionts in shaping the reproductive success of their host depends on their selective environment.