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Significance Wolbachia (wMel strain)-infected Aedes aegypti mosquitoes are refractory to disseminated arboviral infections. Yet previous studies into the mechanism behind Wolbachia-mediated virus blocking have not considered the involvement of lipids, apart from cholesterol, during superinfection. We used liquid chromatography mass spectrometry to study the lipidome in mosquito cells infected with virus, wMel, or superinfected with both virus and wMel. Interestingly, a class of lipids, acyl-carnitines increased during virus infection but remained low with wMel. These findings uncover a previously unknown role for acyl-carnitines in the interaction among virus, wMel, and cells, suggesting a mechanism underlying Wolbachia-mediated pathogen blocking. Importantly, this study supports Wolbachia introgression into A. Aegypti populations as a biocontrol method to reduce arboviral (e.g., DENV) transmission., Wolbachia-infected mosquitoes are refractory to flavivirus infections, but the role of lipids in Wolbachia-mediated virus blocking remains to be elucidated. Here, we use liquid chromatography mass spectrometry to provide a comprehensive picture of the lipidome of Aedes aegypti (Aag2) cells infected with Wolbachia only, either dengue or Zika virus only, and Wolbachia-infected Aag2 cells superinfected with either dengue or Zika virus. This approach identifies a class of lipids, acyl-carnitines, as being down-regulated during Wolbachia infection. Furthermore, treatment with an acyl-carnitine inhibitor assigns a crucial role for acyl-carnitines in the replication of dengue and Zika viruses. In contrast, depletion of acyl-carnitines increases Wolbachia density while addition of commercially available acyl-carnitines impairs Wolbachia production. Finally, we show an increase in flavivirus infection of Wolbachia-infected cells with the addition of acyl-carnitines. This study uncovers a previously unknown role for acyl-carnitines in this tripartite interaction that suggests an important and broad mechanism that underpins Wolbachia-mediated pathogen blocking.

Release and subsequent establishment of Wolbachia-infected Aedes aegypti in native mosquito populations has successfully reduced mosquito-borne disease incidence. While this is promising, further development is required to ensure that this method is scalable and sustainable. Egg release is a beneficial technique that requires reduced onsite resources and increases community acceptance; however, its incidental ecological impacts must be considered to ensure sustainability. In this study, we tested a more environmentally friendly mosquito rearing and release approach through the encapsulation of diet and egg mixtures and the subsequent utilization of waste containers to hatch and release mosquitoes. An ecologically friendly diet mix was specifically developed and tested for use in capsules, and we demonstrated that using either cricket or black soldier fly meal as a substitute for beef liver powder had no adverse effects on fitness or Wolbachia density. We further encapsulated both the egg and diet mixes and demonstrated no loss in viability. To address the potential of increased waste generation through disposable mosquito release containers, we tested reusing commonly found waste containers (aluminum and tin cans, PET, and glass bottles) as an alternative, conducting a case study in Kiribati to assess the concept’s cultural, political, and economic applicability. Our results showed that mosquito emergence and fitness was maintained with a variety of containers, including when tested in the field, compared to control containers, and that there are opportunities to implement this method in the Pacific Islands in a way that is culturally considerate and cost-effective.

Background Biological control programs involving Wolbachia-infected Aedes aegypti are currently deployed in different epidemiological settings. New Caledonia (NC) is an ideal location for the implementation and evaluation of such a strategy as the only proven vector for dengue virus (DENV) is Ae. aegypti and dengue outbreaks frequency and severity are increasing. We report the generation of a NC Wolbachia-infected Ae. aegypti strain and the results of experiments to assess the vector competence and fitness of this strain for future implementation as a disease control strategy in Noumea, NC. Methods/principal findings The NC Wolbachia strain (NC-wMel) was obtained by backcrossing Australian AUS-wMel females with New Caledonian Wild-Type (NC-WT) males. Blocking of DENV, chikungunya (CHIKV), and Zika (ZIKV) viruses were evaluated via mosquito oral feeding experiments and intrathoracic DENV challenge. Significant reduction in infection rates were observed for NC-wMel Ae. aegypti compared to WT Ae. aegypti. No transmission was observed for NC-wMel Ae. aegypti. Maternal transmission, cytoplasmic incompatibility, fertility, fecundity, wing length, and insecticide resistance were also assessed in laboratory experiments. Ae. aegypti NC-wMel showed complete cytoplasmic incompatibility and a strong maternal transmission. Ae. aegypti NC-wMel fitness seemed to be reduced compared to NC-WT Ae. aegypti and AUS-wMel Ae. aegypti regarding fertility and fecundity. However further experiments are required to assess it accurately. Conclusions/significance Our results demonstrated that the NC-wMel Ae. aegypti strain is a strong inhibitor of DENV, CHIKV, and ZIKV infection and prevents transmission of infectious viral particles in mosquito saliva. Furthermore, our NC-wMel Ae. aegypti strain induces reproductive cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, supporting field-releases in Noumea, NC., Author summary Dengue represents a risk for almost half of the world’s population, especially throughout the tropics. In New Caledonia, dengue outbreaks have become more frequent in the past decade along with the recent circulation of chikungunya and Zika viruses. The opportunity to use the biocontrol method involving the release of Wolbachia-infected Ae. aegypti mosquitoes has been investigated as an alternative solution to the traditional control methods, like elimination of larval habitats and pyrethroid insecticide application to kill adults, which are becoming insufficient. A local strain of Ae. aegypti carrying Wolbachia (NC-wMel) has been generated and tested to evaluate its pathogen blocking capacity for the four dengue virus serotypes as well as chikungunya and Zika viruses. The fitness of NC-wMel strain has also been assessed to estimate its ability to compete with the wild-type strain in the field. Noumea city, where a third of the population of New Caledonia resides, has been chosen as the first site to implement the method in New Caledonia. As Ae. aegypti is the only proven vector in New Caledonia, we expect a significant impact on dengue outbreaks occurring in Noumea as soon as a high frequency of NC-wMel is established in the population.

Infection of wMel Wolbachia in Aedes aegypti imparts two signature features that enable its application for biocontrol of dengue. First, the susceptibility of mosquitoes to viruses such as dengue and Zika is reduced. Second, a reproductive manipulation is caused that enables wMel introgression into wild-type mosquito populations. The long-term success of this method relies, in part, on evolution of the wMel genome not compromising the critical features that make it an attractive biocontrol tool. This study compared the wMel Wolbachia genome at the time of initial releases and 1–7 years post-release in Cairns, Australia. Our results show the wMel genome remains highly conserved up to 7 years post-release in gene sequence, content, synteny and structure. This work suggests the wMel genome is stable in its new mosquito host and, therefore, provides reassurance on the potential for wMel to deliver long-term public-health impacts.

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. While there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs.

The insect bacterium Wolbachia pipientis is being introgressed into Aedes aegypti populations as an intervention against the transmission of medically important arboviruses. Here we compare Ae. aegypti mosquitoes infected with wMelCS or wAlbB to the widely used wMel Wolbachia strain on an Australian nuclear genetic background for their susceptibility to infection by dengue virus (DENV) genotypes spanning all four serotypes. All Wolbachia-infected mosquitoes were more resistant to intrathoracic DENV challenge than their wildtype counterparts. Blocking of DENV replication was greatest by wMelCS. Conversely, wAlbB-infected mosquitoes were more susceptible to whole body infection than wMel and wMelCS. We extended these findings via mosquito oral feeding experiments, using viremic blood from 36 acute, hospitalised dengue cases in Vietnam, additionally including wMel and wildtype mosquitoes on a Vietnamese nuclear genetic background. As above, wAlbB was less effective at blocking DENV replication in the abdomen compared to wMel and wMelCS. The transmission potential of all Wolbachia-infected mosquito lines (measured by the presence/absence of infectious DENV in mosquito saliva) after 14 days, was significantly reduced compared to their wildtype counterparts, and lowest for wMelCS and wAlbB. These data support the use of wAlbB and wMelCS strains for introgression field trials and the biocontrol of DENV transmission. Furthermore, despite observing significant differences in transmission potential between wildtype mosquitoes from Australia and Vietnam, no difference was observed between wMel-infected mosquitoes from each background suggesting that Wolbachia may override any underlying variation in DENV transmission potential., Author summary Aedes aegypti transmit a number of medically important arboviruses, including dengue, Zika, chikungunya, Mayaro and yellow fever viruses. Over the past 50 years, the burden of Ae. aegypti-transmitted disease has significantly increased–underscoring how current methods of vector control are unable to cope with this problem. Wolbachia-based biocontrol methods show extreme promise in reducing the global burden of vector-borne disease. The wMel strain, widely being used in field trials around the world, substantially reduces the ability of Ae. aegypti mosquitoes to transmit dengue, Zika, and other viruses. Here we describe a comprehensive comparative study of the viral blocking abilities of wMel to wMelCS and wAlbB which have previously been shown to have stronger viral blocking or an expanded utility in extreme environments, respectively. Using two different methods to measure viral replication and transmission potential, we show that both strains provide improved viral protection over wMel in the lab supporting further examination in field trials. We further compare the transmission potential of wMel in two different genetic backgrounds and find that wMel provides equivalent levels of viral blocking despite differences observed in wildtype mosquitoes, suggesting that viral blocking induced by wMel may override any underlying variation for DENV transmission potential.

The bacterial endosymbiont Wolbachia is a biocontrol tool that inhibits the ability of the Aedes aegypti mosquito to transmit positive-sense RNA viruses such as dengue and Zika. Growing evidence indicates that when Wolbachia strains wMel or wAlbB are introduced into local mosquito populations, human dengue incidence is reduced. Despite the success of this novel intervention, we still do not fully understand how Wolbachia protects mosquitoes from viral infection. Here, we demonstrate that the Wolbachia strain wPip does not inhibit virus infection in Ae. aegypti. We have leveraged this novel finding, and a panel of Ae. aegypti lines carrying virus-inhibitory (wMel and wAlbB) and non-inhibitory (wPip) strains in a common genetic background, to rigorously test a number of hypotheses about the mechanism of Wolbachia-mediated virus inhibition. We demonstrate that, contrary to previous suggestions, there is no association between a strain’s ability to inhibit dengue infection in the mosquito and either its typical density in the midgut or salivary glands, or the degree to which it elevates innate immune response pathways in the mosquito. These findings, and the experimental platform provided by this panel of genetically comparable mosquito lines, clear the way for future investigations to define how Wolbachia prevents Ae. aegypti from transmitting viruses., Author summary Dengue virus, transmitted by the Aedes aegypti mosquito, is one of the fastest-growing infectious diseases, causing an estimated 390 million human infections per year worldwide. Vaccines have limited efficacy and there are no approved therapeutics. This has driven the rise of novel vector control programs, in particular those that use the bacterium, Wolbachia, which prevents transmission of dengue and other human pathogenic viruses when stably introduced into Ae. aegypti populations. Although this is proving to be a highly effective method, the details of how this biocontrol tool works are not well understood. Here we characterise a new Wolbachia strain, wPip, and find that Ae. aegypti carrying wPip are still able to transmit dengue similar to mosquitoes that do not carry Wolbachia. This finding has allowed us to begin a rigorous program of comparative studies to determine which features of a Wolbachia strain determine whether it is antiviral. Understanding these mechanisms will enable us to predict the risk of viral resistance arising against Wolbachia and facilitate preparation of second-generation field release lines.

Wolbachia suppresses the replication of +ssRNA viruses such as dengue and Zika viruses in Aedes aegypti mosquitoes. However, the range of viruses affected by this endosymbiont is yet to be explored. Recently, novel insect-specific viruses (ISVs) have been described from numerous mosquito species and mosquito-derived cell lines. Cell-fusing agent virus (Flaviviridae) and Phasi Charoen-like virus (Bunyaviridae) persistently infect the Ae. aegypti cell line Aag2 which has been used for experimental studies with both the wMel and wMelPop-CLA strains. Wolbachia was found to restrict the replication of CFAV but not the PCLV infection in these lines. Furthermore, an additional Ae. albopictus cell line (RML-12) which contained either wMel or wMelPop-CLA was assessed. While no infectious +ssRNA or dsRNA viruses were detected, a PCLV infection was identified. These observations provide additional evidence to support that insect-specific, +ssRNA viruses can be suppressed in cell culture by Wolbachia but -ssRNA viruses may not., Highlights • Wolbachia suppresses CFAV in Aag2 wMel and wMelPop-CLA cell lines. • PCLV persistently infects the RML-12 cell line. • lbWoLA cachia does not suppress PCLV in Aag2 or RML-12 wMel and wMelPop-Cell lines.

Wolbachia pipientis from Drosophila melanogaster (wMel) is an endosymbiotic bacterium that restricts transmission of human pathogenic flaviviruses and alphaviruses, including dengue, Zika, and chikungunya viruses, when introduced into the mosquito vector Aedes aegypti. To date, wMel-infected Ae. aegypti have been released in field trials in 5 countries to evaluate the effectiveness of this strategy for disease control. Despite the success in establishing wMel-infected mosquitoes in wild populations, and the well-characterized antiviral capabilities of wMel, transinfecting different or additional Wolbachia strains into Ae. aegypti may improve disease impact, and perhaps more importantly, could provide a strategy to account for the possible evolution of resistant arboviruses. Here, we report the successful transinfection of Ae. aegypti with the Wolbachia strains wMelCS (D. melanogaster), wRi (D. simulans) and wPip (Culex quinquefasciatus) and assess the effects on Ae. aegypti fitness, cytoplasmic incompatibility, tissue tropism and pathogen blocking in a laboratory setting. The results demonstrate that wMelCS provides a similar degree of protection against dengue virus as wMel following an infectious blood meal, and significantly reduces viral RNA levels beyond that of wMel following a direct challenge with infectious virus in mosquitoes, with no additional fitness cost to the host. The protection provided by wRi is markedly weaker than that of wMelCS, consistent with previous characterisations of these lines in Drosophila, while wPip was found to substantially reduce the fitness of Ae. aegypti. Thus, we determine wMelCS as a key candidate for further testing in field-relevant fitness tests and viremic blood feeding challenges in a clinical setting to determine if it may represent an alternative Wolbachia strain with more desirable attributes than wMel for future field testing., Author summary Dengue viruses are transmitted by the Aedes aegypti mosquito, with an estimated 390 million human infections occurring per year worldwide. There is no approved antiviral therapeutic, and vaccines described so far have had limited efficacy. Recently, the endosymbiotic bacterium Wolbachia from Drosophila melanogaster (wMel) has been used to infect Ae. aegypti populations as a novel technology for reducing dengue virus transmission. Here we report the generation of three new mosquito lines infected with the Wolbachia strains wMelCS, wRi and wPip. Each line induced cytoplasmic incompatibility and was effectively maternally transmitted, as required for rapid spread through uninfected mosquito populations. Each Wolbachia strain was also found to reside in the salivary glands; a key tissue involved in viral transmission. Perhaps most importantly, wMelCS inhibited dengue virus replication and dissemination in mosquitoes following an infectious blood meal or intrathoracic injection, providing a similar level of protection as that described for wMel. wMelCS therefore warrants further investigation as a potential release strain in future field trials.

Animals experience near constant infection with microorganisms. A significant proportion of these microbiota reside in the alimentary tract. There is a growing appreciation for the roles gut microbiota play in host biology. The gut microbiota of insects, for example, have been shown to help the host overcome pathogen infection either through direct competition or indirectly by stimulating host immunity. These defenses may also be supplemented by coinfecting maternally inherited microbes such as Wolbachia. The presence of Wolbachia in a host can delay and/or reduce death caused by RNA viruses. Whether the gut microbiota of the host interacts with Wolbachia, or vice versa, the precise role of Wolbachia in antiviral protection is not known. In this study, we used 16S rDNA sequencing to characterise changes in gut microbiota composition in Drosophila melanogaster associated with Wolbachia infection and antibiotic treatment. We subsequently tested whether changes in gut composition via antibiotic treatment altered Wolbachia-mediated antiviral properties. We found that both antibiotics and Wolbachia significantly reduced the biodiversity of the gut microbiota without changing the total microbial load. We also showed that changing the gut microbiota composition with antibiotic treatment enhanced Wolbachia density but did not confer greater antiviral protection against Drosophila C virus to the host. We concluded there are significant interactions between Wolbachia and gut microbiota, but changing gut microbiota composition is not likely to be a means through which Wolbachia conveys antiviral protection to its host.

Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. Here we show that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. We further find that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. We propose that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, we suggest that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes., Author Summary Animals need to make gametes–sperm or eggs–in order to reproduce. Gametes are produced from a specialized tissue called the germline that is found within the testes or ovaries. These organs contain a small population of stem cells that are able to both self-renew and differentiate to generate gametes and are thus essential for maintaining gamete production throughout the reproductive lifespan of most animals. Surprisingly, some of the genes that control this process evolve rapidly between Drosophila species. We find for a key germline stem cell regulatory gene, bag of marbles (bam), that its rapid evolution affects only female but not male functions. We further report that the endosymbiont bacterium Wolbachia that infects insects and other species interacts with bam and may be contributing to the wider pattern of rapid evolution of germline stem cell regulatory genes.