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Many insects and other animals carry microbial endosymbionts that influence their reproduction and fitness. These relationships only persist if endosymbionts are reliably transmitted from one host generation to the next. Wolbachia are maternally transmitted endosymbionts found in most insect species, but transmission rates can vary across environments. Maternal transmission of wMel Wolbachia depends on temperature in natural Drosophila melanogaster hosts and in transinfected Aedes aegypti, where wMel is used to block pathogens that cause human disease. In D. melanogaster, wMel transmission declines in the cold as Wolbachia become less abundant in host ovaries and at the posterior pole plasm (the site of germline formation) in mature oocytes. Here, we assess how temperature affects maternal transmission and underlying patterns of Wolbachia localization across 10 Wolbachia strains diverged up to 50 million years-including strains closely related to wMel-and their natural Drosophila hosts. Many Wolbachia maintain high transmission rates across temperatures, despite highly variable (and sometimes low) levels of Wolbachia in the ovaries and at the developing germline in late-stage oocytes. Identifying strains like closely related wMel-like Wolbachia with stable transmission across variable environmental conditions may improve the efficacy of Wolbachia-based biocontrol efforts as they expand into globally diverse environments.

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Several countries have been using Wolbachia deployments to replace highly competent native Aedes aegypti populations with Wolbachia-carrying mosquitoes with lower susceptibility to arboviruses such as dengue, Zika, and chikungunya. In Rio de Janeiro, Wolbachia deployments started in 2015 and still present a moderate introgression with a modest reduction in dengue cases in humans (38%). Here, we evaluated the vector competence of wild-type and wMel-infected Ae. aegypti with a Brazilian genetic background to investigate whether virus leakage could contribute to the observed outcomes in Brazil. We collected the specimens in three areas of Rio de Janeiro with distinct frequencies of mosquitoes with wMel strain and two areas with wild Ae. aegypti. The mosquitoes were orally exposed to two titers of DENV-1 and the saliva of DENV-1-infected Ae. aegypti was microinjected into wMel-free mosquitoes to check their infectivity. When infected with the high DENV-1 titer, the presence of wMel did not avoid viral infection in mosquitoes' bodies and saliva but DENV-1-infected wMel mosquitoes produced lower viral loads than wMel-free mosquitoes. On the other hand, wMel mosquitoes infected with the low DENV-1 titer were less susceptible to virus infection than wMel-free mosquitoes, although once infected, wMel and wMel-free mosquitoes exhibited similar viral loads in the body and the saliva. Our results showed viral leakage in 60% of the saliva of wMel mosquitoes with Brazilian background; thus, sustained surveillance is imperative to monitor the presence of other circulating DENV-1 strains capable of overcoming the Wolbachia blocking phenotype, enabling timely implementation of action plans.

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The introgression of antiviral strains of Wolbachia into Aedes aegypti mosquito populations is a public health intervention for the control of dengue. Plausibly, dengue virus (DENV) could evolve to bypass the antiviral effects of Wolbachia and undermine this approach. Here, we established a serial-passage system to investigate the evolution of DENV in Ae. aegypti mosquitoes infected with the wMel strain of Wolbachia. Using this system, we report on virus genetic outcomes after twenty passages of serotype 1 of DENV (DENV-1). An amino acid substitution, E203K, in the DENV-1 envelope protein was more frequently detected in the consensus sequence of virus populations passaged in wMel-infected Ae. aegypti than wild-type counterparts. Positive selection at residue 203 was reproducible; it occurred in passaged virus populations from independent DENV-1-infected patients and also in a second, independent experimental system. In wild-type mosquitoes and human cells, the 203K variant was rapidly replaced by the progenitor sequence. These findings provide proof of concept that wMel-associated selection of virus populations can occur in experimental conditions. Field-based studies are needed to explore whether wMel imparts selective pressure on DENV evolution in locations where wMel is established.

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BACKGROUND: Dengue virus serotypes (DENV-1 to -4) can be transmitted vertically in Aedes aegpti mosquitoes. Whether infection with the wMel strain of the endosymbiont Wolbachia can reduce the incidence of vertical transmission of DENV from infected females to their offspring is not well understood. METHODS: A laboratory colony of Vietnamese Ae. aegypti, both with and without wMel infection, were infected with DENV-1 by intrathoracic injection (IT) to estimate the rate of vertical transmission (VT) of the virus. VT in the DENV-infected mosquitoes was calculated via the infection rate estimation from mosquito pool data using maximum likelihood estimation (MLE). RESULTS: In 6047 F1 Vietnamese wild-type Ae. aegypti, the MLE of DENV-1 infection was 1.49 per 1000 mosquitoes (95% confidence interval [CI] 0.73-2.74). In 5500 wMel-infected Ae. aegypti, the MLE infection rate was 0 (95% CI 0-0.69). The VT rates between mosquito lines showed a statistically significant difference. CONCLUSIONS: The results reinforce the view that VT is a rare event in wild-type mosquitoes and that infection with wMel is effective in reducing VT.

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(1) Background: The deployment of the bacterium Wolbachia to reduce arbovirus transmission is ongoing in several countries worldwide. When Wolbachia-carrying Aedes aegypti are released and established in the field, females may feed on dengue-infected hosts. The effects of simultaneous exposure on life-history traits of Ae. aegypti to Wolbachia wMel strain and dengue-1 virus DENV-1 remain unclear. (2) Methods: We monitored 4 groups (mosquitoes with either DENV-1 or Wolbachia, coinfected with DENV-1 and Wolbachia, as well as negative controls) to estimate Ae. aegypti survival, oviposition success, fecundity, collapsing and fertility of quiescent eggs for 12 weeks. (3) Results: Neither DENV-1 nor Wolbachia had a significant impact on mosquito survival nor on mosquito fecundity, although the last parameter showed a tendency to decrease with ageing. There was a significant decrease in oviposition success in individuals carrying Wolbachia. Wolbachia infection and storage time significantly increased egg collapse parameter on the egg viability assay, while DENV-1 had a slight protective effect on the first four weeks of storage. (4) Conclusions: Despite limitations, our results contribute to better understanding of the tripartite interaction of virus, bacteria and mosquito that may take place in field conditions and aid in guaranteeing the Wolbachia strategy success.

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A broad array of endosymbionts radiate through host populations via vertical transmission, yet much remains unknown concerning the cellular basis, diversity, and routes underlying this transmission strategy. Here, we address these issues, by examining the cellular distributions of Wolbachia strains that diverged up to 50 million years ago in the oocytes of 18 divergent Drosophila species. This analysis revealed 3 Wolbachia distribution patterns: (1) a tight clustering at the posterior pole plasm (the site of germline formation); (2) a concentration at the posterior pole plasm, but with a significant bacteria population distributed throughout the oocyte; and (3) a distribution throughout the oocyte, with none or very few located at the posterior pole plasm. Examination of this latter class indicates Wolbachia accesses the posterior pole plasm during the interval between late oogenesis and the blastoderm formation. We also find that 1 Wolbachia strain in this class concentrates in the posterior somatic follicle cells that encompass the pole plasm of the developing oocyte. In contrast, strains in which Wolbachia concentrate at the posterior pole plasm generally exhibit no or few Wolbachia in the follicle cells associated with the pole plasm. Taken together, these studies suggest that for some Drosophila species, Wolbachia invade the germline from neighboring somatic follicle cells. Phylogenomic analysis indicates that closely related Wolbachia strains tend to exhibit similar patterns of posterior localization, suggesting that specific localization strategies are a function of Wolbachia-associated factors. Previous studies revealed that endosymbionts rely on 1 of 2 distinct routes of vertical transmission: continuous maintenance in the germline (germline-to-germline) or a more circuitous route via the soma (germline-to-soma-to-germline). Here, we provide compelling evidence that Wolbachia strains infecting Drosophila species maintain the diverse arrays of cellular mechanisms necessary for both of these distinct transmission routes. This characteristic may account for its ability to infect and spread globally through a vast range of host insect species.

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BACKGROUND: Releases of Wolbachia (wMel)-infected Aedes aegypti mosquitoes significantly reduced the incidence of virologically confirmed dengue in a previous cluster randomised trial in Yogyakarta City, Indonesia. Following the trial, wMel releases were extended to the untreated control areas, to achieve city-wide coverage of Wolbachia. OBJECTIVE: In this predefined analysis, we evaluated the impact of the wMel deployments in Yogyakarta on dengue hemorrhagic fever (DHF) case notifications and on the frequency of perifocal insecticide spraying by public health teams. METHODS: Monthly counts of DHF cases notified to the Yogyakarta District Health Office between January 2006 and May 2022 were modelled as a function of time-varying local wMel treatment status (fully- and partially-treated vs untreated, and by quintile of wMel prevalence). The frequency of insecticide fogging in wMel-treated and untreated areas was analysed using negative binomial regression. RESULTS: Notified DHF incidence was 83% lower in fully treated vs untreated periods (IRR 0.17 [95% CI 0.14, 0.20]), and 78% lower in areas with 80-100% wMel prevalence compared to areas with 0-20% wMel (IRR 0.23 [0.17, 0.30]). A similar intervention effect was observed at 60-80% wMel prevalence as at 80-100% prevalence (76% vs 78% efficacy, respectively). Pre-intervention, insecticide fogging occurred at similar frequencies in areas later randomised to wMel-treated and untreated arms of the trial. After wMel deployment, fogging occurred significantly less frequently in treated areas (IRR 0.17 [0.10, 0.30]). CONCLUSIONS: Deployments of wMel-infected Aedes aegypti mosquitoes resulted in an 83% reduction in the application of perifocal insecticide spraying, consistent with lower dengue case notifications in wMel-treated areas. These results show that the Wolbachia intervention effect demonstrated previously in a cluster randomised trial was also measurable from routine surveillance data.

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The Wolbachia insect control method, employed by the World Mosquito Program (WMP), relies on introgressing Wolbachia through target Aedes aegypti populations to reduce the incidence of dengue. Since 2010, the WMP has been producing Wolbachia-infected mosquitoes at numerous sites across the globe for release in 11 countries. As the technology has matured, greater focus has been placed on mosquito production at larger central facilities for transport to remote release sites, both domestically and internationally. Of particular note is the production of Wolbachia-infected mosquitoes at the WMP's Australian production facility for successful international deployments in Fiji, Vanuatu, Kiribati and Sri Lanka. This requires careful management of both production and supply-chain processes to ensure that the quality of the mosquito eggs, specifically the hatch rate and Wolbachia infection rate, is maintained. To ensure the cost-effectiveness and scalability of the Wolbachia method, these processes will be further refined to facilitate deployment from large centralised production facilities.

La méthode de contrôle des insectes mise en oeuvre par le World Mosquito Program (WMP) recourt à la bactérie Wolbachia et repose sur l'introgression de cette dernière par le biais des populations cibles d'Aedes aegypti dans le but de réduire l'incidence de la dengue. Depuis 2010, le WMP produit des moustiques infectés par Wolbachia à partir de nombreux sites répartis dans le monde entier en vue de leur lâcher dans 11 pays. Cette technologie s'étant bien développée, l'accent est désormais mis sur la production de moustiques dans des établissements centralisés de plus grande envergure afin de les expédier vers des sites de lâcher éloignés, tant sur le territoire national qu'à l'échelle internationale. Il importe de souligner la production de moustiques infectés par Wolbachia conduite par le WMP sur son site australien et leur déploiement international couronné de succès aux Fidji, au Vanuatu, à Kiribati et au Sri Lanka. Cela nécessite une gestion rigoureuse des procédures aussi bien lors de la production que tout au long de la chaîne d'approvisionnement afin de veiller au maintien de la qualité des oeufs de moustiques et plus spécifiquement du taux d'éclosion et du taux d'infection par Wolbachia. Afin d'assurer la rentabilité et l'évolutivité de la méthode basée sur Wolbachia, ces procédures seront encore affinées afin de faciliter le déploiement à partir de sites de production à grande échelle centralisés.

El método de control de insectos con Wolbachia que emplea el World Mosquito Program (WMP) reposa en la introgresión de Wolbachia a través de poblaciones de Aedes aegypti para reducir la incidencia del dengue. Desde 2010, el WMP viene generando mosquitos infectados por Wolbachia en numerosas instalaciones repartidas por todo el globo, mosquitos que después son liberados en 11 países. A medida que la tecnología ha ido madurando, la tendencia ha sido cada vez más la de concentrar la producción de mosquitos en grandes instalaciones centrales y desde allí enviarlos a los remotos lugares de suelta, ya estén en el mismo país o en otros países. Especialmente destacable es la producción de mosquitos infectados por Wolbachia en las instalaciones que el WMP tiene en Australia, utilizadas con éxito como centro de operaciones para aplicar el método en otros países como Fiji, Vanuatu, Kiribati y Sri Lanka. Ello exige una cuidadosa gestión de los procesos tanto de producción como de la cadena de suministro, que garantice un nivel constante de calidad de los huevos de mosquito, y más concretamente de las tasas de eclosión y de infección por Wolbachia. A fin de asegurar que el método ofrezca una buena relación costo-eficacia y sea reproducible a mayor escala, esos procesos serán perfeccionados para facilitar el despliegue de los insectos a partir de grandes instalaciones de producción centralizada.

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Wolbachia, an intracellular maternally transmitted endosymbiont, has been shown to interfere with the replication of dengue virus in Aedes aegypti mosquitoes. The Wolbachia-transinfected Ae. aegypti has been currently released in many countries to test its effectiveness in preventing the transmission of dengue virus. ICMR-Vector Control Research Centre in collaboration with World Mosquito Program Monash University, Australia, has generated two new Wolbachia-introgressed Ae. aegypti Puducherry (Pud) lines via backcrossing Ae. aegypti females of Australian (Aus) strains, infected with wMel and wAlbB Wolbachia with wild-type Ae. aegypti Puducherry (Pud) males. Wolbachia infections are known to induce a fitness cost and confer benefit on the host mosquito populations that will influence spread of the Wolbachia into native wild mosquito populations during the field release. Hence, the induced fitness cost or benefit/advantage in the two newly generated Ae. aegypti (Pud) lines was assessed in the laboratory in comparison with the wild-type Ae. aegypti (Pud) strain. In addition, maternal transmission (MT) efficiency, induced cytoplasmic incompatibility (CI), and insecticide resistance status of the two (Pud) lines were determined to assess the likely frequency of wMel and wAlbB infections in the native wild population after field invasion. The study shows that wMel and wAlbB infections did not induce any fitness cost on the two newly generated (Pud) lines. Rather, in terms of wing length, fecundity, egg hatch rate, and adult survival, the Wolbachia introgression conferred fitness benefits on the (Pud) lines compared to uninfected Wolbachia free wild Ae. aegypti population. wMel and wAlbB exhibited a high maternal transmission (99-100%) and induced nearly complete (98-100%) cytoplasmic incompatibility. Both the (Pud) lines were resistant to deltamethrin, malathion, DDT, and temephos, and the level of resistance was almost the same between the two lines as in the wild type. Overall, the stable association of wMel and wAlbB established with Ae. aegypti and the reproductive advantages of the (Pud) lines encourage a pilot release in the field for population replacement potential.

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Endosymbioses influence host physiology, reproduction, and fitness, but these relationships require efficient microbe transmission between host generations to persist. Maternally transmitted Wolbachia are the most common known endosymbionts,1 but their frequencies vary widely within and among host populations for unknown reasons.2,3 Here, we integrate genomic, cellular, and phenotypic analyses with mathematical models to provide an unexpectedly simple explanation for global wMel Wolbachia prevalence in Drosophila melanogaster. Cooling temperatures decrease wMel cellular abundance at a key stage of host oogenesis, producing temperature-dependent variation in maternal transmission that plausibly explains latitudinal clines of wMel frequencies on multiple continents. wMel sampled from a temperate climate targets the germline more efficiently in the cold than a recently differentiated tropical variant (∼2,200 years ago), indicative of rapid wMel adaptation to climate. Genomic analyses identify a very narrow list of wMel alleles-most notably, a derived stop codon in the major Wolbachia surface protein WspB-that underlie thermal sensitivity of cellular Wolbachia abundance and covary with temperature globally. Decoupling temperate wMel and host genomes further reduces transmission in the cold, a pattern that is characteristic of host-microbe co-adaptation to a temperate climate. Complex interactions among Wolbachia, hosts, and the environment (GxGxE) mediate wMel cellular abundance and maternal transmission, implicating temperature as a key determinant of Wolbachia spread and equilibrium frequencies, in conjunction with Wolbachia effects on host fitness and reproduction.4,5 Our results motivate the strategic use of locally selected wMel variants for Wolbachia-based biocontrol efforts, which protect millions of individuals from arboviruses that cause human disease.6.

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Endosymbionts can influence host reproduction and fitness to favor their maternal transmission. For example, endosymbiotic Wolbachia bacteria often cause cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-modified sperm. Infected females can rescue CI, providing them a relative fitness advantage. Wolbachia-induced CI strength varies widely and tends to decrease as host males age. Since strong CI drives Wolbachia to high equilibrium frequencies, understanding how fast and why CI strength declines with male age is crucial to explaining age-dependent CI's influence on Wolbachia prevalence. Here, we investigate if Wolbachia densities and/or CI gene (cif) expression covary with CI-strength variation and explore covariates of age-dependent Wolbachia-density variation in two classic CI systems. wRi CI strength decreases slowly with Drosophila simulans male age (6%/day), but wMel CI strength decreases very rapidly (19%/day), yielding statistically insignificant CI after only 3 days of Drosophila melanogaster adult emergence. Wolbachia densities and cif expression in testes decrease as wRi-infected males age, but both surprisingly increase as wMel-infected males age, and CI strength declines. We then tested if phage lysis, Octomom copy number (which impacts wMel density), or host immune expression covary with age-dependent wMel densities. Only host immune expression correlated with density. Together, our results identify how fast CI strength declines with male age in two model systems and reveal unique relationships between male age, Wolbachia densities, cif expression, and host immunity. We discuss new hypotheses about the basis of age-dependent CI strength and its contributions to Wolbachia prevalence. IMPORTANCEWolbachia bacteria are the most common animal-associated endosymbionts due in large part to their manipulation of host reproduction. Many Wolbachia cause cytoplasmic incompatibility (CI) that kills uninfected host eggs. Infected eggs are protected from CI, favoring Wolbachia spread in natural systems and in transinfected mosquito populations where vector-control groups use strong CI to maintain pathogen-blocking Wolbachia at high frequencies for biocontrol of arboviruses. CI strength varies considerably in nature and declines as males age for unknown reasons. Here, we determine that CI strength weakens at different rates with age in two model symbioses. Wolbachia density and CI gene expression covary with wRi-induced CI strength in Drosophila simulans, but neither explain rapidly declining wMel-induced CI in aging D. melanogaster males. Patterns of host immune gene expression suggest a candidate mechanism behind age-dependent wMel densities. These findings inform how age-dependent CI may contribute to Wolbachia prevalence in natural systems and potentially in transinfected systems.

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Heritable symbionts have diverse effects on the physiology, reproduction and fitness of their hosts. Maternally transmitted Wolbachia are one of the most common endosymbionts in nature, infecting about half of all insect species. We test the hypothesis that Wolbachia alter host behaviour by assessing the effects of 14 different Wolbachia strains on the locomotor activity of nine Drosophila host species. We find that Wolbachia alter the activity of six different host genotypes, including all hosts in our assay infected with wRi-like Wolbachia strains (wRi, wSuz and wAur), which have rapidly spread among Drosophila species in about the last 14 000 years. While Wolbachia effects on host activity were common, the direction of these effects varied unpredictably and sometimes depended on host sex. We hypothesize that the prominent effects of wRi-like Wolbachia may be explained by patterns of Wolbachia titre and localization within host somatic tissues, particularly in the central nervous system. Our findings support the view that Wolbachia have wide-ranging effects on host behaviour. The fitness consequences of these behavioural modifications are important for understanding the evolution of host-symbiont interactions, including how Wolbachia spread within host populations.

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In this paper, we propose a dengue transmission model of SIR(S)-SI type that accounts for two sex-structured mosquito populations: the wild mosquitoes (males and females that are Wolbachia-free), and those deliberately infected with either wMel or wMelPop strain of Wolbachia. This epidemiological model has four possible outcomes: with or without Wolbachia and with or without dengue. To reach the desired outcome, with Wolbachia and without dengue, we employ the dynamic optimization approach and then design optimal programs for releasing Wolbachia-carrying male and female mosquitoes. Our discussion is focused on advantages and drawbacks of two Wolbachia strains, wMelPop and wMel, that are recommended for dengue prevention and control. On the one hand, the wMel strain guarantees a faster population replacement, ensures durable Wolbachia persistence in the wild mosquito population, and requiters fewer releases. On the other hand, the wMelPop strain displays better results for averting dengue infections in the human population.

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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.

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Heritable symbionts can modify a range of ecologically important host traits, including behavior. About half of all insect species are infected with maternally transmitted Wolbachia, a bacterial endosymbiont known to alter host reproduction, nutrient acquisition, and virus susceptibility. Here, we broadly test the hypothesis that Wolbachia modifies host behavior by assessing the effects of eight different Wolbachia strains on the temperature preference of six Drosophila melanogaster subgroup species. Four of the seven host genotypes infected with A-group Wolbachia strains (wRi in Drosophila simulans, wHa in D. simulans, wSh in Drosophila sechellia, and wTei in Drosophila teissieri) prefer significantly cooler temperatures relative to uninfected genotypes. Contrastingly, when infected with divergent B-group wMau, Drosophila mauritiana prefers a warmer temperature. For most strains, changes to host temperature preference do not alter Wolbachia titer. However, males infected with wSh and wTei tend to experience an increase in titer when shifted to a cooler temperature for 24 h, suggesting that Wolbachia-induced changes to host behavior may promote bacterial replication. Our results indicate that Wolbachia modifications to host temperature preference are likely widespread, which has important implications for insect thermoregulation and physiology. Understanding the fitness consequences of these Wolbachia effects is crucial for predicting evolutionary outcomes of host-symbiont interactions, including how Wolbachia spreads to become common.IMPORTANCE Microbes infect a diversity of species, influencing the performance and fitness of their hosts. Maternally transmitted Wolbachia bacteria infect most insects and other arthropods, making these bacteria some of the most common endosymbionts in nature. Despite their global prevalence, it remains mostly unknown how Wolbachia influence host physiology and behavior to proliferate. We demonstrate pervasive effects of Wolbachia on Drosophila temperature preference. Most hosts infected with A-group Wolbachia prefer cooler temperatures, whereas the one host species infected with divergent B-group Wolbachia prefers warmer temperatures, relative to uninfected genotypes. Changes to host temperature preference generally do not alter Wolbachia abundance in host tissues, but for some A-group strains, adult males have increased Wolbachia titer when shifted to a cooler temperature. This suggests that Wolbachia-induced changes to host behavior may promote bacterial replication. Our results help elucidate the impact of endosymbionts on their hosts amid the global Wolbachia pandemic.

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BACKGROUND: The World Mosquito Program uses Wolbachia pipientis for the biocontrol of arboviruses transmitted by Aedes aegypti mosquitoes. Diagnostic testing for Wolbachia in laboratory colonies and in field-caught mosquito populations has typically employed PCR. New, simpler methods to diagnose Wolbachia infection in mosquitoes are required for large-scale operational use. METHODS: Field-collected Ae. aegypti mosquitoes from North Queensland were tested using primers designed to detect the Wolbachia wsp gene, specific to the strain wMel. The results were analysed by colour change in the reaction mix. Furthermore, to confirm the efficiency of the LAMP assay, the results were compared to the gold-standard qPCR test. RESULTS: A novel loop-mediated isothermal amplification (LAMP) colorimetric test for the wMel strain of Wolbachia was designed, developed and validated for use in a high-throughput setting. Against the standard qPCR test, the analytical sensitivity, specificity and diagnostic metrics were: sensitivity (99.6%), specificity (92.2%), positive predictive value (97.08%) and negative predictive value (99.30%). CONCLUSIONS: We describe an alternative, novel and high-throughput method for diagnosing wMel Wolbachia infections in mosquitoes. This assay should support Wolbachia surveillance in both laboratory and field populations of Ae. aegypti.

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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.

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The biological control of mosquito transmission by the use of the naturally occurring insect-specific bacterial endosymbiont Wolbachia has been successfully tested in small field trials. The approach has been translated successfully to larger field sites in Townsville, Australia and expanded to more than 10 countries through the Eliminate Dengue Program. The broader application of the program beyond limiting the transmission of dengue and including other Aedes aegypti borne mosquitoes has seen the program growing into a global not-for-profit initiative to be known as the World Mosquito Program.

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The wMel strain of Wolbachia can reduce the permissiveness of Aedes aegypti mosquitoes to disseminated arboviral infections. Here, we report that wMel-infected Ae. aegypti (Ho Chi Minh City background), when directly blood-fed on 141 viremic dengue patients, have lower dengue virus (DENV) transmission potential and have a longer extrinsic incubation period than their wild-type counterparts. The wMel-infected mosquitoes that are field-reared have even greater relative resistance to DENV infection when fed on patient-derived viremic blood meals. This is explained by an increased susceptibility of field-reared wild-type mosquitoes to infection than laboratory-reared counterparts. Collectively, these field- and clinically relevant findings support the continued careful field-testing of wMel introgression for the biocontrol of Ae. aegypti-born arboviruses.

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Iron is required for organismal growth. Therefore, limiting iron availability may be a key part of the host's innate immune response to various pathogens, for example, in Drosophila infected with Zygomycetes. One way the host can transiently reduce iron bioavailability is by ferritin overexpression. To study the effects of neuronal-specific ferritin overexpression on survival and neurodegeneration we generated flies simultaneously over-expressing transgenes for both ferritin subunits in all neurons. We used two independent recombinant chromosomes bearing UAS-Fer1HCH, UAS-Fer2LCH transgenes and obtained qualitatively different levels of late-onset behavioral and lifespan declines. We subsequently discovered that one parental strain had been infected with a virulent form of the bacterial endosymbiont Wolbachia, causing widespread neuronal apoptosis and premature death. This phenotype was exacerbated by ferritin overexpression and was curable by antibiotic treatment. Neuronal ferritin overexpression in uninfected flies did not cause evident neurodegeneration but resulted in a late-onset behavioral decline, as previously reported for ferritin overexpression in glia. The results suggest that ferritin overexpression in the central nervous system of flies is tolerated well in young individuals with adverse manifestations appearing only late in life or under unrelated pathophysiological conditions.