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Abstract Background Aedes aegypti, the main arboviral mosquito vector, is attracted to human dwellings and makes use of human-generated breeding sites. Past research has shown that bacterial communities associated with such sites undergo compositional shifts as larvae develop and that exposure to different bacteria during larval stages can have an impact on mosquito development and life-history traits. Based on these facts, we hypothesized that female Ae. aegypti shape the bacteria communities of breeding sites during oviposition as a form of niche construction to favor offspring fitness. Results To test this hypothesis, we first verified that gravid females can act as mechanical vectors of bacteria. We then elaborated an experimental scheme to test the impact of oviposition on breeding site microbiota. Five different groups of experimental breeding sites were set up with a sterile aqueous solution of larval food, and subsequently exposed to (1) the environment alone, (2) surface-sterilized eggs, (3) unsterilized eggs, (4) a non-egg laying female, or (5) oviposition by a gravid female. The microbiota of these differently treated sites was assessed by amplicon-oriented DNA sequencing once the larvae from the sites with eggs had completed development and formed pupae. Microbial ecology analyses revealed significant differences between the five treatments in terms of diversity. In particular, between-treatment shifts in abundance profiles were detected, showing that females induce a significant decrease in microbial alpha diversity through oviposition. In addition, indicator species analysis pinpointed bacterial taxa with significant predicting values and fidelity coefficients for the samples in which single females laid eggs. Furthermore, we provide evidence regarding how one of these indicator taxa, Elizabethkingia, exerts a positive effect on the development and fitness of mosquito larvae. Conclusions Ovipositing females impact the composition of the microbial community associated with a breeding site, promoting certain bacterial taxa over those prevailing in the environment. Among these bacteria, we found known mosquito symbionts and showed that they can improve offspring fitness if present in the water where eggs are laid. We deem this oviposition-mediated bacterial community shaping as a form of niche construction initiated by the gravid female.

Abstract 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

Abstract Background Aedes aegypti control programs have failed to restrain mosquito population expansion and, consequently, the spread of diseases such as dengue, Zika, and Chikungunya. Wolbachia infection of mosquitoes is a new and promising complementary tool for the control of arbovirus transmission. The use of Wolbachia-infected mosquitoes, mass reared using human blood, is currently being tested in several countries. However, the use of human blood for mass rearing mosquitoes, and thus expansion of this strategy, is problematic. With the aim of overcoming this problem, we tested the effect of different types of blood source on the fitness parameters of female Ae. aegypti and the Wolbachia titer over generations to be able to guarantee the suitability of an alternative source to human blood for mass rearing Wolbachia-infected mosquitoes. Methods We investigated and compared essential parameters of the vector capacity of laboratory strains of Ae. aegypti with and without Wolbachia that fed on blood of different types of host (human, guinea pig, and mouse). The parameters analyzed were fecundity, fertility, pupation dynamics, and adult survival. Also, we tested whether it is possible to maintain mosquitoes with Wolbachia on mouse blood over generations without losing the bacterium titer. Results The average number of eggs per female, egg viability and pupation dynamics in the Wolbachia-infected mosquito (wMelBr) strain were similar, regardless of the blood source. The F1 progenies of females that fed on mouse blood or human blood were analyzed. The longevity of males was lower than that of females. F1 female survival differed depending on the presence of Wolbachia in the mother. In subsequent generations analyzed up until F35, the relative Wolbachia density was even higher when mosquitoes fed on mouse blood in comparison to human blood. Conclusions Taken together, our results provide no evidence that the different types of blood influenced the fitness of the Wolbachia-infected mosquitoes. The presence of the bacterium in the colonies of Wolbachia-infected Ae. aegypti after 35 generations under the conditions evaluated indicates that they can be maintained on mouse blood. Based on these results, we show that it is possible to use mouse blood to feed female mosquitoes when using human blood for this purpose is problematic.

Abstract Background Aedes aegypti is a major disease vector in urban habitats, involved in the transmission of dengue, chikungunya and Zika. Despite innumerous attempts to contain disease outbreaks, there are neither efficient vaccines nor definite vector control methods nowadays. In recent years, an innovative strategy to control arboviruses, which exploits the endosymbiotic bacterium Wolbachia pipientis, emerged with great expectations. The success of the method depends on many aspects, including Wolbachia’s cytoplasmic incompatibility and pathogen interference phenotypes, as well as its effect on host fitness. In this work, we investigated the influence the Wolbachia strain wMel exerts on embryo development and egg viability and speculate on its field release use. Methods Wild-type (Br or Rockefeller) and Wolbachia-harboring specimens (wMelBr) were blood-fed and submitted to synchronous egg laying for embryo development assays. Samples were analyzed for morphological markers, developmental endpoint and egg resistance to desiccation (ERD). Quiescent egg viability over time was also assessed. Results wMelBr samples completed embryogenesis 2–3 hours later than wild-type. This delay was also observed through the onset of both morphological and physiological markers, respectively by the moments of germband extension and ERD acquisition. Following the end of embryonic development, wMelBr eggs were slightly less resistant to desiccation and showed reduced viability levels, which rapidly decayed after 40 days into quiescence, from approximately 75% to virtually 0% in less than a month. Conclusions Our data revealed that the wMel strain of Wolbachia slightly delays embryogenesis and also affects egg quality, both through reduced viability and desiccation resistance. These findings suggest that, although embryonic fitness is somehow compromised by wMel infection, an efficient host reproductive manipulation through cytoplasmic incompatibility seems sufficient to overcome these effects in nature and promote bacterial invasion, as shown by successful ongoing field implementation.

Abstract 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.

Abstract Background Mosquito-borne diseases are rapidly spreading to vast territories, putting at risk most of the world’s population. A key player in this scenario is Aedes aegypti, a hematophagous species which hosts and transmits viruses causing dengue and other serious illnesses. Since vector control strategies relying only on insecticides have proven unsustainable, an alternative method involving the release of Wolbachia-harboring individuals has emerged. Its successful implementation vastly depends on how fit the released individuals are in the natural habitat, being able to mate with wild populations and to spread Wolbachia to subsequent generations. In mosquitoes, an important aspect of reproductive fitness is the acoustic communication between males and females, which translates to interactions between harmonic frequencies in close proximity flight. This study aimed to characterize the flight tone produced by individuals harboring Wolbachia, also evaluating their ability to establish stable acoustic interactions. Methods Wild-type (WT) and Wolbachia-harboring specimens (wMelBr) were thorax-tethered to blunt copper wires and placed at close proximity to sensitive microphones. Wing-beat frequencies (WBFs) were characterized at fundamental and harmonic levels, for both single individuals and couples. Harmonic interactions in homogeneous and heterogeneous couples of WT and wMelBr variants were identified, categorized and quantified accordingly. Results In tethered ‘solo’ flights, individuals harboring Wolbachia developed WBFs, differing slightly, in a sex-dependent way, from those of the WT strain. To test the ability to form harmonic ‘duets’, tethered couples of wMelBr and WT individuals were shuffled in different sex pairs and had their flight tones analyzed. All couple types, with WT and/or wMelBr individuals, were able to interact acoustically in the frequency range of 1300–1500 Hz, which translates to the convergence between male’s second harmonic and female’s third. No significant differences were found in the proportions of interacting couples between the pair types. Surprisingly, spectrograms also revealed the convergence between alternative harmonic frequencies, inside and outside the species putative hearing threshold. Conclusions Wolbachia infection leads to small sex-dependent changes on the flight tones of Ae. aegypti, but it does not seem to prevent the stereotyped harmonic interaction between males and females. Therefore, when released in the natural habitat to breed with native individuals, Wolbachia-harboring individuals shall be fit enough to meet the criteria of acoustically-related mating behavior and promote bacteria dispersion effectively.

Abstract Background Wolbachia pipientis is a common endosymbiotic bacterium of arthropods that strongly inhibits dengue virus (DENV) infection and transmission in the primary vector, the mosquito Aedes aegypti. For that reason, Wolbachia-infected Ae. aegypti are currently being released into the field as part of a novel strategy to reduce DENV transmission. However, there is evidence that DENV can be transmitted vertically from mother to progeny, and this may help the virus persist in nature in the absence of regular human transmission. The effect of Wolbachia infection on this process had not previously been examined. Results We challenged Ae. aegypti with different Brazilian DENV isolates either by oral feeding or intrathoracic injection to ensure disseminated infection. We examined the effect of Wolbachia infection on the prevalence of DENV infection, and viral load in the ovaries. For orally infected mosquitoes, Wolbachia decreased the prevalence of infection by 71.29%, but there was no such effect when the virus was injected. Interestingly, regardless of the method of infection, Wolbachia infection strongly reduced DENV load in the ovaries. We then looked at the effect of Wolbachia on vertical transmission, where we observed only very low rates of vertical transmission. There was a trend towards lower rates in the presence of Wolbachia, with overall maximum likelihood estimate of infection rates of 5.04 per 1000 larvae for mosquitoes without Wolbachia, and 1.93 per 1000 larvae for Wolbachia-infected mosquitoes, after DENV injection. However, this effect was not statistically significant. Conclusions Our data support the idea that vertical transmission of DENV is rare in nature, even in the absence of Wolbachia. Indeed, we observed that vertical transmission rates were low even when the midgut barrier was bypassed, which might help to explain why we only observed a trend towards lower vertical transmission rates in the presence of Wolbachia. Nevertheless, the low prevalence of disseminated DENV infection and lower DENV load in the ovaries supports the hypothesis that the presence of Wolbachia in Ae. aegypti would have an effect on the vertical transmission of DENV in the field.

An amendment to this paper has been published and can be accessed via the original article.

Abstract Background The bacterium Wolbachia is a promising agent for the biological control of vector-borne diseases as some strains have the ability to block the transmission of key human disease-causing pathogens. Fast, accurate and inexpensive methods of differentiating between infected and uninfected insects will be of critical importance as field-based trials of Wolbachia-based bio-control become increasingly common. Findings We have developed a specific and sensitive method of detecting Wolbachia based on the isothermal DNA amplification. This technique can be performed in an ordinary heat block without the need for gel-based visualisation, and is effective for a wide variety of insect hosts. Conclusion Here we present the development of a rapid, highly sensitive and inexpensive method to detect Wolbachia in a variety of insect hosts, including key mosquito disease vectors.