The Native Wolbachia Symbionts Limit Transmission of Dengue Virus in Aedes albopictus

Background

The chikungunya (CHIK) outbreak that struck La Reunion Island in 2005 was preceded by few human cases of Dengue (DEN), but which surprisingly did not lead to an epidemic as might have been expected in a non-immune population. Both arboviral diseases are transmitted to humans by two main mosquito species, Aedes aegypti and Aedes albopictus. In the absence of the former, Ae. albopictus was the only species responsible for viral transmission on La Reunion Island. This mosquito is naturally super-infected with two Wolbachia strains, wAlbA and wAlbB. While Wolbachia does not affect replication of CHIK virus (CHIKV) in Ae. albopictus, a similar effect was not observed with DEN virus (DENV).

Methods/Principal Findings

To understand the weak vectorial status of Ae. albopictus towards DENV, we used experimental oral infections of mosquitoes from La Reunion Island to characterize the impact of Wolbachia on DENV infection. Viral loads and Wolbachia densities were measured by quantitative PCR in different organs of Ae. albopictus where DENV replication takes place after ingestion. We found that: (i) Wolbachia does not affect viral replication, (ii) Wolbachia restricts viral density in salivary glands, and (iii) Wolbachia limits transmission of DENV, as infectious viral particles were only detected in the saliva of Wolbachia-uninfected Ae. albopictus, 14 days after the infectious blood-meal.

Conclusions

We show that Wolbachia does not affect the replication of DENV in Ae. albopictus. However, Wolbachia is able to reduce viral infection of salivary glands and limit transmission, suggesting a role of Wolbachia in naturally restricting the transmission of DENV in Ae. albopictus from La Reunion Island. The extension of this conclusion to other Ae. albopictus populations should be investigated.     

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.     

Understanding the mechanism of Chikungunya virus vector competence in three species of mosquitoes

Chikungunya virus (CHIKV) is primarily transmitted by Aedes spp. mosquitoes. The present study investigated vector competence for CHIKV in Aedes aegypti and Aedes albopictus mosquitoes found in Madurai, South India. The role of receptor proteins on midguts contributing to permissiveness of CHIKV to Aedes spp. mosquitoes was also undertaken. Mosquitoes were orally infected with CHIKV DRDE‐06. Infection of midguts and dissemination to heads was confirmed by immunofluorescence assay at different time points. A plaque assay was performed from mosquito homogenates at different time points to study CHIKV replication. Presence of putative CHIKV receptor proteins on mosquito midgut epithelial cells was detected by virus overlay protein binding assay (VOPBA). The identity of these proteins was established using mass spectrometry. CHIKV infection of Ae. aegypti and Ae. albopictus midguts and dissemination to heads was observed to be similar. A plaque assay performed with infected mosquito homogenates revealed that CHIKV replication dynamics was similar in Aedes sp. mosquitoes until 28 days post infection. VOPBA performed with mosquito midgut membrane proteins revealed that prohibitin could serve as a putative CHIKV receptor on Aedes mosquito midguts, whereas an absence of CHIKV binding protein/s on Culex quinquefasciatus midguts can partially explain the non‐permissiveness of these mosquitoes to infection.     

Exposure to chikungunya virus and adult longevity in Aedes aegypti (L.) and Aedes albopictus (Skuse)

Chikungunya virus (CHIKV) recently emerged as a global threat to public health through its adaptation to the cosmopolitan mosquito Aedes albopictus Skuse. Aedes albopictus is highly susceptible to the emergent strain of CHIKV, relative to the historical vector of CHIKV, Aedes aegypti (L.). We hypothesized that the high susceptibility of Ae. albopictus to CHIKV may have a cost in terms of longevity and fecundity among infected vs non‐infected mosquitoes, relative to Ae. aegypti. We performed a longevity experiment comparing Ae. aegypti and Ae. albopictus exposed to the emergent strain of CHIKV (LR‐2006OPY1). We found a small but significant decrease in longevity of Ae. albopictus, but not Ae. aegypti, in response to exposure to CHIKV. We did not observe significant differences in numbers of eggs laid by either species in response to exposure. Longevity and body titer of infected Ae. albopictus were significantly negatively correlated, such that individuals that lived longer had lower viral body titers when they died. The cost of exposure, while not high, suggests there may be physiological constraints in the evolution of viral infectiousness in its insect vector.     

Chikungunya virus impacts the diversity of symbiotic bacteria in mosquito vector

Mosquitoes transmit numerous arboviruses including dengue and chikungunya virus (CHIKV). In recent years, mosquito species Aedes albopictus has expanded in the Indian Ocean region and was the principal vector of chikungunya outbreaks in La Reunion and neighbouring islands in 2005 and 2006. Vector‐associated bacteria have recently been found to interact with transmitted pathogens. For instance, Wolbachia modulates the replication of viruses or parasites. However, there has been no systematic evaluation of the diversity of the entire bacterial populations within mosquito individuals particularly in relation to virus invasion. Here, we investigated the effect of CHIKV infection on the whole bacterial community of Ae. albopictus. Taxonomic microarrays and quantitative PCR showed that members of Alpha‐ and Gammaproteobacteria phyla, as well as Bacteroidetes, responded to CHIKV infection. The abundance of bacteria from the Enterobacteriaceae family increased with CHIKV infection, whereas the abundance of known insect endosymbionts like Wolbachia and Blattabacterium decreased. Our results clearly link the pathogen propagation with changes in the dynamics of the bacterial community, suggesting that cooperation or competition occurs within the host, which may in turn affect the mosquito traits like vector competence.     

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.     

A Wolbachia wMel Transinfection in Aedes albopictus Is Not Detrimental to Host Fitness and Inhibits Chikungunya Virus

Background

Wolbachia inherited intracellular bacteria can manipulate the reproduction of their insect hosts through cytoplasmic incompatibility (CI), and certain strains have also been shown to inhibit the replication or dissemination of viruses. Wolbachia strains also vary in their relative fitness effects on their hosts and this is a particularly important consideration with respect to the potential of newly created transinfections for use in disease control.

Methodology/Principal Findings

In Aedes albopictus mosquitoes transinfected with the wMel strain from Drosophila melanogaster, which we previously reported to be unable to transmit dengue in lab challenges, no significant detrimental effects were observed on egg hatch rate, fecundity, adult longevity or male mating competitiveness. All these parameters influence the population dynamics of Wolbachia, and the data presented are favourable with respect to the aim of taking wMel to high population frequency. Challenge with the chikungunya (CHIKV) virus, for which Ae. albopictus is an important vector, was conducted and the presence of wMel abolished CHIKV dissemination to the saliva.

Conclusions/significance

Taken together, these data suggest that introducing wMel into natural Ae. albopictus populations using bidirectional CI could be an efficient strategy for preventing or reducing the transmission of arboviruses by this species.     

Two Chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different patterns of infection in the mosquito, Aedes albopictus

Background

A Chikungunya (CHIK) outbreak hit La Réunion Island in 2005–2006. The implicated vector was Aedes albopictus. Here, we present the first study on the susceptibility of Ae. albopictus populations to sympatric CHIKV isolates from La Réunion Island and compare it to other virus/vector combinations.

Methodology and Findings

We orally infected 8 Ae. albopictus collections from La Réunion and 3 from Mayotte collected in March 2006 with two Chikungunya virus (CHIKV) from La Réunion: (i) strain 05.115 collected in June 2005 with an Alanine at the position 226 of the glycoprotein E1 and (ii) strain 06.21 collected in November 2005 with a substitution A226V. Two other CHIKV isolates and four additional mosquito strains/species were also tested. The viral titer of the infectious blood-meal was 10⁷ plaque forming units (pfu)/mL. Dissemination rates were assessed by immunofluorescent staining on head squashes of surviving females 14 days after infection. Rates were at least two times higher with CHIKV 06.21 compared to CHIKV 05.115. In addition, 10 individuals were analyzed every day by quantitative RT-PCR. Viral RNA was quantified on (i) whole females and (ii) midguts and salivary glands of infected females. When comparing profiles, CHIKV 06.21 produced nearly 2 log more viral RNA copies than CHIKV 05.115. Furthermore, females infected with CHIKV 05.115 could be divided in two categories: weakly susceptible or strongly susceptible, comparable to those infected by CHIKV 06.21. Histological analysis detected the presence of CHIKV in salivary glands two days after infection. In addition, Ae. albopictus from La Réunion was as efficient vector as Ae. aegypti and Ae. albopictus from Vietnam when infected with the CHIKV 06.21.

Conclusions

Our findings support the hypothesis that the CHIK outbreak in La Réunion Island was due to a highly competent vector Ae. albopictus which allowed an efficient replication and dissemination of CHIKV 06.21.     

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.     

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.     

Wolbachia strain wAlbA blocks Zika virus transmission in Aedes aegypti

Transinfections of the maternally transmitted endosymbiont Wolbachia pipientis can reduce RNA virus replication and prevent transmission by Aedes aegypti, and also have the capacity to invade wild-type populations, potentially reaching and maintaining high infection frequencies. Levels of virus transmission blocking are positively correlated with Wolbachia intracellular density. Despite reaching high densities in Ae. aegypti, transinfections of wAlbA, a strain native to Aedes albopictus, showed no blocking of Semliki Forest Virus in previous intrathoracic injection challenges. To further characterize wAlbA blocking in Ae. aegypti, adult females were intrathoracically challenged with Zika (ZIKV) and dengue viruses, and then fed a ZIKV-containing bloodmeal. No blocking was observed with either virus when challenged by intrathoracic injection. However, when ZIKV was delivered orally, wAlbA-infected females showed a significant reduction in viral replication and dissemination compared with uninfected controls, as well as a complete absence of virus in saliva. Although other Wolbachia strains have been shown to cause more robust viral blocking in Ae. aegypti, these findings demonstrate that, in principle, wAlbA could be used to reduce virus transmission in this species. Moreover, the results highlight the potential for underestimation of the strength of virus-blocking when based on intrathoracic injection compared with more natural oral challenges.     

Chikungunya Virus and Aedes Mosquitoes: Saliva Is Infectious as soon as Two Days after Oral Infection

Background

Aedes aegypti and Aedes albopictus are potential vectors of chikungunya virus (CHIKV). The recent CHIKV outbreaks were caused by a new variant characterized by a mutation in the E1 glycoprotein gene (E1-226V) which has favored a better transmissibility by Ae. albopictus. As Ae. albopictus tends to replace Ae. aegypti in many regions, one question remained: is Ae. albopictus as efficient as Ae. aegypti to transmit the variant E1-226V of CHIKV?

Methodology and Findings

We infected orally both species with the variant E1-226V and estimated the infection, the viral dissemination, and the transmission rate by real time RT-PCR. Additionally, we used an in vitro assay to determine the amount of virus delivered by mosquitoes in their saliva. We found that Ae. aegypti as well as Ae. albopictus ensured a high replication of the virus which underwent an efficient dissemination as detectable in the salivary glands at day 2 post-infection (pi). Infectious CHIKV particles were delivered by salivary glands from day 2 with a maximum at day 6 pi for Ae. albopictus (10^3.3 PFU) and day 7 pi for Ae. aegypti (10^2.5 PFU).

Conclusions

Ae. albopictus is slightly more efficient than Ae. aegypti to transmit the variant E1-226V of CHIKV. These results will help to design an efficient vector control to limit transmission as soon as the first human cases are diagnosed.     

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.     

SYTO11 staining vs FISH staining: a comparison of two methods to stain Wolbachia pipientis in cell cultures

Aims: The Aedes albopictus C7‐10 cell line was infected with Wolbachia strains wRi and wAlbB to create C7‐10R and C7‐10B cell lines, respectively. We compared two different methods, fluorescence in situ hybridization staining and SYTO11 staining, to describe these new Wolbachia infections in C7‐10. Methods and Results: Both staining methods were as efficient to stain Wolbachia. A formula was developed to quantify Wolbachia infection. The infection levels in C7‐10B and C7‐10R differed. The live stain SYTO11 was found to be useful to visualize Wolbachia in replicating host cells. Its potential cytotoxic effect at high concentration was investigated. Conclusions: C7‐10 supported two Wolbachia infections, constituting new tools to study Wolbachia–host interactions. The different infection levels suggest that wRi and wAlbB have different requirements for their survival in C7‐10 host cell line. Observation of SYTO11‐stained live cells gave new insights on Wolbachia segregation pattern during host cell mitosis. Significance and Impact of the Study: Wolbachia‐induced phenotypes in their arthropod and worm hosts could potentially be used to control pest populations. However, the mechanisms underlying these phenotypes are difficult to study because of Wolbachia’s intracellular lifestyle. The Wolbachia infections in C7‐10 described here could be used as in vitro models to investigate Wolbachia biology.     

Induction of cell migration by transient Wolbachia pipientis infection in Aedes albopictus cell line

Wolbachia is a genus of maternally transmitted bacteria having an endosymbiotic relationship with arthropod and nematode species. These bacteria manipulate host development, sex-determination, and reproduction. In addition, they are known to potentially suppress vector-borne diseases by interfering with pathogen transmission. Although the occurrence of Wolbachia infection in insects has been known, the underlying mechanisms that mediate their interactions remain unclear. To examine the influence of Wolbachia adaptation on the host, we infected wAlbA and wAlbB strain from Aedes albopictus into the C6/36 cell line derived from Ae. albopictus. The transient Wolbachia infection was characterized by induction of cell migration without cell proliferation. The production of nitrite and reactive oxygen species (ROS) was induced by transient Wolbachia infection. Cells with transient Wolbachia infection exhibited elevated expression of Toll-like receptor 6 (TLR6) and myeloid differentiation primary response 88 (Myd88). Conversely, the expression of TLR2, TLR4, TLR7, Cactus, Ankyrin, and Argonaute2 (AGO2) was inhibited upon Wolbachia infection. These results suggest that Wolbachia has an influence on the cell migration ability as well as host innate immune response in vitro. Considering these results, transient Wolbachia strain transfer in C6/36 cells might be an important approach for studying Wolbachia-host interactions and might help gain a deeper understanding of the early adaptation of Wolbachia in the original host insect.     

Proteasome activity in a naïve mosquito cell line infected with <Emphasis Type="Italic">Wolbachia pipientis w</Emphasis>AlbB

We used Wolbachia pipientis strain wAlbB from Aedes albopictus Aa23 cells to infect clonal Ae. albopictus TK-6 cells, which are resistant to 5-bromodeoxyuridine. Infected TK-6 cells were cultured in medium containing 5-bromodeoxyuridine to select against Aa23 cells that might have persisted in the inoculum. Infected TK-6 lines retained the Wolbachia infection for 5 mo, indicating that their metabolic processes support Wolbachia growth and multiplication. To investigate early events after Wolbachia infection, we labeled infected cells with ³⁵S[methionine/cysteine]. Patterns of labeled proteins on sodium dodecyl sulfate gels were similar in control and infected cells, with the exception of a 29-kDa protein. Tandem mass spectrometry revealed that the 29-kDa band included α and β subunits of the 26S proteasome. Independent confirmation of the up-regulation of the proteasome was established by probing Western blots with a monoclonal antibody to the proteasome-associated co-factor, ubiquitin. Wolbachia’s loss of metabolic pathways for the synthesis of most amino acids and retention of pathways for their uptake and metabolism suggest that proteasome activation provides a mechanism whereby controlled degradation of intracellular host proteins would increase availability of amino acids to support establishment and maintenance of the Wolbachia infection.     

Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia

Background

Mosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia.

Methods and Findings

CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and Aedes mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in Ae. albopictus cells (C6/36). Both CHIKV strains infected Ae. albopictus mosquitoes at a higher rate than Ae. aegypti, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected Ae. aegypti at higher rates.

Conclusions

The greater ability of the ECSA strain to replicate in Ae. albopictus may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where Ae. albopictus predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and Aedes species.     

Male mating performance and cytoplasmic incompatibility in a wPip Wolbachia trans‐infected line of Aedes albopictus (Stegomyia albopicta)

Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae) is a maternally inherited endosymbiont of a large number of insects and other arthropods that induces various effects on host reproductive biology. Among these, cytoplasmic incompatibility (CI) is a form of sterility induced in eggs produced by mating between infected males and females uninfected or infected by an incompatible Wolbachia strain. This phenomenon has been proposed as a potential way to produce functionally sterile males to be used in genetic control programmes. In this paper, we report on experiments carried out to evaluate the mating performances of males of an Aedes albopictus (Stegomyia albopicta) (Diptera: Culicidae) line (ARwP), harbouring a new Wolbachia infection [the wPip strain from Culex pipiens Linnaeus (Diptera: Culicidae)], in comparison with naturally infected males (SR line). ARwP males did not differ from SR males with regard to insemination capacity. Mating competitiveness did not differ significantly between lines in either laboratory or greenhouse conditions. Moreover, crosses with SR females were characterized by a 100% CI regardless of ARwP male age. All of these findings suggest that ARwP males may represent a very efficient tool for control programmes against Ae. albopictus based on the release of functionally sterile males.     

Are Aedes albopictus or other mosquito species from northern Italy competent to sustain new arboviral outbreaks?

The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae), native to Southeast Asia, has extended its geographical distribution to invade new temperate and tropical regions. This species was introduced in 1990 to Italy and has since become the main pest in urban settings. It was incriminated as a principal vector in the first European outbreak of chikungunya virus (CHIKV) in the province of Ravenna (Italy) in 2007. This outbreak was associated with CHIKV E1-226V, efficiently transmitted by Ae. albopictus . The occurrence of this outbreak in a temperate country led us to estimate the potential of Ae. albopictus to transmit CHIKV and dengue virus (DENV), and to determine the susceptibility to CHIKV of other mosquito species collected in northern Italy. Experimental infections showed that Ae. albopictus exhibited high disseminated infection rates for CHIKV (75.0% in Alessandria; 90.3% in San Lazzaro) and low disseminated infection rates for DENV-2 (14.3% in San Lazzaro; 38.5% in Alessandria). Moreover, Ae. albopictus was able to attain a high level of viral replication, with CHIKV detectable in the salivary glands at day 2 after infection. In addition, the other three mosquito species, Anopheles maculipennis Meigen, Aedes vexans vexans (Meigen) and Culex pipiens L., showed variable susceptibilities to infection with CHIKV, of 0%, 7.7% and 0–33%, respectively. This information on vector competence is crucial in assessing the risk for an outbreak of CHIKV or DENV in Italy.     

Transinfection and growth discrepancy of Drosophila Wolbachia strain wMel in cell lines of the mosquito Aedes albopictus

Aim: The Wolbachia strain wMel can protect Drosophila melanogaster against pathogenic RNA viruses. To analyse the potential of this inhibitory effect against arboviruses vectorized by these mosquitoes, we here first transinfected the Aedes albopictus Aa23 and C6/36 cell lines with the Wolbachia strain wMel and then monitored their infection dynamics. Methods and Results: Wolbachia strain wMel was transferred into A. albopictus Aa23 and C6/36 cell lines using the shell vial technique. The presence of the bacterium in the transinfected cells was monitored by quantitative PCR and fluorescence in situ hybridization. Bacteria could be detected in the cytoplasm of both the Aa23 and C6/36 cell lines. However, the dynamics and stability of the bacterial infection differed depending on the initial cell background. The Aa23 cell line, which had been treated with a tetracycline antibiotic 2 years previously to eliminate its natural Wolbachia wAlbB‐infecting strain, lost the introduced Wolbachia wMel strain after 12 passages postinfection. In contrast, the C6/36 cell line, which had originally been aposymbiotic, displayed a stable infection with Wolbachia wMel. The bacterial density in C6/36 was greater than that of the A. albopictus RML12 cell line from which the wMel strain had originated. Conclusions: Transient or persistent transinfection of A. albopictus Aa23 and C6/36 cell lines with Wolbachia wMel strain was achieved. The results indicate the influence of the genetic background of mosquito cells in maintaining Wolbachia originating from a distant dipteral host. Significance and Impact of the Study: The cell model built here can now be used to investigate the viral inhibitory effect of the Wolbachia wMel strain against arboviruses such as dengue and chikungunya, which are transmitted by the mosquito A. albopictus.