Establishment of a Wolbachia Superinfection in Aedes aegypti Mosquitoes as a Potential Approach for Future Resistance Management

Wolbachia pipientis is an endosymbiotic bacterium estimated to chronically infect between 40–75% of all arthropod species. Aedes aegypti, the principle mosquito vector of dengue virus (DENV), is not a natural host of Wolbachia. The transinfection of Wolbachia strains such as wAlbB, wMel and wMelPop-CLA into Ae. aegypti has been shown to significantly reduce the vector competence of this mosquito for a range of human pathogens in the laboratory. This has led to wMel-transinfected Ae. aegypti currently being released in five countries to evaluate its effectiveness to control dengue disease in human populations. Here we describe the generation of a superinfected Ae. aegypti mosquito line simultaneously infected with two avirulent Wolbachia strains, wMel and wAlbB. The line carries a high overall Wolbachia density and tissue localisation of the individual strains is very similar to each respective single infected parental line. The superinfected line induces unidirectional cytoplasmic incompatibility (CI) when crossed to each single infected parental line, suggesting that the superinfection would have the capacity to replace either of the single constituent infections already present in a mosquito population. No significant differences in fitness parameters were observed between the superinfected line and the parental lines under the experimental conditions tested. Finally, the superinfected line blocks DENV replication more efficiently than the single wMel strain when challenged with blood meals from viremic dengue patients. These results suggest that the deployment of superinfections could be used to replace single infections and may represent an effective strategy to help manage potential resistance by DENV to field deployments of single infected strains.     

Detection of Arboviruses and Other Micro-Organisms in Experimentally Infected Mosquitoes Using Massively Parallel Sequencing

Human disease incidence attributed to arbovirus infection is increasing throughout the world, with effective control interventions limited by issues of sustainability, insecticide resistance and the lack of effective vaccines. Several promising control strategies are currently under development, such as the release of mosquitoes trans-infected with virus-blocking Wolbachia bacteria. Implementation of any control program is dependent on effective virus surveillance and a thorough understanding of virus-vector interactions. Massively parallel sequencing has enormous potential for providing comprehensive genomic information that can be used to assess many aspects of arbovirus ecology, as well as to evaluate novel control strategies. To demonstrate proof-of-principle, we analyzed Aedes aegypti or Aedes albopictus experimentally infected with dengue, yellow fever or chikungunya viruses. Random amplification was used to prepare sufficient template for sequencing on the Personal Genome Machine. Viral sequences were present in all infected mosquitoes. In addition, in most cases, we were also able to identify the mosquito species and mosquito micro-organisms, including the bacterial endosymbiont Wolbachia. Importantly, naturally occurring Wolbachia strains could be differentiated from strains that had been trans-infected into the mosquito. The method allowed us to assemble near full-length viral genomes and detect other micro-organisms without prior sequence knowledge, in a single reaction. This is a step toward the application of massively parallel sequencing as an arbovirus surveillance tool. It has the potential to provide insight into virus transmission dynamics, and has applicability to the post-release monitoring of Wolbachia in mosquito populations.     

The Effect of Virus-Blocking Wolbachia on Male Competitiveness of the Dengue Vector Mosquito,Aedes aegypti

Background

The bacterial endosymbiont Wolbachia blocks the transmission of dengue virus by its vector mosquito Aedes aegypti, and is currently being evaluated for control of dengue outbreaks. Wolbachia induces cytoplasmic incompatibility (CI) that results in the developmental failure of offspring in the cross between Wolbachia-infected males and uninfected females. This increases the relative success of infected females in the population, thereby enhancing the spread of the beneficial bacterium. However, Wolbachia spread via CI will only be feasible if infected males are sufficiently competitive in obtaining a mate under field conditions. We tested the effect of Wolbachia on the competitiveness of A. aegypti males under semi-field conditions.

Methodology/Principal Findings

In a series of experiments we exposed uninfected females to Wolbachia-infected and uninfected males simultaneously. We scored the competitiveness of infected males according to the proportion of females producing non-viable eggs due to incompatibility. We found that infected males were equally successful to uninfected males in securing a mate within experimental tents and semi-field cages. This was true for males infected by the benign wMel Wolbachia strain, but also for males infected by the virulent wMelPop (popcorn) strain. By manipulating male size we found that larger males had a higher success than smaller underfed males in the semi-field cages, regardless of their infection status.

Conclusions/Significance

The results indicate that Wolbachia infection does not reduce the competitiveness of A. aegypti males. Moreover, the body size effect suggests a potential advantage for lab-reared Wolbachia-males during a field release episode, due to their better nutrition and larger size. This may promote Wolbachia spread via CI in wild mosquito populations and underscores its potential use for disease control.     

Identification of Wolbachia Strains in Mosquito Disease Vectors

Wolbachia bacteria are common endosymbionts of insects, and some strains are known to protect their hosts against RNA viruses and other parasites. This has led to the suggestion that releasing Wolbachia-infected mosquitoes could prevent the transmission of arboviruses and other human parasites. We have identified Wolbachia in Kenyan populations of the yellow fever vector Aedes bromeliae and its relative Aedes metallicus, and in Mansonia uniformis and Mansonia africana, which are vectors of lymphatic filariasis. These Wolbachia strains cluster together on the bacterial phylogeny, and belong to bacterial clades that have recombined with other unrelated strains. These new Wolbachia strains may be affecting disease transmission rates of infected mosquito species, and could be transferred into other mosquito vectors as part of control programs.     

Competition for Amino Acids Between Wolbachia and the Mosquito Host,Aedes aegypti

The endosymbiont Wolbachia represents a promising method of dengue control, as it reduces the ability of the primary vector, the mosquito Aedes aegypti, to transmit viruses. When mosquitoes infected with the virulent Wolbachia strain wMelPop are fed non-human blood, there is a drastic reduction in mosquito fecundity and egg viability. Wolbachia has a reduced genome and is clearly dependent on its host for a wide range of nutritional needs. The fitness defects seen in wMelPop-infected A. aegypti could be explained by competition between the mosquito and the symbiont for essential blood meal nutrients, the profiles of which are suboptimal in non-human blood. Here, we examine cholesterol and amino acids as candidate molecules for competition, as they have critical roles in egg structural development and are known to vary between blood sources. We found that Wolbachia infection reduces total cholesterol levels in mosquitoes by 15–25 %. We then showed that cholesterol supplementation of a rat blood meal did not improve fecundity or egg viability deficits. Conversely, amino acid supplementation of sucrose before and after a sheep blood meal led to statistically significant increases in fecundity of approximately 15–20 eggs per female and egg viability of 30–40 %. This mosquito system provides the first empirical evidence of competition between Wolbachia and a host over amino acids and may suggest a general feature of Wolbachia–insect associations. These competitive processes could affect many aspects of host physiology and potentially mosquito fitness, a key concern for Wolbachia-based mosquito biocontrol.     

Application of wMelPop Wolbachia Strain to Crash Local Populations of Aedes aegypti

The endosymbiotic bacteria Wolbachia pipientis (wMel strain) has been successfully established in several populations of Aedes aegypti, the primary dengue vector. The virulent Wolbachia strain wMelPop is known to cause several pathological impacts (increased egg mortality, life shortening, etc.) reducing overall fitness in the mosquito Ae. aegypti. Increased egg mortality could substantially reduce egg banks in areas with a lengthy monsoonal dry season, and be employed to eliminate local populations. We tested this application under semi-field cage conditions. First, we determined that wMelPop infection significantly reduced the survival of desiccation-resistant eggs of the dengue vector Ae. aegypti, with shade and temperature having a significant impact; nearly all wMelPop-infected eggs failed to hatch after 6 and 10 weeks in summer and winter conditions, respectively. In laboratory selection experiments we found that egg desiccation resistance can be increased by selection, and that this effect of wMelPop infection is due to the nuclear background of the host rather than Wolbachia. We then conducted an invasion of wMelPop within a semi-field cage using sustained weekly releases of wMelPop infected mosquitoes, with fixation achieved after 9 weeks. The egg populations wMelPop infected and an uninfected control were then subjected to a simulated prolonged monsoonal dry season (2.5 months) before flooding to induce hatching. The wMelPop infected eggs suffered significantly greater mortality than the controls, with only 0.67% and 4.35% of respective infected and uninfected eggs held in 99% shade hatching after 80 days. These studies suggest that wMelPop could be used to locally eliminate populations of Ae. aegypti that are exposed to prolonged dry conditions, particularly if combined with vector control.     

A Virulent Wolbachia Infection Decreases the Viability of the Dengue Vector Aedes aegypti during Periods of Embryonic Quiescence

A new approach for dengue control has been proposed that relies on life-shortening strains of the obligate intracellular bacterium Wolbachia pipientis to modify mosquito population age structure and reduce pathogen transmission. Previously we reported the stable transinfection of the major dengue vector Aedes aegypti with a life-shortening Wolbachia strain (wMelPop-CLA) from the vinegar fly Drosophila melanogaster. Here, we report a further characterization of the phenotypic effects of this virulent Wolbachia infection on several life-history traits of Ae. aegypti. Minor costs of wMelPop-CLA infection for pre-imaginal survivorship, development and adult size were found. However, we discovered that the wMelPop-CLA infection dramatically decreased the viability of desiccated Ae. aegypti eggs over time. Similarly, the reproductive fitness of wMelPop-CLA infected Ae. aegypti females declined with age. These results reveal a general pattern associated with wMelPop-CLA induced pathogenesis in this mosquito species, where host fitness costs increase during aging of both immature and adult life-history stages. In addition to influencing the invasion dynamics of this particular Wolbachia strain, we suggest that the negative impact of wMelPop-CLA on embryonic quiescence may have applied utility as a tool to reduce mosquito population size in regions with pronounced dry seasons or in regions that experience cool winters.     

Replacing a Native Wolbachia with a Novel Strain Results in an Increase in Endosymbiont Load and Resistance to Dengue Virus in a Mosquito Vector

Wolbachia is a maternally transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce pathogen interference and spread into mosquito vector populations makes it possible to develop Wolbachia as a biological control agent for vector-borne disease control. Although Wolbachia induces resistance to dengue virus (DENV), filarial worms, and Plasmodium in mosquitoes, species like Aedes polynesiensis and Aedes albopictus, which carry native Wolbachia infections, are able to transmit dengue and filariasis. In a previous study, the native wPolA in Ae. polynesiensis was replaced with wAlbB from Ae. albopictus, and resulted in the generation of the transinfected “MTB” strain with low susceptibility for filarial worms. In this study, we compare the dynamics of DENV serotype 2 (DENV-2) within the wild type “APM” strain and the MTB strain of Ae. polynesiensis by measuring viral infection in the mosquito whole body, midgut, head, and saliva at different time points post infection. The results show that wAlbB can induce a strong resistance to DENV-2 in the MTB mosquito. Evidence also supports that this resistance is related to a dramatic increase in Wolbachia density in the MTB''s somatic tissues, including the midgut and salivary gland. Our results suggests that replacement of a native Wolbachia with a novel infection could serve as a strategy for developing a Wolbachia-based approach to target naturally infected insects for vector-borne disease control.     

Mitochondrial DNA variants help monitor the dynamics of Wolbachia invasion into host populations

Wolbachia is the most widespread endosymbiotic bacterium of insects and other arthropods that can rapidly invade host populations. Deliberate releases of Wolbachia into natural populations of the dengue fever mosquito, Aedes aegypti, are used as a novel biocontrol strategy for dengue suppression. Invasion of Wolbachia through the host population relies on factors such as high fidelity of the endosymbiont transmission and limited immigration of uninfected individuals, but these factors can be difficult to measure. One way of acquiring relevant information is to consider mitochondrial DNA (mtDNA) variation alongside Wolbachia in field-caught mosquitoes. Here we used diagnostic mtDNA markers to differentiate infection-associated mtDNA haplotypes from those of the uninfected mosquitoes at release sites. Unique haplotypes associated with Wolbachia were found at locations outside Australia. We also performed mathematical and qualitative analyses including modelling the expected dynamics of the Wolbachia and mtDNA variants during and after a release. Our analyses identified key features in haplotype frequency patterns to infer the presence of imperfect maternal transmission of Wolbachia, presence of immigration and possibly incomplete cytoplasmic incompatibility. We demonstrate that ongoing screening of the mtDNA variants should provide information on maternal leakage and immigration, particularly in releases outside Australia. As we demonstrate in a case study, our models to track the Wolbachia dynamics can be successfully applied to temporal studies in natural populations or Wolbachia release programs, as long as there is co-occurring mtDNA variation that differentiates infected and uninfected populations.     

Native Wolbachia from Aedes albopictus Blocks Chikungunya Virus Infection In Cellulo

Wolbachia, a widespread endosymbiont of terrestrial arthropods, can protect its host against viral and parasitic infections, a phenotype called "pathogen blocking". However, in some cases Wolbachia may have no effect or even enhance pathogen infection, depending on the host-Wolbachia-pathogen combination. The tiger mosquito Aedes albopictus is naturally infected by two strains of Wolbachia, wAlbA and wAlbB, and is a competent vector for different arboviruses such as dengue virus (DENV) and Chikungunya virus (CHIKV). Interestingly, it was shown in some cases that Ae. albopictus native Wolbachia strains are able to inhibit DENV transmission by limiting viral replication in salivary glands, but no such impact was measured on CHIKV replication in vivo. To better understand the Wolbachia/CHIKV/Ae. albopictus interaction, we generated a cellular model using Ae. albopictus derived C6/36 cells that we infected with the wAlbB strain. Our results indicate that CHIKV infection is negatively impacted at both RNA replication and virus assembly/secretion steps in presence of wAlbB. Using FISH, we observed CHIKV and wAlbB in the same mosquito cells, indicating that the virus is still able to enter the cell in the presence of the bacterium. Further work is needed to decipher molecular pathways involved in Wolbachia-CHIKV interaction at the cellular level, but this cellular model can be a useful tool to study the mechanism behind virus blocking phenotype induced by Wolbachia. More broadly, this underlines that despite Wolbachia antiviral potential other complex interactions occur in vivo to determine mosquito vector competence in Ae. albopictus.     

Combining the Sterile Insect Technique with the Incompatible Insect Technique: I-Impact of Wolbachia Infection on the Fitness of Triple- and Double-Infected Strains of Aedes albopictus

The mosquito species Aedes albopictus is a major vector of the human diseases dengue and chikungunya. Due to the lack of efficient and sustainable methods to control this mosquito species, there is an increasing interest in developing and applying the sterile insect technique (SIT) and the incompatible insect technique (IIT), separately or in combination, as population suppression approaches. Ae. albopictus is naturally double-infected with two Wolbachia strains, wAlbA and wAlbB. A new triple Wolbachia-infected strain (i.e., a strain infected with wAlbA, wAlbB, and wPip), known as HC and expressing strong cytoplasmic incompatibility (CI) in appropriate matings, was recently developed. In the present study, we compared several fitness traits of three Ae. albopictus strains (triple-infected, double-infected and uninfected), all of which were of the same genetic background (“Guangzhou City, China”) and were reared under the same conditions. Investigation of egg-hatching rate, survival of pupae and adults, sex ratio, duration of larval stages (development time from L₁ to pupation), time to emergence (development time from L₁ to adult emergence), wing length, female fecundity and adult longevity indicated that the presence of Wolbachia had only a minimal effect on host fitness. Based on this evidence, the HC strain is currently under consideration for mass rearing and application in a combined SIT-IIT strategy to control natural populations of Ae. albopictus in mainland China.     

Constraints on the use of lifespan-shortening Wolbachia to control dengue fever

Dengue fever, a viral disease spread by the mosquito Aedes aegypti, affects 50–100 million people a year in many tropical countries. Because the virus must incubate within mosquito hosts for two weeks before being able to transmit the infection, shortening the lifespan of mosquitoes may curtail dengue transmission. We developed a continuous time reaction-diffusion model of the spatial spread of Wolbachia through a population of A. aegypti. This model incorporates the lifespan-shortening effects of Wolbachia on infected A. aegypti and the fitness advantage to infected females due to cytoplasmic incompatibility (CI). We found that local establishment of the Wolbachia infection can occur if the fitness advantage due to CI exceeds the fitness reduction due to lifespan-shortening effects, in accordance with earlier results concerning fecundity reduction. However, spatial spread is possible only if the fitness advantage due to CI is twice as great as the fitness reduction due to lifespan shortening effects. Moreover, lifespan-shortening and fecundity-reduction can have different effects on the speed of wave-retreat. Using data from the literature, we estimated all demographic parameters for infected and uninfected mosquitoes and computed the velocities of spread of infection. Our most optimistic estimates suggest that the spatial spread of lifespan-shortening Wolbachia may be so slow that efficient spatial spread would require a prohibitively large number of point releases. However, as these estimates of demographic parameters may not accurately reflect natural conditions, further research is necessary to corroborate these predictions.     

Infertility and fecundity loss of Wolbachia-infected Aedes aegypti hatched from quiescent eggs is expected to alter invasion dynamics

The endosymbiotic bacterium Wolbachia shows viral blocking in its mosquito host, leading to its use in arboviral disease control. Releases with Wolbachia strains wMel and wAlbB infecting Aedes aegypti have taken place in several countries. Mosquito egg survival is a key factor influencing population persistence and this trait is also important when eggs are stored prior to releases. We therefore tested the viability of mosquitoes derived from Wolbachia wMel and wAlbB-infected as well as uninfected eggs after long-term storage under diurnal temperature cycles of 11–19°C and 22–30°C. Eggs stored at 11–19°C had higher hatch proportions than those stored at 22–30°C. Adult Wolbachia density declined when they emerged from eggs stored for longer, which was associated with incomplete cytoplasmic incompatibility (CI) when wMel-infected males were crossed with uninfected females. Females from stored eggs at both temperatures continued to show perfect maternal transmission of Wolbachia, but storage reduced the fecundity of both wMel and wAlbB-infected females relative to uninfected mosquitoes. Furthermore, we found a very strong negative impact of the wAlbB infection on the fertility of females stored at 22–30°C, with almost 80% of females hatching after 11 weeks of storage being infertile. Our findings provide guidance for storing Wolbachia-infected A. aegypti eggs to ensure high fitness adult mosquitoes for release. Importantly, they also highlight the likely impact of egg quiescence on the population dynamics of Wolbachia-infected populations in the field, and the potential for Wolbachia to suppress mosquito populations through cumulative fitness costs across warm and dry periods, with expected effects on dengue transmission.     

The oxidizing agent, paraquat, is more toxic to Wolbachia than to mosquito host cells

Cultured cells provide an important in vitro system for examining metabolic interactions between the intracellular bacterium, Wolbachia pipientis, and its insect hosts. To test whether Wolbachia-associated changes in antioxidant activities could provide a tool to select for infected cells, we tested the effects of paraquat (PQ) on Aedes albopictus mosquito cells. Like mammalian cells, mosquito cells tolerate PQ over a wide range of concentrations, and for considerable lengths of time, depending on cell density at the time of treatment. When mosquito cells were plated at low density and allowed to grow in the presence of PQ, we measured an LC₅₀ of approximately 1–2 μM. Unexpectedly, cells persistently infected with Wolbachia strain wStr, from the planthopper Laodelphax striatellus, grew to higher densities in the presence of 1.5 μM PQ than in its absence. This effect of PQ was similar to the improved growth of host cells that occurs in the presence of antibiotics that suppress the Wolbachia infection. A more detailed examination of growth and metabolic sensitivity indicated that wStr is about 10-fold more sensitive to PQ than the mosquito host cells. Microscopic examination confirmed that Wolbachia levels were reduced in PQ-treated cells, and DNA estimates based on the polymerase chain reaction (PCR) indicated that Wolbachia abundance decreased by approximately 100-fold over a 10-d period. Although Wolbachia genomes encode superoxide dismutase, inspection of annotated genomes indicates that they lack several genes encoding products that ameliorate oxidative damage, including catalase, which converts the PQ byproduct, hydrogen peroxide, to molecular oxygen and water. We suggest that loss of multiple genes that participate in repair of oxidative damage accounts for increased sensitivity of Wolbachia to PQ, relative to its host cells.     

Wolbachia enhances insect‐specific flavivirus infection in Aedes aegypti mosquitoes

Mosquitoes transmit a diverse group of human flaviviruses including West Nile, dengue, yellow fever, and Zika viruses. Mosquitoes are also naturally infected with insect‐specific flaviviruses (ISFs), a subgroup of the family not capable of infecting vertebrates. Although ISFs are not medically important, they are capable of altering the mosquito's susceptibility to flaviviruses and may alter host fitness. Wolbachia is an endosymbiotic bacterium of insects that when present in mosquitoes limits the replication of co‐infecting pathogens, including flaviviruses. Artificially created Wolbachia‐infected Aedes aegypti mosquitoes are being released into the wild in a series of trials around the globe with the hope of interrupting dengue and Zika virus transmission from mosquitoes to humans. Our work investigated the effect of Wolbachia on ISF infection in wild‐caught Ae. aegypti mosquitoes from field release zones. All field mosquitoes were screened for the presence of ISFs using general degenerate flavivirus primers and their PCR amplicons sequenced. ISFs were found to be common and widely distributed in Ae. aegypti populations. Field mosquitoes consistently had higher ISF infection rates and viral loads compared to laboratory colony material indicating that environmental conditions may modulate ISF infection in Ae. aegypti. Surprisingly, higher ISF infection rates and loads were found in Wolbachia‐infected mosquitoes compared to the Wolbachia‐free mosquitoes. Our findings demonstrate that the symbiont is capable of manipulating the mosquito virome and that Wolbachia‐mediated viral inhibition is not universal for flaviviruses. This may have implications for the Wolbachia‐based DENV control strategy if ISFs confer fitness effects or alter mosquito susceptibility to other flaviviruses.     

The Potential Use of Wolbachia-Based Mosquito Biocontrol Strategies for Japanese Encephalitis

Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted by the infectious bite of Culex mosquitoes. The virus causes the development of the disease Japanese encephalitis (JE) in a small proportion of those infected, predominantly affecting children in eastern and southern Asia. Annual JE incidence estimates range from 50,000–175,000, with 25%–30% of cases resulting in mortality. It is estimated that 3 billion people live in countries in which JEV is endemic. The virus exists in an enzootic transmission cycle, with mosquitoes transmitting JEV between birds as reservoir hosts and pigs as amplifying hosts. Zoonotic infection occurs as a result of spillover events from the main transmission cycle. The reservoir avian hosts include cattle egrets, pond herons, and other species of water birds belonging to the family Ardeidae. Irrigated rice fields provide an ideal breeding ground for mosquitoes and attract migratory birds, maintaining the transmission of JEV. Although multiple vaccines have been developed for JEV, they are expensive and require multiple doses to maintain efficacy and immunity. As humans are a “dead-end” host for the virus, vaccination of the human population is unlikely to result in eradication. Therefore, vector control of the principal mosquito vector, Culex tritaeniorhynchus, represents a more promising strategy for reducing transmission. Current vector control strategies include intermittent irrigation of rice fields and space spraying of insecticides during outbreaks. However, Cx. Tritaeniorhynchus is subject to heavy exposure to pesticides in rice fields, and as a result, insecticide resistance has developed. In recent years, significant advancements have been made in the potential use of the bacterial endosymbiont Wolbachia for mosquito biocontrol. The successful transinfection of Wolbachia strains from Drosophila flies to Aedes (Stegomyia) mosquitoes has resulted in the generation of “dengue-refractory” mosquito lines. The successful establishment of Wolbachia in wild Aedes aegypti populations has recently been demonstrated, and open releases in dengue-endemic countries are ongoing. This review outlines the current control methods for JEV in addition to highlighting the potential use of Wolbachia-based biocontrol strategies to impact transmission. JEV and dengue virus are both members of the Flavivirus genus, and the successful establishment of Drosophila Wolbachia strains in Cx. Tritaeniorhynchus, as the principal vector of JEV, is predicted to significantly impact JEV transmission.     

Depletion of host cell riboflavin reduces Wolbachia levels in cultured mosquito cells

Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC₅₀ of approximately 12 μg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.     

Wolbachia infection does not alter attraction of the mosquito Aedes (Stegomyia) aegypti to human odours

The insect endosymbiont Wolbachia pipientis (Rickettsiales: Rickettsiaceae) is undergoing field trials around the world to determine if it can reduce transmission of dengue virus from the mosquito Stegomyia aegypti to humans. Two different Wolbachia strains have been released to date. The primary effect of the wMel strain is pathogen protection whereby infection with the symbiont limits replication of dengue virus inside the mosquito. A second strain, wMelPop, induces pathogen protection, reduces the adult mosquito lifespan and decreases blood feeding success in mosquitoes after 15 days of age. Here we test whether Wolbachia infection affects mosquito attraction to host odours in adults aged 5 and 15 days. We found no evidence of reduced odour attraction of mosquitoes, even for those infected with the more virulent wMelPop. This bodes well for fitness and competitiveness in the field given that the mosquitoes must find hosts to reproduce for the biocontrol method to succeed.     

Symbionts Commonly Provide Broad Spectrum Resistance to Viruses in Insects: A Comparative Analysis of Wolbachia Strains

In the last decade, bacterial symbionts have been shown to play an important role in protecting hosts against pathogens. Wolbachia, a widespread symbiont in arthropods, can protect Drosophila and mosquito species against viral infections. We have investigated antiviral protection in 19 Wolbachia strains originating from 16 Drosophila species after transfer into the same genotype of Drosophila simulans. We found that approximately half of the strains protected against two RNA viruses. Given that 40% of terrestrial arthropod species are estimated to harbour Wolbachia, as many as a fifth of all arthropods species may benefit from Wolbachia-mediated protection. The level of protection against two distantly related RNA viruses – DCV and FHV – was strongly genetically correlated, which suggests that there is a single mechanism of protection with broad specificity. Furthermore, Wolbachia is making flies resistant to viruses, as increases in survival can be largely explained by reductions in viral titer. Variation in the level of antiviral protection provided by different Wolbachia strains is strongly genetically correlated to the density of the bacteria strains in host tissues. We found no support for two previously proposed mechanisms of Wolbachia-mediated protection — activation of the immune system and upregulation of the methyltransferase Dnmt2. The large variation in Wolbachia''s antiviral properties highlights the need to carefully select Wolbachia strains introduced into mosquito populations to prevent the transmission of arboviruses.