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Background: Introgression of genetic material from species of the insect bacteria Wolbachia into populations of Aedes aegypti mosquitoes has been shown in randomised and non-randomised trials to reduce the incidence of dengue; however, evidence for the real-world effectiveness of large-scale deployments of Wolbachia-infected mosquitoes for arboviral disease control in endemic settings is still scarce. A large Wolbachia (wMel strain) release programme was implemented in 2017 in Rio de Janeiro, Brazil. We aimed to assess the effect of this programme on the incidence of dengue and chikungunya in the city., Methods: 67 million wMel-infected mosquitoes were released across 28 489 locations over an area of 86·8 km 2 in Rio de Janeiro between Aug 29, 2017 and Dec 27, 2019. Following releases, mosquitoes were trapped and the presence of wMel was recorded. In this spatiotemporal modelling study, we assessed the effect of the release programme on the incidence of dengue and chikungunya. We used spatiotemporally explicit mathematical models applied to geocoded dengue cases (N=283 270) from 2010 to 2019 and chikungunya cases (N=57 705) from 2016 to 2019., Findings: On average, 32% of mosquitoes collected from the release zones between 1 month and 29 months after the initial release tested positive for wMel. Reduced wMel introgression occurred in locations and seasonal periods in which cases of dengue and chikungunya were historically high, with a decrease to 25% of mosquitoes testing positive for wMel during months in which disease incidence was at its highest. Despite incomplete introgression, we found that the releases were associated with a 38% (95% CI 32-44) reduction in the incidence of dengue and a 10% (4-16) reduction in the incidence of chikungunya., Interpretation: Stable establishment of wMel in the geographically diverse, urban setting of Rio de Janeiro seems to be more complicated than has been observed elsewhere. However, even intermediate levels of wMel seem to reduce the incidence of disease caused by two arboviruses. These findings will help to guide future release programmes., Funding: Bill & Melinda Gates Foundation and the European Research Council., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Arboviruses transmitted by Aedes aegypti including dengue, Zika, and chikungunya are a major global health problem, with over 2.5 billion at risk for dengue alone. There are no licensed antivirals for these infections, and safe and effective vaccines are not yet widely available. Thus, prevention of arbovirus transmission by vector modification is a novel approach being pursued by multiple researchers. However, the field needs high-quality evidence derived from randomized, controlled trials upon which to base the implementation and maintenance of vector control programs. Here, we report the EVITA Dengue trial design (DMID 17-0111), which assesses the efficacy in decreasing arbovirus transmission of an innovative approach developed by the World Mosquito Program for vector modification of Aedes mosquitoes by Wolbachia pipientis., Methods: DMID 17-0111 is a cluster-randomized trial in Belo Horizonte, Brazil, with clusters defined by primary school catchment areas. Clusters (n = 58) will be randomized 1:1 to intervention (release of Wolbachia-infected Aedes aegypti mosquitoes) vs. control (no release). Standard vector control activities (i.e., insecticides and education campaigns for reduction of mosquito breeding sites) will continue as per current practice in the municipality. Participants (n = 3480, 60 per cluster) are children aged 6-11 years enrolled in the cluster-defining school and living within the cluster boundaries who will undergo annual serologic surveillance for arboviral infection. The primary objective is to compare sero-incidence of arboviral infection between arms., Discussion: DMID 17-0111 aims to determine the efficacy of Wolbachia-infected mosquito releases in reducing human infections by arboviruses transmitted by Aedes aegypti and will complement the mounting evidence for this method from large-scale field releases and ongoing trials. The trial also represents a critical step towards robustness and rigor for how vector control methods are assessed, including the simultaneous measurement and correlation of entomologic and epidemiologic outcomes. Data from this trial will inform further the development of novel vector control methods., Trial Registration: ClinicalTrials.gov NCT04514107 . Registered on 17 August 2020 Primary sponsor: National Institute of Health, National Institute of Allergy and Infectious Diseases., (© 2022. The Author(s).)

In this review we examine how exploiting the Wolbachia-mosquito relationship has become an increasingly popular strategy for controlling arbovirus transmission. Field deployments of Wolbachia-infected mosquitoes have led to significant decreases in dengue virus incidence via high levels of mosquito population suppression and replacement, emphasizing the success of Wolbachia approaches. Here, we examine how improved knowledge of Wolbachia-host interactions has provided key insight into the mechanisms of the essential phenotypes of pathogen blocking and cytoplasmic incompatibility. And we discuss recent studies demonstrating that extrinsic factors, such as ambient temperature, can modulate Wolbachia density and maternal transmission. Finally, we assess the prospects of using Wolbachia to control other vectors and agricultural pest species., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The introduction of the bacterium Wolbachia (wMel strain) into Aedes aegypti mosquitoes reduces their capacity to transmit dengue and other arboviruses. Evidence of a reduction in dengue case incidence following field releases of wMel-infected Ae. aegypti has been reported previously from a cluster randomised controlled trial in Indonesia, and quasi-experimental studies in Indonesia and northern Australia., Methodology/principal Findings: Following pilot releases in 2015-2016 and a period of intensive community engagement, deployments of adult wMel-infected Ae. aegypti mosquitoes were conducted in Niterói, Brazil during 2017-2019. Deployments were phased across four release zones, with a total area of 83 km2 and a residential population of approximately 373,000. A quasi-experimental design was used to evaluate the effectiveness of wMel deployments in reducing dengue, chikungunya and Zika incidence. An untreated control zone was pre-defined, which was comparable to the intervention area in historical dengue trends. The wMel intervention effect was estimated by controlled interrupted time series analysis of monthly dengue, chikungunya and Zika case notifications to the public health surveillance system before, during and after releases, from release zones and the control zone. Three years after commencement of releases, wMel introgression into local Ae. aegypti populations was heterogeneous throughout Niterói, reaching a high prevalence (>80%) in the earliest release zone, and more moderate levels (prevalence 40-70%) elsewhere. Despite this spatial heterogeneity in entomological outcomes, the wMel intervention was associated with a 69% reduction in dengue incidence (95% confidence interval 54%, 79%), a 56% reduction in chikungunya incidence (95%CI 16%, 77%) and a 37% reduction in Zika incidence (95%CI 1%, 60%), in the aggregate release area compared with the pre-defined control area. This significant intervention effect on dengue was replicated across all four release zones, and in three of four zones for chikungunya, though not in individual release zones for Zika., Conclusions/significance: We demonstrate that wMel Wolbachia can be successfully introgressed into Ae. aegypti populations in a large and complex urban setting, and that a significant public health benefit from reduced incidence of Aedes-borne disease accrues even where the prevalence of wMel in local mosquito populations is moderate and spatially heterogeneous. These findings are consistent with the results of randomised and non-randomised field trials in Indonesia and northern Australia, and are supportive of the Wolbachia biocontrol method as a multivalent intervention against dengue, chikungunya and Zika., Competing Interests: The authors have declared that no competing interest exist.

The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti , Aedes albopictus , and Culex quinquefasciatus . We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.

Pathogens may manipulate their human and mosquito hosts to enhance disease transmission. Dengue, caused by four viral serotypes, is the fastest-growing transmissible disease globally resulting in 50-100 million infections annually. Transmission of the disease relies on the interaction between humans and the vector Aedes aegypti and is largely dependent on the odor-mediated host seeking of female mosquitoes. In this study, we use activity monitors to demonstrate that dengue virus-1 affects the locomotion and odor-mediated behavior of Ae. aegypti, reflecting the progression of infection within the mosquito. Mosquitoes 4-6 days post-infection increase locomotion, but do not alter their odor-driven host-seeking response. In contrast, females 14-16 days post-infection are less active, yet more sensitive to human odors as assessed by behavioral and electrophysiological assays. Such an increase in physiological and behavioral sensitivity is reflected by the antennal-specific increase in abundance of neural signaling transcripts in 14 days post-infection females, as determined by transcriptome analysis. This suggests that the sensitivity of the mosquito peripheral olfactory system is altered by the dengue virus by enhancing the overall neural responsiveness of the antenna, rather than the selective regulation of chemosensory-related genes. Our study reveals that dengue virus-1 enhances vector-related behaviors in the early stages post-infection that aid in avoiding predation and increasing spatial exploration. On the other hand, at the later stages of infection, the virus enhances the host-seeking capacity of the vector, thereby increasing the risk of virus transmission. A potential mechanism is discussed., Competing Interests: The authors have declared that no competing interests exist.

Mayaro virus (MAYV), a sylvatic arbovirus belonging to the Togaviridae family and Alphavirus genus, is responsible for an increasing number of outbreaks in several countries of Central and South America. Despite Haemagogus janthinomys being identified as the main vector of MAYV, laboratory studies have already demonstrated the competence of Aedes aegypti to transmit MAYV. It has also been demonstrated that the Wolbachia w Mel strain is able to impair the replication and transmission of MAYV in Ae. aegypti . In Ae. aegypti , the small interfering RNA (siRNA) pathway is an important antiviral mechanism; however, it remains unclear whether siRNA pathway acts against MAYV infection in Ae. aegypti . The main objective of this study was to determine the contribution of the siRNA pathway in the control of MAYV infection. Thus, we silenced the expression of AGO2, an essential component of the siRNA pathway, by injecting dsRNA-targeting AGO2 (dsAGO2). Our results showed that AGO2 is required to control MAYV replication upon oral infection in Wolbachia -free Ae. aegypti . On the other hand, we found that Wolbachia -induced resistance to MAYV in Ae. aegypti is independent of the siRNA pathway. Our study brought new information regarding the mechanism of viral protection, as well as on Wolbachia mediated interference.

Background: Rio de Janeiro and Niterói municipalities in southeastern Brazil experience large dengue epidemics every 2 to 5 years, with >100,000 cases notified in epidemic years. Costs of vector control and direct and indirect costs due to the Aedes -borne diseases dengue, chikungunya and Zika were estimated to total $650 million USD in 2016, but traditional vector control strategies have not been effective in preventing arboviral disease outbreaks. The Wolbachia method is a novel and self-sustaining approach for the biological control of arboviral diseases, in which the transmission potential of Ae. aegypti mosquitoes is reduced by stably transfecting them with the Wolbachia bacterium. This paper describes a study protocol for evaluating the effect of large-scale non-randomised releases of Wolbachia mosquitoes on the incidence of dengue, Zika and chikungunya in the municipalities of Niterói and Rio de Janeiro. This follows a lead-in period since 2014 involving intensive community engagement, regulatory and public approval, entomological surveys, and small-scale pilot releases. Method: The planned releases during 2017-2019 cover a combined area of 121 km2 with a resident population of 1.1 million, across the two cities. Untreated areas with comparable historical dengue profiles and sociodemographic characteristics have been identified a priori as comparative control areas in each municipality. The proposed pragmatic epidemiological approach combines a controlled interrupted time series analysis of routinely notified suspected and laboratory-confirmed arboviral cases, together with monitoring of arbovirus activity utilising outbreak signals routinely used in public health disease surveillance. Discussion: If the current project is successful, this model for control of arboviral disease through Wolbachia releases can be expanded nationally and regionally., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Durovni B et al.)

Background: Rio de Janeiro and Niterói are neighbouring cities in southeastern Brazil which experience large dengue epidemics every 2 to 5 years, with >100,000 cases notified in epidemic years. Costs of vector control and direct and indirect costs due to the Aedes -borne diseases dengue, chikungunya and Zika were estimated to total $650 million USD in 2016, but traditional vector control strategies have not been effective in preventing mosquito-borne disease outbreaks. The Wolbachia method is a novel and self-sustaining approach for the biological control of Aedes -borne diseases, in which the transmission potential of Aedes aegypti mosquitoes is reduced by stably transfecting them with the Wolbachia bacterium ( w Mel strain). This paper describes a study protocol for evaluating the effect of large-scale non-randomised releases of Wolbachia- -infected mosquitoes on the incidence of dengue, Zika and chikungunya in the two cities of Niterói and Rio de Janeiro. This follows a lead-in period since 2014 involving intensive community engagement, regulatory and public approval, entomological surveys, and small-scale pilot releases. Method: The Wolbachia releases during 2017-2019 covered a combined area of 170 km 2 with a resident population of 1.2 million, across Niterói and Rio de Janeiro. Untreated areas with comparable historical dengue profiles and demographic characteristics have been identified a priori as comparative control areas in each city. The proposed pragmatic epidemiological approach combines a controlled interrupted time series analysis of routinely notified suspected and laboratory-confirmed dengue and chikungunya cases, together with monitoring of Aedes -borne disease activity utilising outbreak signals routinely used in public health disease surveillance. Discussion: If the current project is successful, this model for control of mosquito-borne disease through Wolbachia releases can be expanded nationally and regionally., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Durovni B et al.)

Background: Wolbachia's ability to restrict arbovirus transmission makes it a promising tool to combat mosquito-transmitted diseases. Wolbachia-infected Aedes aegypti are currently being released in locations such as Brazil, which regularly experience concurrent outbreaks of different arboviruses. A. aegypti can become co-infected with, and transmit multiple arboviruses with one bite, which can complicate patient diagnosis and treatment., Methodology/principle Findings: Using experimental oral infection of A. aegypti and then RT-qPCR, we examined ZIKV/DENV-1 and ZIKV/DENV-3 co-infection in Wolbachia-infected A. aegypti and observed that Wolbachia-infected mosquitoes experienced lower prevalence of infection and viral load than wildtype mosquitoes, even with an extra infecting virus. Critically, ZIKV/DENV co-infection had no significant impact on Wolbachia's ability to reduce viral transmission. Wolbachia infection also strongly altered expression levels of key immune genes Defensin C and Transferrin 1, in a virus-dependent manner., Conclusions/significance: Our results suggest that pathogen interference in Wolbachia-infected A. aegypti is not adversely affected by ZIKV/DENV co-infection, which suggests that Wolbachia-infected A. aegypti will likely prove suitable for controlling mosquito-borne diseases in environments with complex patterns of arbovirus transmission., Competing Interests: The authors have declared that no competing interests exist.

New possibilities for vector-borne disease control are revealed by Duvall et al. (2019), who link host-seeking behavioral modulation in Aedes aegypti to neuropeptide Y (NPY)-like receptor 7. Small-molecule screening yields agonist compounds able to activate NPYLR7 and suppress attraction to hosts., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Dengue virus (DENV) is an arbovirus transmitted to humans by Aedes mosquitoes 1 . In the insect vector, the small interfering RNA (siRNA) pathway is an important antiviral mechanism against DENV 2-5 . However, it remains unclear when and where the siRNA pathway acts during the virus cycle. Here, we show that the siRNA pathway fails to efficiently silence DENV in the midgut of Aedes aegypti although it is essential to restrict systemic replication. Accumulation of DENV-derived siRNAs in the midgut reveals that impaired silencing results from a defect downstream of small RNA biogenesis. Notably, silencing triggered by endogenous and exogenous dsRNAs remained effective in the midgut where known components of the siRNA pathway, including the double-stranded RNA (dsRNA)-binding proteins Loquacious and r2d2, had normal expression levels. We identified an Aedes-specific paralogue of loquacious and r2d2, hereafter named loqs2, which is not expressed in the midgut. Loqs2 interacts with Loquacious and r2d2 and is required to control systemic replication of DENV and also Zika virus. Furthermore, ectopic expression of Loqs2 in the midgut of transgenic mosquitoes is sufficient to restrict DENV replication and dissemination. Together, our data reveal a mechanism of tissue-specific regulation of the mosquito siRNA pathway controlled by Loqs2.

Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W-) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism.

Mosquito species, members of the genera Aedes, Anopheles and Culex, are the major vectors of human pathogens including protozoa (Plasmodium sp.), filariae and of a variety of viruses (causing dengue, chikungunya, yellow fever, West Nile). There is lack of efficient methods and tools to treat many of the diseases caused by these major human pathogens, since no efficient vaccines or drugs are available; even in malaria where insecticide use and drug therapies have reduced incidence, 219 million cases still occurred in 2010. Therefore efforts are currently focused on the control of vector populations. Insecticides alone are insufficient to control mosquito populations since reduced susceptibility and even resistance is being observed more and more frequently. There is also increased concern about the toxic effects of insecticides on non-target (even beneficial) insect populations, on humans and the environment. During recent years, the role of symbionts in the biology, ecology and evolution of insect species has been well-documented and has led to suggestions that they could potentially be used as tools to control pests and therefore diseases. Wolbachia is perhaps the most renowned insect symbiont, mainly due to its ability to manipulate insect reproduction and to interfere with major human pathogens thus providing new avenues for pest control. We herein present recent achievements in the field of mosquito-Wolbachia symbiosis with an emphasis on Aedes albopictus. We also discuss how Wolbachia symbiosis can be harnessed for vector control as well as the potential to combine the sterile insect technique and Wolbachia-based approaches for the enhancement of population suppression programs., (Copyright © 2013 International Atomic Energy Agency 2013. Published by Elsevier B.V. All rights reserved.)

The obligate endosymbiont Wolbachia pipientis is found in a wide range of invertebrates where they are best known for manipulating host reproduction. Recent studies have shown that Wolbachia also can modulate the lifespan of host insects and interfere with the development of human pathogens in mosquito vectors. Despite considerable study, very little is known about the molecular interactions between Wolbachia and its hosts that might mediate these effects. Using microarrays, we show that the microRNA (miRNA) profile of the mosquito, Aedes aegypti, is significantly altered by the wMelPop-CLA strain of W. pipientis. We found that a host miRNA (aae-miR-2940) is induced after Wolbachia infection in both mosquitoes and cell lines. One target of aae-miR-2940 is the Ae. aegypti metalloprotease gene. Interestingly, expression of the target gene was induced after Wolbachia infection, ectopic expression of the miRNA independent of Wolbachia, or transfection of an artificial mimic of the miRNA into mosquito cells. We also confirmed the interaction of aae-miR-2940 with the target sequences using GFP as a reporter gene. Silencing of the metalloprotease gene in both Wolbachia-infected cells and adult mosquitoes led to a significant reduction in Wolbachia density, as did inhibition of the miRNA in cells. These results indicate that manipulation of the mosquito metalloprotease gene via aae-miR-2940 is crucial for efficient maintenance of the endosymbiont. This report shows how Wolbachia alters the host miRNA profile and provides insight into the mechanisms of host manipulation used by this widespread endosymbiont.

Introduction of the life-shortening strain of Wolbachia pipientis, wMelPop, into the key dengue vector, Aedes aegypti, and the anti-pathogen effects in Wolbachia-infected hosts highlights the need for more research into its interactions with its original host, Drosophila melanogaster, and the novel mosquito host. The visual difference in darkness between the eggs of wMelPop Wolbachia-infected and uninfected mosquito hosts after egg deposition led to further investigation into melanization levels of the insects. Both D. melanogaster and A. aegypti infected with wMelPop showed increased levels of melanization, especially in females. This result was also seen in D. melanogaster and Drosophila simulans infected with the closely related wMel strain. D. simulans infected with other strains of Wolbachia did not display this difference. HPLC analysis of hemolymph from mosquitoes showed that this difference was not due to dopamine levels in the host as they were no different in wMelPop-infected and control mosquitoes before or after blood feeding., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Wolbachia is an intracellular bacterium that has been stably transinfected into the mosquito vector of dengue, Aedes aegypti. This inherited infection causes a range of metabolic and phenotypic alterations in the mosquito, which might be related to neuronal abnormalities. In order to determine if these alterations were caused by the manipulation of neuroamines by this bacterium, we studied the expression of genes involved in the dopamine biosynthetic pathway and also measured the amount of dopamine in infected and uninfected mosquitoes of different ages. Wolbachia-infected mosquitoes exhibit greater expression of some genes related to the melanization pathway, but not for those directly linked to dopamine production. Although dopamine levels were higher in Wolbachia-positive mosquitoes this was not consistent across all insect ages nor was it related to the previously described Wolbachia induced "bendy" and "shaky" phenotypes.

Vector-born diseases cause millions of deaths every year globally. Alternatives for the control of diseases such as malaria and dengue fever are urgently needed and the use of transgenic mosquitoes that block parasite/virus is a sound strategy to be used within control programs. However, prior to use transgenic mosquitoes as control tools, it is important to study their fitness since different biological aspects might influence their ability to disseminate and compete with wild populations. We previously reported the construction of four transgenic Aedes fluviatilis mosquito lines expressing a Plasmodium- blocking molecule (mutated bee venom phospholipase A(2)-mPLA(2)). Presently we studied two aspects of their fitness: body size, that has been used as a fitness-related status, and the expression of major enzymes classes involved in the metabolism of xenobiotics, including insecticides. Body size analysis (recorded by geometric wing morphometrics) indicated that both male and female mosquitoes were larger than the non-transgenic counterparts, suggesting that this characteristic might have an impact on their overall fitness. By contrast, no significant difference in the activity of enzymes related to metabolic insecticide resistance was detected in transgenic mosquitoes. The implication on fitness advantage of these features, towards the implementation of this strategy, is further discussed.

Mosquito-borne diseases such as dengue fever, chikungunya or malaria affect millions of people each year and control solutions are urgently needed. An international research program is currently being developed that relies on the introduction of the bacterial endosymbiont Wolbachia pipientis into Aedes aegypti to control dengue transmission. In order to prepare for open-field testing releases of Wolbachia-infected mosquitoes, an intensive social research and community engagement program was undertaken in Cairns, Northern Australia. The most common concern expressed by the diverse range of community members and stakeholders surveyed was the necessity of assuring the safety of the proposed approach for humans, animals and the environment. To address these concerns a series of safety experiments were undertaken. We report in this paper on the experimental data obtained, discuss the limitations of experimental risk assessment and focus on the necessity of including community concerns in scientific research.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases., (Copyright 2009 Elsevier Inc. All rights reserved.)

Background: Mosquitoes are vectors of many serious pathogens in tropical and sub-tropical countries. Current control strategies almost entirely rely upon insecticides, which increasingly face the problems of high cost, increasing mosquito resistance and negative effects on non-target organisms. Alternative strategies include the proposed use of inherited life-shortening agents, such as the Wolbachia bacterium. By shortening mosquito vector lifespan, Wolbachia could potentially reduce the vectorial capacity of mosquito populations. We have recently been able to stably transinfect Aedes aegypti mosquitoes with the life-shortening Wolbachia strain wMelPop, and are assessing various aspects of its interaction with the mosquito host to determine its likely impact on pathogen transmission as well as its potential ability to invade A. aegypti populations., Methodology/principal Findings: Here we have examined the probing behavior of Wolbachia-infected mosquitoes in an attempt to understand both the broader impact of Wolbachia infection on mosquito biology and, in particular, vectorial capacity. The probing behavior of wMelPop-infected mosquitoes at four adult ages was examined and compared to uninfected controls during video-recorded feeding trials on a human hand. Wolbachia-positive insects, from 15 days of age, showed a drastic increase in the time spent pre-probing and probing relative to uninfected controls. Two other important features for blood feeding, saliva volume and apyrase content of saliva, were also studied., Conclusions/significance: As A. aegypti infected with wMelPop age, they show increasing difficulty in completing the process of blood feeding effectively and efficiently. Wolbachia-infected mosquitoes on average produced smaller volumes of saliva that still contained the same amount of apyrase activity as uninfected mosquitoes. These effects on blood feeding behavior may reduce vectorial capacity and point to underlying physiological changes in Wolbachia-infected mosquitoes.

In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions. [ABSTRACT FROM AUTHOR]

Mosquito breeding sites are complex aquatic environments with wide microbial diversity and physicochemical parameters that can change over time during the development of immature insect stages. Changes in biotic and abiotic conditions in water can alter life-history traits of adult mosquitos but this area remains understudied. Here, using microbial genomic and metabolomics analyses, we explored the metabolites associated with Aedes aegypti breeding sites as well as the potential contribution of Klebsiella sp., symbiotic bacteria highly associated with mosquitoes. We sought to address whether breeding sites have a signature metabolic profile and understand the metabolite contribution of the bacteria in the aquatic niches where Ae. aegypti larvae develop. An analysis of 32 mosquito-associated bacterial genomes, including Klebsiella , allowed us to identify gene clusters involved in primary metabolic pathways. From them, we inferred metabolites that could impact larval development (e.g., spermidine), as well as influence the quality assessment of a breeding site by a gravid female (e.g., putrescine), if produced by bacteria in the water. We also detected significant variance in metabolite presence profiles between water samples representing a decoupled oviposition event (oviposition by single females and manually deposited eggs) versus a control where no mosquito interactions occurred (PERMANOVA: p < 0.05; R 2 = 24.64% and R 2 = 30.07%). Five Klebsiella metabolites were exclusively linked to water samples where oviposition and development occurred. These data suggest metabolomics can be applied to identify compounds potentially used by female Ae. aegypti to evaluate the quality of a breeding site. Elucidating the physiological mechanisms by which the females could integrate these sensory cues while ovipositing constitutes a growing field of interest, which could benefit from a more depurated list of candidate molecules. [ABSTRACT FROM AUTHOR]

Traditional methods of vector control have proven insufficient to reduce the alarming incidence of dengue, Zika, and chikungunya in endemic countries. The bacterium symbiont Wolbachia has emerged as an efficient pathogen-blocking and self-dispersing agent that reduces the vectorial potential of Aedes aegypti populations and potentially impairs arboviral disease transmission. In this work, we report the results of a large-scale Wolbachia intervention in Ilha do Governador, Rio de Janeiro, Brazil. w Mel-infected adults were released across residential areas between August 2017 and March 2020. Over 131 weeks, including release and post-release phases, we monitored the w Mel prevalence in field specimens and analyzed introgression profiles of two assigned intervention areas, RJ1 and RJ2. Our results revealed that w Mel successfully invaded both areas, reaching overall infection rates of 50–70% in RJ1 and 30–60% in RJ2 by the end of the monitoring period. At the neighborhood-level, w Mel introgression was heterogeneous in both RJ1 and RJ2, with some profiles sustaining a consistent increase in infection rates and others failing to elicit the same. Correlation analysis revealed a weak overall association between RJ1 and RJ2 (r = 0.2849, p = 0.0236), and an association at a higher degree when comparing different deployment strategies, vehicle or backpack-assisted, within RJ1 (r = 0.4676, p < 0.0001) or RJ2 (r = 0.6263, p < 0.0001). The frequency knockdown resistance (kdr) alleles in w Mel-infected specimens from both areas were consistently high over this study. Altogether, these findings corroborate that w Mel can be successfully deployed at large-scale as part of vector control intervention strategies and provide the basis for imminent disease impact studies in Southeastern Brazil. [ABSTRACT FROM AUTHOR]

Dengue represents a serious threat to human health, with billions of people living at risk of the disease. Wolbachia pipientis is a bacterial endosymbiont common to many insect species. Wolbachia transinfections in mosquito disease vectors have great value for disease control given the bacterium’s ability to spread into wild mosquito populations, and to interfere with infections of pathogens, such as dengue virus. Aedes fluviatilis is a mosquito with a widespread distribution in Latin America, but its status as a dengue vector has not been clarified. Ae. fluviatilis is also naturally infected by the wFlu Wolbachia strain, which has been demonstrated to enhance infection with the avian malarial parasite Plasmodium gallinaceum. We performed experimental infections of Ae. fluviatilis with DENV-2 and DENV-3 isolates from Brazil via injection or oral feeding to provide insight into its competence for the virus. We also examined the effect of the native Wolbachia infection on the virus using a mosquito line where the wFlu infection had been cleared by antibiotic treatment. Through RT-qPCR, we observed that Ae. fluviatilis could become infected with both viruses via either method of infection, although at a lower rate than Aedes aegypti, the primary dengue vector. We then detected DENV-2 and DENV-3 in the saliva of injected mosquitoes, and observed that injection of DENV-3-infected saliva produced subsequent infections in naïve Ae. aegypti. However, across our data we observed no difference in prevalence of infection and viral load between Wolbachia-infected and -uninfected mosquitoes, suggesting that there is no effect of wFlu on dengue virus. Our results highlight that Ae. fluviatilis could potentially serve as a dengue vector under the right circumstances, although further testing is required to determine if this occurs in the field. [ABSTRACT FROM AUTHOR]

Wolbachia pipientis, a maternally transmitted bacterium that colonizes arthropods, may affect the general aspects of insect physiology, particularly reproduction. Wolbachia is a natural endosymbiont of Aedes fluviatilis, whose effects in embryogenesis and reproduction have not been addressed so far. In this context, we investigated the correlation between glucose metabolism and morphological alterations during A. fluviatilis embryo development in Wolbachia-positive (W+) and Wolbachia-negative (W−) mosquito strains. While both strains do not display significant morphological and larval hatching differences, larger differences were observed in hexokinase activity and glycogen contents during early and mid-stages of embryogenesis, respectively. To investigate if glycogen would be required for parasite-host interaction, we reduced Glycogen Synthase Kinase-3 (GSK-3) levels in adult females and their eggs by RNAi. GSK-3 knock-down leads to embryonic lethality, lower levels of glycogen and total protein and Wolbachia reduction. Therefore, our results suggest that the relationship between A. fluviatilis and Wolbachia may be modulated by glycogen metabolism. [ABSTRACT FROM AUTHOR]

Wolbachia pipientis is a maternally transmitted symbiotic bacterium that mainly colonizes arthropods, potentially affecting different aspects of the host’s physiology, e.g., reproduction, immunity, and metabolism. It has been shown that Wolbachia modulates glycogen metabolism in mosquito Aedes fluviatilis (Ae. fluviatilis). Glycogen synthesis is controlled by the enzyme GSK3, which is also involved in immune responses in both vertebrate and invertebrate organisms. Here we investigated the mechanisms behind immune changes mediated by glycogen synthase kinase β (GSK3β) in the symbiosis between Ae. fluviatilis and W. pipientis using a GSK3β inhibitor or RNAi-mediated gene silencing. GSK3β inhibition or knockdown increased glycogen content and Wolbachia population, together with a reduction in Relish2 and gambicin transcripts. Furthermore, knockdown of Relish2 or Caspar revealed that the immunodeficiency pathway acts to control Wolbachia numbers in the host. In conclusion, we describe for the first time the involvement of GSK3β in Ae. fluviatilis immune response, acting to control the Wolbachia endosymbiotic population. [ABSTRACT FROM AUTHOR]

Wolbachia pipientis is a maternally transmitted bacterium that mostly colonizes arthropods, including the mosquito Aedes fluviatilis , potentially affecting different aspects of host physiology. This intracellular bacterium prefers gonadal tissue cells, interfering with the reproductive cycle of insects, arachnids, crustaceans, and nematodes. Wolbachia's ability to modulate the host's reproduction is related to its success in prevalence and frequency. Infecting oocytes is essential for vertical propagation, ensuring its presence in the germline. The mosquito Ae. fluviatilis is a natural host for this bacterium and therefore represents an excellent experimental model in the effort to understand host-symbiont interactions and the mutual metabolic regulation. The aim of this study was to comparatively describe metabolic changes in naturally Wolbachia -infected and uninfected ovaries of Ae. fluviatilis during the vitellogenic period of oogenesis, thus increasing the knowledge about Wolbachia parasitic/symbiotic mechanisms. [Display omitted] • Wolbachia is fundamental to maintained the immune-metabolic equilibrium during oogenesis of the Ae. fluviatilis mosquito. • The relative content of Wolbachia pipientis increases during oogenesis. • Forty-eight hours after blood feeding has a significant development reduction of ovaries morphological in mosquitoes without Wolbachia. [ABSTRACT FROM AUTHOR]