Changing domesticity of Aedes aegypti in northern peninsular Malaysia: reproductive consequences and potential epidemiological implications

Background

The domestic dengue vector Aedes aegypti mosquitoes breed in indoor containers. However, in northern peninsular Malaysia, they show equal preference for breeding in both indoor and outdoor habitats. To evaluate the epidemiological implications of this peridomestic adaptation, we examined whether Ae. aegypti exhibits decreased survival, gonotrophic activity, and fecundity due to lack of host availability and the changing breeding behavior.

Methodology/Principal Findings

This yearlong field surveillance identified Ae. aegypti breeding in outdoor containers on an enormous scale. Through a sequence of experiments incorporating outdoors and indoors adapting as well as adapted populations, we observed that indoors provided better environment for the survival of Ae. aegypti and the observed death patterns could be explained on the basis of a difference in body size. The duration of gonotrophic period was much shorter in large-bodied females. Fecundity tended to be greater in indoor acclimated females. We also found increased tendency to multiple feeding in outdoors adapted females, which were smaller in size compared to their outdoors breeding counterparts.

Conclusion/Significance

The data presented here suggest that acclimatization of Ae. aegypti to the outdoor environment may not decrease its lifespan or gonotrophic activity but rather increase breeding opportunities (increased number of discarded containers outdoors), the rate of larval development, but small body sizes at emergence. Size is likely to be correlated with disease transmission. In general, small size in Aedes females will favor increased blood-feeding frequency resulting in higher population sizes and disease occurrence.     

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.     

Female-specific flightless (fsRIDL) phenotype for control of Aedes albopictus

Background

Aedes albopictus, the Asian tiger mosquito, is a vector of several arboviruses including dengue and chikungunya, and is also a significant nuisance mosquito. It is one of the most invasive of mosquitoes with a relentlessly increasing geographic distribution. Conventional control methods have so far failed to control Ae. albopictus adequately. Novel genetics-based strategies offer a promising alternative or aid towards efficient control of this mosquito.

Methodology/Principal Findings

We describe here the isolation, characterisation and use of the Ae. albopictus Actin-4 gene to drive a dominant lethal gene in the indirect flight muscles of Ae. albopictus, thus inducing a conditional female-specific late-acting flightless phenotype. We also show that in this context, the Actin-4 regulatory regions from both Ae. albopictus and Ae. aegypti can be used to provide conditional female-specific flightlessness in either species.

Conclusion/Significance

With the disease-transmitting females incapacitated, the female flightless phenotype encompasses a genetic sexing mechanism and would be suitable for controlling Ae. albopictus using a male-only release approach as part of an integrated pest management strategy.     

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.     

Genetic Structure and Wolbachia Genotyping in Naturally Occurring Populations of Aedes albopictus across Contiguous Landscapes of Orissa,India

Background

Aedes albopictus has recently been implicated as a major vector in the emergence of dengue and chikungunya in several parts of India, like Orissa, which is gradually gaining endemicity for arboviral diseases. Ae. albopictus is further known to be naturally infected with Wolbachia (maternally inherited bacterium), which causes cytoplasmic incompatibility (CI) in mosquitoes leading to sperm-egg incompatibility inducing the death of embryo. Knowledge of genetic diversity of Ae. albopictus, along with revealing the type of Wolbachia infection in Ae. albopictus is important to explore the genetic and biological characteristics of Ae. albopictus, prior to exploring the uses of CI-based vector control strategies. In this study, we assessed the population genetic structure and the pattern of Wolbachia infection in Ae. albopictus mosquitoes of Orissa.

Methods and Results

Ae. albopictus mosquitoes were collected from 15 districts representing the four physiographical regions of Orissa from 2010–2012, analyzed for genetic variability at seven microsatellite loci and genotyped for Wolbachia strain detection using wsp gene primers. Most microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structure among all geographic populations (F_ST = 0.088). Genetic diversity was high (F_ST = 0.168) in Coastal Plains populations when compared with other populations, which was also evident from cluster analyses that showed most Coastal Plains populations consisted of a separate genetic cluster. Genotyping analyses revealed that Wolbachia-infected Ae. albopictus field populations of Orissa were mostly superinfected with wAlbA and wAlbB strains. Wolbachia superinfection was more pronounced in the Coastal Plain populations.

Conclusion

High genetic structure and Wolbachia superinfection, observed in the Coastal Plain populations of Orissa suggested it to be genetically and biologically more unique than other populations, and hence could influence their vectorial attributes. Such high genetic diversity observed among Coastal Plains populations could be attributed to multiple introductions of Ae. albopictus in this region.     

High Efficiency of Temperate Aedes albopictus to Transmit Chikungunya and Dengue Viruses in the Southeast of France

Background

Since 2005, cases of chikungunya (CHIK) were caused by an unusual vector, Aedes albopictus. This mosquito, present in Europe since 1979, has gained importance since its involvement in the first CHIK outbreak in Italy in 2007. The species is capable of transmitting experimentally 26 arboviruses. However, the vectorial status of its temperate populations has remained little investigated. In 2010, autochthonous cases of CHIK and dengue (DEN) were reported in southeastern France. We evaluated the potential of a French population of Ae. albopictus in the transmission of both viruses.

Methodology and Principal Findings

We used two strains of each virus, CHIK and DEN: one strain was isolated from an imported case, and one from an autochthonous case. We used as controls Aedes aegypti from India and Martinique, the source of the imported cases of CHIK and DEN, respectively. We showed that Ae. albopictus from Cagnes-sur-Mer (AL-CSM) was as efficient as the typical tropical vector Ae. aegypti from India to experimentally transmit both CHIK strains isolated from patients in Fréjus, with around 35–67% of mosquitoes delivering up to 14 viral particles at day 3 post-infection (pi). The unexpected finding came from the high efficiency of AL-CSM to transmit both strains of DENV-1 isolated from patients in Nice. Almost 67% of Ae. albopictus AL-CSM which have ensured viral dissemination were able to transmit at day 9 pi when less than 21% of the typical DEN vector Ae. aegypti from Martinique could achieve transmission.

Conclusions/Significance

Temperate Ae. albopictus behaves differently compared to its counterpart from tropical regions, where recurrent epidemic outbreaks occur. Its potential responsibility for outbreaks in Europe should not be minimized.     

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.     

Urbanization Increases Aedes albopictus Larval Habitats and Accelerates Mosquito Development and Survivorship

Introduction

Aedes albopictus is a very invasive and aggressive insect vector that causes outbreaks of dengue fever, chikungunya disease, and yellow fever in many countries. Vector ecology and disease epidemiology are strongly affected by environmental changes. Urbanization is a worldwide trend and is one of the most ecologically modifying phenomena. The purpose of this study is to determine how environmental changes due to urbanization affect the ecology of Aedes albopictus.

Methods

Aquatic habitats and Aedes albopictus larval population surveys were conducted from May to November 2013 in three areas representing rural, suburban, and urban settings in Guangzhou, China. Ae. albopictus adults were collected monthly using BG-Sentinel traps. Ae. albopictus larva and adult life-table experiments were conducted with 20 replicates in each of the three study areas.

Results

The urban area had the highest and the rural area had the lowest number of aquatic habitats that tested positive for Ae. albopictus larvae. Densities in the larval stages varied among the areas, but the urban area had almost two-fold higher densities in pupae and three-fold higher in adult populations compared with the suburban and rural areas. Larvae developed faster and the adult emergence rate was higher in the urban area than in suburban and rural areas. The survival time of adult mosquitoes was also longer in the urban area than it was in suburban and rural areas. Study regions, surface area, water depth, water clearance, surface type, and canopy coverage were important factors associated with the presence of Ae. albopictus larvae.

Conclusions

Urbanization substantially increased the density, larval development rate, and adult survival time of Ae. albopictus, which in turn potentially increased the vector capacity, and therefore, disease transmissibility. Mosquito ecology and its correlation with dengue virus transmission should be compared in different environmental settings.     

Next Generation Sequencing Reveals Regulation of Distinct Aedes microRNAs during Chikungunya Virus Development

Background

Application of genomics and Next Generation sequencing has led to the identification of new class of cellular functional molecules, namely, small RNAs. Of the several classes of ncRNAs (non-coding RNA), microRNAs have been demonstrated to exert determinative influence on various cellular processes. It is becoming abundantly clear that host/vector/pathogen encoded microRNAs impact eventual pathogenesis. In this context, the participation of vector based microRNAs in disease transmission and pathogen development is being investigated intensively. A few studies have highlighted the role of vector encoded microRNAs in pathogen infection. We conducted this study to evaluate the role of host miRNAs upon CHIKV (Chikungunya Virus) infection in an important vector, Aedes albopictus.

Findings

We identified 88 and 79 known miRNAs in uninfected and CHIKV infected Ae. albopictus Singh''s cell line respectively. We further identified nine novel miRNAs in Ae. albopictus. Comparison of the two libraries revealed differential expression of 77 common miRNAs between them. CHIKV infection specifically altered the miRNA profile of a specific set of eight miRNAs. Putative targets of these regulated miRNAs were identified and classified into their pathways.

Conclusions

In our study we have identified and described the profiles of various miRNAs upon CHIKV infection in Ae. albopictus. This investigation provides an insight about cellular modification by miRNAs during CHIKV infection and the results provide leads for identifying potential candidates for vector based antiviral strategies.     

Orally Co-Infected Aedes albopictus from La Reunion Island,Indian Ocean,Can Deliver Both Dengue and Chikungunya Infectious Viral Particles in Their Saliva

Background

First described in humans in 1964, reports of co-infections with dengue (DENV) and chikungunya (CHIKV) viruses are increasing, particularly after the emergence of chikungunya (CHIK) in the Indian Ocean in 2005–2006 due to a new variant highly transmitted by Aedes albopictus. In this geographic area, a dengue (DEN) outbreak transmitted by Ae. albopictus took place shortly before the emergence of CHIK and co-infections were reported in patients. A co-infection in humans can occur following the bite of two mosquitoes infected with one virus or to the bite of a mosquito infected with two viruses. Co-infections in mosquitoes have never been demonstrated in the field or in the laboratory. Thus, we question about the ability of a mosquito to deliver infectious particles of two different viruses through the female saliva.

Methodology/Principal Findings

We orally exposed Ae. albopictus from La Reunion Island with DENV-1 and CHIKV isolated respectively during the 2004–2005 and the 2005–2006 outbreaks on this same island. We were able to show that Ae. albopictus could disseminate both viruses and deliver both infectious viral particles concomitantly in its saliva. We also succeeded in inducing a secondary infection with CHIKV in mosquitoes previously inoculated with DENV-1.

Conclusions/Significance

In this study, we underline the ability of Ae. albopictus to be orally co-infected with two different arboviruses and furthermore, its capacity to deliver concomitantly infectious particles of CHIKV and DENV in saliva. This finding is of particular concern as Ae. albopictus is still expanding its geographical range in the tropical as well as in the temperate regions. Further studies are needed to try to elucidate the molecular/cellular basis of this phenomenon.     

Age-Dependent Effects of Oral Infection with Dengue Virus on Aedes aegypti (Diptera: Culicidae) Feeding Behavior,Survival, Oviposition Success and Fecundity

Background

Aedes aegypti is the main vector of dengue, a disease that is increasing its geographical range as well as incidence rates. Despite its public health importance, the effect of dengue virus (DENV) on some mosquito traits remains unknown. Here, we investigated the impact of DENV-2 infection on the feeding behavior, survival, oviposition success and fecundity of Ae. aegypti females.

Methods/Principal Findings

After orally-challenging Ae. aegypti females with a DENV-2 strain using a membrane feeder, we monitored the feeding behavior, survival, oviposition success and fecundity throughout the mosquito lifespan. We observed an age-dependent cost of DENV infection on mosquito feeding behavior and fecundity. Infected individuals took more time to ingest blood from anesthetized mice in the 2^nd and 3^rd weeks post-infection, and also longer overall blood-feeding times in the 3^rd week post-infection, when females were around 20 days old. Often, infected Ae. aegypti females did not lay eggs and when they were laid, smaller number of eggs were laid compared to uninfected controls. A reduction in the number of eggs laid per female was evident starting on the 3^rd week post-infection. DENV-2 negatively affected mosquito lifespan, since overall the longevity of infected females was halved compared to that of the uninfected control group.

Conclusions

The DENV-2 strain tested significantly affected Ae. aegypti traits directly correlated with vectorial capacity or mosquito population density, such as feeding behavior, survival, fecundity and oviposition success. Infected mosquitoes spent more time ingesting blood, had reduced lifespan, laid eggs less frequently, and when they did lay eggs, the clutches were smaller than uninfected mosquitoes.     

Genetic structure of the tiger mosquito, Aedes albopictus, in Cameroon (Central Africa)

Background

Aedes albopictus (Skuse, 1884) (Diptera: Culicidae), a mosquito native to Asia, has recently invaded all five continents. In Central Africa it was first reported in the early 2000s, and has since been implicated in the emergence of arboviruses such as dengue and chikungunya in this region. Recent genetic studies of invasive species have shown that multiple introductions are a key factor for successful expansion in new areas. As a result, phenotypic characters such as vector competence and insecticide susceptibility may vary within invasive pest species, potentially affecting vector efficiency and pest management. Here we assessed the genetic variability and population genetics of Ae. albopictus isolates in Cameroon (Central Africa), thereby deducing their likely geographic origin.

Methods and Results

Mosquitoes were sampled in 2007 in 12 localities in southern Cameroon and analyzed for polymorphism at six microsatellite loci and in two mitochondrial DNA regions (ND5 and COI). All the microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structureamong geographic populations (F_ST = 0.068, P<0.0001). Analysis of mtDNA sequences revealed four haplotypes each for the COI and ND5 genes, with a dominant haplotype shared by all Cameroonian samples. The weak genetic variation estimated from the mtDNA genes is consistent with the recent arrival of Ae. albopictus in Cameroon. Phylogeographic analysis based on COI polymorphism indicated that Ae. albopictus populations from Cameroon are related to tropical rather than temperate or subtropical outgroups.

Conclusion

The moderate genetic diversity observed among Cameroonian Ae. albopictus isolates is in keeping with recent introduction and spread in this country. The genetic structure of natural populations points to multiple introductions from tropical regions.     

Aedes (Stegomyia) albopictus (Skuse): A Potential Vector of Zika Virus in Singapore

Background

Zika virus (ZIKV) is a little known arbovirus until it caused a major outbreak in the Pacific Island of Yap in 2007. Although the virus has a wide geographic distribution, most of the known vectors are sylvatic Aedes mosquitoes from Africa where the virus was first isolated. Presently, Ae. aegypti is the only known vector to transmit the virus outside the African continent, though Ae. albopictus has long been a suspected vector. Currently, Ae. albopictus has been shown capable of transmitting more than 20 arboviruses and its notoriety as an important vector came to light during the recent chikungunya pandemic. The vulnerability of Singapore to emerging infectious arboviruses has stimulated our interest to determine the competence of local Ae. albopictus to transmit ZIKV.

Methodology/Principal Findings

To determine the competence of Ae. albopictus to ZIKV, we orally infected local mosquito strains to a Ugandan strain virus. Fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 80–85%RH. Twelve mosquitoes were then sampled daily from day one to seven and on day 10 and 14 post infection (pi). Zika virus titre in the midgut and salivary glands of each mosquito were determined using tissue culture infectious dose₅₀ assay, while transmissibility of the virus was determined by detecting viral antigen in the mosquito saliva by qRT-PCR. High dissemination and transmission rate of ZIKV were observed. By day 7-pi, all mosquitoes have disseminated infection and 73% of these mosquitoes have ZIKV in their saliva. By day 10-pi, all mosquitoes were potentially infectious.

Conclusions/Significance

The study highlighted the potential of Ae. albopictus to transmit ZIKV and the possibility that the virus could be established locally. Nonetheless, the threat of ZIKV can be mitigated by existing dengue and chikungunya control program being implemented in Singapore.     

Is Switzerland Suitable for the Invasion of Aedes albopictus?

Background

Over the last 30 years, the Asian tiger mosquito, Aedes albopictus, has rapidly spread around the world. The European distribution comprises the Mediterranean basin with a first appearance in Switzerland in 2003. Early identification of the most suitable areas in Switzerland allowing progressive invasion by this species is considered crucial to suggest adequate surveillance and control plans.

Methodology/Principal Findings

We identified the most suitable areas for invasion and establishment of Ae. albopictus in Switzerland. The potential distribution areas linked to the current climatic suitability were assessed using remotely sensed land surface temperature data recorded by the MODIS satellite sensors. Suitable areas for adult survival and overwintering of diapausing eggs were also identified for future climatic conditions, considering two different climate change scenarios (A1B, A2) for the periods 2020–2049 and 2045–2074. At present, the areas around Lake Geneva in western Switzerland provide suitable climatic conditions for Ae. albopictus. In northern Switzerland, parts of the Rhine valley, around Lake Constance, as well as the surroundings of Lake Neuchâtel, appear to be suitable for the survival at least of adult Ae. albopictus. However, these areas are characterized by winters currently being too cold for survival and development of diapausing eggs. In southern Switzerland, Ae. albopictus is already well-established, especially in the Canton of Ticino. For the years 2020–2049, the predicted possible spread of the tiger mosquito does not differ significantly from its potential current distribution. However, important expansions are obtained if the period is extended to the years 2045–2074, when Ae. albopictus may invade large new areas.

Conclusions/Significance

Several parts of Switzerland provide suitable climatic conditions for invasion and establishment of Ae. albopictus. The current distribution and rapid spread in other European countries suggest that the tiger mosquito will colonize new areas in Switzerland in the near future.     

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.     

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.     

piggybac- and PhiC31-Mediated Genetic Transformation of the Asian Tiger Mosquito,Aedes albopictus (Skuse)

Background

The Asian tiger mosquito, Aedes albopictus (Skuse), is a vector of several arboviruses including dengue and chikungunya. This highly invasive species originating from Southeast Asia has travelled the world in the last 30 years and is now established in Europe, North and South America, Africa, the Middle East and the Caribbean. In the absence of vaccine or antiviral drugs, efficient mosquito control strategies are crucial. Conventional control methods have so far failed to control Ae. albopictus adequately.

Methodology/Principal Findings

Germline transformation of Aedes albopictus was achieved by micro-injection of embryos with a piggyBac-based transgene carrying a 3xP3-ECFP marker and an attP site, combined with piggyBac transposase mRNA and piggyBac helper plasmid. Five independent transgenic lines were established, corresponding to an estimated transformation efficiency of 2–3%. Three lines were re-injected with a second-phase plasmid carrying an attB site and a 3xP3-DsRed2 marker, combined with PhiC31 integrase mRNA. Successful site-specific integration was observed in all three lines with an estimated transformation efficiency of 2–6%.

Conclusions/Significance

Both piggybac- and site-specific PhiC31-mediated germline transformation of Aedes albopictus were successfully achieved. This is the first report of Ae. albopictus germline transformation and engineering, a key step towards studying and controlling this species using novel molecular techniques and genetic control strategies.     

Comparative Host Feeding Patterns of the Asian Tiger Mosquito,Aedes albopictus,in Urban and Suburban Northeastern USA and Implications for Disease Transmission

Background

Aedes albopictus is an invasive species which continues expanding its geographic range and involvement in mosquito-borne diseases such as chikungunya and dengue. Host selection patterns by invasive mosquitoes are critically important because they increase endemic disease transmission and drive outbreaks of exotic pathogens. Traditionally, Ae. albopictus has been characterized as an opportunistic feeder, primarily feeding on mammalian hosts but occasionally acquiring blood from avian sources as well. However, limited information is available on their feeding patterns in temperate regions of their expanded range. Because of the increasing expansion and abundance of Ae. albopictus and the escalating diagnoses of exotic pathogens in travelers returning from endemic areas, we investigated the host feeding patterns of this species in newly invaded areas to further shed light on its role in disease ecology and assess the public health threat of an exotic arbovirus outbreak.

Methodology/Principal Findings

We identified the vertebrate source of 165 blood meals in Ae. albopictus collected between 2008 and 2011 from urban and suburban areas in northeastern USA. We used a network of Biogents Sentinel traps, which enhance Ae. albopictus capture counts, to conduct our collections of blooded mosquitoes. We also analyzed blooded Culex mosquitoes collected alongside Ae. albopictus in order to examine the composition of the community of blood sources. We found no evidence of bias since as expected Culex blood meals were predominantly from birds (n = 149, 93.7%) with only a small proportion feeding on mammals (n = 10, 6.3%). In contrast, Aedes albopictus fed exclusively on mammalian hosts with over 90% of their blood meals derived from humans (n = 96, 58.2%) and domesticated pets (n = 38, 23.0% cats; and n = 24, 14.6% dogs). Aedes albopictus fed from humans significantly more often in suburban than in urban areas (χ², p = 0.004) and cat-derived blood meals were greater in urban habitats (χ², p = 0.022). Avian-derived blood meals were not detected in any of the Ae. albopictus tested.

Conclusions/Significance

The high mammalian affinity of Ae. albopictus suggests that this species will be an efficient vector of mammal- and human-driven zoonoses such as La Crosse, dengue, and chikungunya viruses. The lack of blood meals obtained from birds by Ae. albopictus suggest that this species may have limited exposure to endemic avian zoonoses such as St. Louis encephalitis and West Nile virus, which already circulate in the USA. However, growing populations of Ae. albopictus in major metropolitan urban and suburban centers, make a large autochthonous outbreak of an arbovirus such as chikungunya or dengue viruses a clear and present danger. Given the difficulties of Ae. albopictus suppression, we recommend that public health practitioners and policy makers install proactive measures for the imminent mitigation of an exotic pathogen outbreak.     

Persistent Wolbachia and Cultivable Bacteria Infection in the Reproductive and Somatic Tissues of the Mosquito Vector Aedes albopictus

Background

Commensal and symbiotic microbes have a considerable impact on the behavior of many arthropod hosts, including hematophagous species that transmit pathogens causing infectious diseases to human and animals. Little is known about the bacteria associated with mosquitoes other than the vectorized pathogens. This study investigated Wolbachia and cultivable bacteria that persist through generations in Ae. albopictus organs known to host transmitted arboviruses, such as dengue and chikungunya.

Methodology/Principal Findings

We used culturing, diagnostic and quantitative PCR, as well as in situ hybridization, to detect and locate bacteria in whole individual mosquitoes and in dissected tissues. Wolbachia, cultivable bacteria of the genera Acinetobacter, Comamonas, Delftia and Pseudomonas co-occurred and persisted in the bodies of both males and females of Ae. albopictus initially collected in La Réunion during the chikungunya outbreak, and maintained as colonies in insectaries. In dissected tissues, Wolbachia and the cultivable Acinetobacter can be detected in the salivary glands. The other bacteria are commonly found in the gut. Quantitative PCR estimates suggest that Wolbachia densities are highest in ovaries, lower than those of Acinetobacter in the gut, and approximately equal to those of Acinetobacter in the salivary glands. Hybridization using specific fluorescent probes successfully localized Wolbachia in all germ cells, including the oocytes, and in the salivary glands, whereas the Acinetobacter hybridizing signal was mostly located in the foregut and in the anterior midgut.

Conclusions/Significance

Our results show that Proteobacteria are distributed in the somatic and reproductive tissues of mosquito where transmissible pathogens reside and replicate. This location may portend the coexistence of symbionts and pathogens, and thus the possibility that competition or cooperation phenomena may occur in the mosquito vector Ae. albopictus. Improved understanding of the vectorial system, including the role of bacteria in the vector''s biology and competence, could have major implications for understanding viral emergences and for disease control.     

Glacial History of a Modern Invader: Phylogeography and Species Distribution Modelling of the Asian Tiger Mosquito Aedes albopictus

Background

The tiger mosquito, Aedes albopictus, is one of the 100 most invasive species in the world and a vector of human diseases. In the last 30 years, it has spread from its native range in East Asia to Africa, Europe, and the Americas. Although this modern invasion has been the focus of many studies, the history of the species’ native populations remains poorly understood. Here, we aimed to assess the role of Pleistocene climatic changes in shaping the current distribution of the species in its native range.

Methodology/Principal Findings

We investigated the phylogeography, historical demography, and species distribution of Ae. albopictus native populations at the Last Glacial Maximum (LGM). Individuals from 16 localities from East Asia were analyzed for sequence variation at two mitochondrial genes. No phylogeographic structure was observed across the study area. Demographic analyses showed a signature of population expansion that started roughly 70,000 years BP. The occurrence of a continuous and climatically suitable area comprising Southeast China, Indochinese Peninsula, and Sundaland during LGM was indicated by species distribution modelling.

Conclusions/Significance

Our results suggest an evolutionary scenario in which, during the last glacial phase, Ae. albopictus did not experience a fragmentation phase but rather persisted in interconnected populations and experienced demographic growth. The wide ecological flexibility of the species probably played a crucial role in its response to glacial-induced environmental changes. Currently, there is little information on the impact of Pleistocene climatic changes on animal species in East Asia. Most of the studies focused on forest-associated species and suggested cycles of glacial fragmentation and post-glacial expansion. The case of Ae. albopictus, which exhibits a pattern not previously observed in the study area, adds an important piece to our understanding of the Pleistocene history of East Asian biota.