Effect of Wolbachia on Replication of West Nile Virus in a Mosquito Cell Line and Adult Mosquitoes

Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.     

Limited Dengue Virus Replication in Field-Collected Aedes aegypti Mosquitoes Infected with Wolbachia

Introduction

Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus.

Methodology/Principal Findings

Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes.

Conclusions/Significance

Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.     

Human Probing Behavior of Aedes aegypti when Infected with a Life-Shortening Strain of Wolbachia

Background

Mosquitoes are vectors of many serious pathogens in tropical and sub-tropical countries. Current control strategies almost entirely rely upon insecticides, which increasingly face the problems of high cost, increasing mosquito resistance and negative effects on non-target organisms. Alternative strategies include the proposed use of inherited life-shortening agents, such as the Wolbachia bacterium. By shortening mosquito vector lifespan, Wolbachia could potentially reduce the vectorial capacity of mosquito populations. We have recently been able to stably transinfect Aedes aegypti mosquitoes with the life-shortening Wolbachia strain wMelPop, and are assessing various aspects of its interaction with the mosquito host to determine its likely impact on pathogen transmission as well as its potential ability to invade A. aegypti populations.

Methodology/Principal Findings

Here we have examined the probing behavior of Wolbachia-infected mosquitoes in an attempt to understand both the broader impact of Wolbachia infection on mosquito biology and, in particular, vectorial capacity. The probing behavior of wMelPop-infected mosquitoes at four adult ages was examined and compared to uninfected controls during video-recorded feeding trials on a human hand. Wolbachia-positive insects, from 15 days of age, showed a drastic increase in the time spent pre-probing and probing relative to uninfected controls. Two other important features for blood feeding, saliva volume and apyrase content of saliva, were also studied.

Conclusions/Significance

As A. aegypti infected with wMelPop age, they show increasing difficulty in completing the process of blood feeding effectively and efficiently. Wolbachia-infected mosquitoes on average produced smaller volumes of saliva that still contained the same amount of apyrase activity as uninfected mosquitoes. These effects on blood feeding behavior may reduce vectorial capacity and point to underlying physiological changes in Wolbachia-infected mosquitoes.     

Wolbachia Infection Reduces Blood-Feeding Success in the Dengue Fever Mosquito, Aedes aegypti

Background

The mosquito Aedes aegypti was recently transinfected with a life-shortening strain of the endosymbiont Wolbachia pipientis (wMelPop) as the first step in developing a biocontrol strategy for dengue virus transmission. In addition to life-shortening, the wMelPop-infected mosquitoes also exhibit increased daytime activity and metabolic rates. Here we sought to quantify the blood-feeding behaviour of Wolbachia-infected females as an indicator of any virulence or energetic drain associated with Wolbachia infection.

Methodology/Principal Findings

In a series of blood-feeding trials in response to humans, we have shown that Wolbachia-infected mosquitoes do not differ in their response time to humans, but that as they age they obtain fewer and smaller blood meals than Wolbachia-uninfected controls. Lastly, we observed a behavioural characteristic in the Wolbachia infected mosquitoes best described as a “bendy” proboscis that may explain the decreased biting success.

Conclusions/Significance

Taken together the evidence suggests that wMelPop infection may be causing tissue damage in a manner that intensifies with mosquito age and that leads to reduced blood-feeding success. These behavioural changes require further investigation with respect to a possible physiological mechanism and their role in vectorial capacity of the insect. The selective decrease of feeding success in older mosquitoes may act synergistically with other Wolbachia-associated traits including life-shortening and viral protection in biocontrol strategies.     

Impact of Wolbachia on Infection with Chikungunya and Yellow Fever Viruses in the Mosquito Vector Aedes aegypti

Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 10⁴ times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.     

Wolbachia infection in Aedes aegypti mosquitoes alters blood meal excretion and delays oviposition without affecting trypsin activity

Blood feeding in Aedes aegypti is essential for reproduction, but also permits the mosquito to act as a vector for key human pathogens such as the Zika and dengue viruses. Wolbachia pipientis is an endosymbiotic bacterium that can manipulate the biology of Aedes aegypti mosquitoes, making them less competent hosts for many pathogens. Yet while Wolbachia affects other aspects of host physiology, it is unclear whether it influences physiological processes associated with blood meal digestion. To that end, we examined the effects of wMel Wolbachia infection in Ae. aegypti, on survival post-blood feeding, blood meal excretion, rate of oviposition, expression levels of key genes involved in oogenesis, and activity levels of trypsin blood digestion enzymes. We observed that wMel infection altered the rate and duration of blood meal excretion, delayed the onset of oviposition and was associated with a greater number of eggs being laid later. wMel-infected Ae. aegypti also had lower levels of key yolk protein precursor genes necessary for oogenesis. However, all of these effects occurred without a change in trypsin activity. These results suggest that Wolbachia infection may disrupt normal metabolic processes associated with blood feeding and reproduction in Ae. aegypti.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.     

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.     

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.     

Wolbachia strain wAlbB maintains high density and dengue inhibition following introduction into a field population of Aedes aegypti

Aedes aegypti mosquitoes carrying the wAlbB Wolbachia strain show a reduced capacity to transmit dengue virus. wAlbB has been introduced into wild Ae. aegypti populations in several field sites in Kuala Lumpur, Malaysia, where it has persisted at high frequency for more than 2 years and significantly reduced dengue incidence. Although these encouraging results indicate that wAlbB releases can be an effective dengue control strategy, the long-term success depends on wAlbB maintaining high population frequencies and virus transmission inhibition, and both could be compromised by Wolbachia–host coevolution in the field. Here, wAlbB-carrying Ae. aegypti collected from the field 20 months after the cessation of releases showed no reduction in Wolbachia density or tissue distribution changes compared to a wAlbB laboratory colony. The wAlbB strain continued to induce complete unidirectional cytoplasmic incompatibility, showed perfect maternal transmission under laboratory conditions, and retained its capacity to inhibit dengue. Additionally, a field-collected wAlbB line was challenged with Malaysian dengue patient blood, and showed significant blocking of virus dissemination to the salivary glands. These results indicate that wAlbB continues to inhibit currently circulating strains of dengue in field populations of Ae. aegypti, and provides additional support for the continued scale-up of Wolbachia wAlbB releases for dengue control.This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases’.     

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.     

Variation in Esterase Activity Among Different <Emphasis Type="Italic">Aedes aegypti</Emphasis> L. Populations from the Dooars and Terai Regions of West Bengal,India

Mosquitoes are responsible for transmitting the most important vector-borne diseases like malaria, dengue etc. across the world, especially in tropical countries. The most prominent species of the genus Aedes, A. aegypti and A. albopictus act as vectors for numerous viral infections. Because of non-availability of vaccine for dengue fever, vector control stands the only approach to prevent the viral transmission. As per recommendations by World Health Organization (2015), the main insecticides used for mosquito control are DDT, malathion, chlorpyrifos, temephos, bendiocarb and synthetic pyrethroids. Excessive and unwanted usage of insecticides not only increases vectors’ resistance to insecticides, but also results in cross resistance to other insecticides. In mosquitoes, metabolic detoxifications of insecticides by detoxifying enzymes are the main strategy to withstand recurrent exposure to synthetic insecticides. Esterases are one of the three gene families of detoxification enzymes involved in metabolic detoxification of insecticide and confers resistance for organophosphorus insecticide (OP) as well as for carbamate insecticide. In the present study, the activity of α- and β-esterases have been evaluated in different A. aegypti populations collected from the Dooars and Terai regions of West Bengal, India. The activity of α-esterases was 1.2–3.1 fold and β-esterases was 2.0–23.0 fold higher than laboratory control population. The electro-phoregrams of α-esterases showed expression of 11 isozymes from two gene loci and for β-esterases showed expression of 09 isozymes from two gene loci in all the field populations of A. aegypti. The results showed a significant variation of esterase activity and isozyme expression among different population indicating variability in biochemical susceptibility among the populations. The present work presents first hand data on biochemical resistance of A. aegypti in this region and may be used as an integral component for planning and evaluation of vector-borne diseases and integrated vector management programmes.     

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.     

From Lab to Field: The Influence of Urban Landscapes on the Invasive Potential of Wolbachia in Brazilian Aedes aegypti Mosquitoes

Background

The symbiotic bacterium Wolbachia is currently being trialled as a biocontrol agent in several countries to reduce dengue transmission. Wolbachia can invade and spread to infect all individuals within wild mosquito populations, but requires a high rate of maternal transmission, strong cytoplasmic incompatibility and low fitness costs in the host in order to do so. Additionally, extensive differences in climate, field-release protocols, urbanization level and human density amongst the sites where this bacterium has been deployed have limited comparison and analysis of Wolbachia’s invasive potential.

Methodology/Principal Findings

We examined key phenotypic effects of the wMel Wolbachia strain in laboratory Aedes aegypti mosquitoes with a Brazilian genetic background to characterize its invasive potential. We show that the wMel strain causes strong cytoplasmic incompatibility, a high rate of maternal transmission and has no evident detrimental effect on host fecundity or fertility. Next, to understand the effects of different urban landscapes on the likelihood of mosquito survival, we performed mark-release-recapture experiments using Wolbachia-uninfected Brazilian mosquitoes in two areas of Rio de Janeiro where Wolbachia will be deployed in the future. We characterized the mosquito populations in relation to the socio-demographic conditions at these sites, and at three other future release areas. We then constructed mathematical models using both the laboratory and field data, and used these to describe the influence of urban environmental conditions on the likelihood that the Wolbachia infection frequency could reach 100% following mosquito release. We predict successful invasion at all five field sites, however the conditions by which this occurs vary greatly between sites, and are strongly influenced by the size of the local mosquito population.

Conclusions/Significance

Through analysis of laboratory, field and mathematical data, we show that the wMel strain of Wolbachia possesses the characteristics required to spread effectively in different urban socio-demographic environments in Rio de Janeiro, including those where mosquito releases from the Eliminate Dengue Program will take place.     

Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida,USA

The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two‐step PCR assay to detect Wolbachia in wild‐collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop‐mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia‐positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.     

Wolbachia induces male-specific mortality in the mosquito Culex pipiens (LIN strain)

Background

Wolbachia are maternally inherited endosymbionts that infect a diverse range of invertebrates, including insects, arachnids, crustaceans and filarial nematodes. Wolbachia are responsible for causing diverse reproductive alterations in their invertebrate hosts that maximize their transmission to the next generation. Evolutionary theory suggests that due to maternal inheritance, Wolbachia should evolve toward mutualism in infected females, but strict maternal inheritance means there is no corresponding force to select for Wolbachia strains that are mutualistic in males.

Methodology/Principal findings

Using cohort life-table analysis, we demonstrate that in the mosquito Culex pipiens (LIN strain), Wolbachia-infected females show no fitness costs due to infection. However, Wolbachia induces up to a 30% reduction in male lifespan.

Conclusions/significance

These results indicate that the Wolbachia infection of the Culex pipiens LIN strain is virulent in a sex-specific manner. Under laboratory situations where mosquitoes generally mate at young ages, Wolbachia strains that reduce male survival could evolve by drift because increased mortality in older males is not a significant selective force.     

Assessment of fitness and vector competence of a New Caledonia wMel Aedes aegypti strain before field-release

BackgroundBiological 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 findingsThe 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/significanceOur 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.     

Wolbachia Infection Modifies the Profile,Shuttling and Structure of MicroRNAs in a Mosquito Cell Line

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of the miRNA profile of a mosquito cell line from Aedes aegypti. We characterized the miRNA composition of the nucleus and the cytoplasm of uninfected cells and compared it with the one of cells infected with the endosymbiotic bacterium Wolbachia strain wMelPop-CLA. We found an overall increase of small RNAs between 18 and 28 nucleotides in both cellular compartments in Wolbachia-infected cells and identified specific miRNAs induced and/or suppressed by the Wolbachia infection. We discuss the mechanisms that the cell may use to shuttle miRNAs between the cytoplasm and the nucleus. In addition, we identified piRNAs that changed their abundance in response to Wolbachia infection. The miRNAs and piRNAs identified in this study provide promising leads for investigations into the host-endosymbiont interactions and for better understanding of how Wolbachia manipulates the host miRNA machinery in order to facilitate its persistent replication in infected cells.