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.     

Characterization of the ribosomal proteins from mosquito (Aedes albopictus) cells

Proteins from the large and small subunits of Aedes albopictus (mosquito) cytoplasmic ribosomes were characterized by two-dimensional polyacrylamide gel electrophoresis. The small subunit contained 28-31 proteins ranging in molecular mass from 10 to 49 kDa. The large subunit contained 36-39 proteins that ranged in molecular mass from 11 to 53 kDa. The largest protein on the small subunit, S1, was the predominant phosphorylated ribosomal protein. Under long-term labelling conditions, L4 and L33 were also phosphorylated. Peptide mapping by partial proteolysis indicated that Ae. albopictus S1 may share partial amino acid homology with the phosphorylated ribosomal protein S6 from Drosophila melanogaster. Unlike Drosophila S6, however, Aedes S1 was not dephosphorylated during heat shock. Treatment of mosquito cells with the insect molting hormone 20-hydroxyecdysone did not affect phosphorylation of ribosomal proteins.     

Heterochromatin in cells derived from Aedes albopictus (Skuse) mosquito tissue culture

Results obtained with the DNA d-r method in cultured cells from A. albopictus show that this technique brings out the location of the kinetochore as well as the nucleolar associated heterochromatin. It is also speculated that the kinetochore heterochromatin may be involved in somatic pairing, in the attachment of chromosomes to the nuclear membrane and in chromosome breakage.     

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.     

Isolation of microsatellite markers in the tiger mosquito Aedes albopictus (Skuse)

Among the numerous molecular markers available in population genetics, microsatellites are one of the most powerful tools developed in recent years. This paper describes the isolation of six polymorphic microsatellite loci in the tiger mosquito Aedes albopictus using an enriched genomic library technique. Such loci should be an efficient tool in population genetic studies for this mosquito species.     

Generation of a novel Wolbachia infection in Aedes albopictus (Asian tiger mosquito) via embryonic microinjection

Genetic strategies that reduce or block pathogen transmission by mosquitoes are being investigated as a means to augment current control measures. Strategies of vector suppression and replacement are based upon intracellular Wolbachia bacteria, which occur naturally in many insect populations. Maternally inherited Wolbachia have evolved diverse mechanisms to manipulate host insect reproduction and promote infection invasion. One mechanism is cytoplasmic incompatibility (CI) through which Wolbachia promotes infection spread by effectively sterilizing uninfected females. In a prior field test, releases of Wolbachia-infected males were used to suppress a field population of Culex pipiens. An additional strategy would employ Wolbachia as a vehicle to drive desired transgenes into vector populations (population replacement). Wolbachia-based population suppression and population replacement strategies require an ability to generate artificial Wolbachia associations in mosquitoes. Here, we demonstrate a technique for transferring Wolbachia (transfection) in a medically important mosquito species: Aedes albopictus (Asian tiger mosquito). Microinjection was used to transfer embryo cytoplasm from a double-infected Ae. albopictus line into an aposymbiotic line. The resulting mosquito line is single-infected with the wAlbB Wolbachia type. The artificially generated infection type is not known to occur naturally and displays a new CI crossing type and the first known example of bidirectional CI in Aedes mosquitoes. We discuss the results in relation to applied mosquito control strategies and the evolution of Wolbachia infections in Ae. albopictus.     

Structural characterization of the micropatterned egg plastron in the mosquito,Aedes albopictus

Eggs of the Asian tiger mosquito Aedes albopictus are fully covered with an air‐covering plastron network that enables them to stay below the surface of the water. Scanning electron microscopy revealed that the chorionic surface was covered with clusters of globular tubercles of different sizes. Histologically, the eggshell provides a typical water‐repellent microarchitecture consisting of an outer exochorion, a membranous endochorion and an intermediate pillar layer. The eggshell has a distinct chorionic tubercle that increases the surface area for gas exchange to enhance respiration capacity. In particular, the chorionic wall gives rise to a hexagonal pattern with smooth and elevated boundaries. In A. albopictus, a set of micropatterns for each hexagon was specified, and each central tubercle was surrounded by 20 small peripheral tubercles. Our fine structural analysis revealed that the partitioning of the surface into numerous hexagonal chorionic sculptures is associated with the Voronoi partition (tessellation), based on the distance to a point in a specific area of the plane. Therefore, the micropatterned surface of the mosquito eggshell appears to not only resist wetting by hydrostatic pressure but also provide resistance to lysate deposition by the biofouling process.     

Genetic evidence for the origin of Aedes aegypti,the yellow fever mosquito,in the southwestern Indian Ocean

Aedes aegypti is among the best‐studied mosquitoes due to its critical role as a vector of human pathogens and ease of laboratory rearing. Until now, this species was thought to have originated in continental Africa, and subsequently colonized much of the world following the establishment of global trade routes. However, populations of this mosquito on the islands in the southwestern Indian Ocean (SWIO), where the species occurs with its nearest relatives referred to as the Aegypti Group, have received little study. We re‐evaluated the evolutionary history of Ae. aegypti and these relatives, using three data sets: nucleotide sequence data, 18,489 SNPs and 12 microsatellites. We found that: (a) the Aegypti Group diverged 16 MYA (95% HPD: 7–28 MYA) from its nearest African/Asian ancestor; (b) SWIO populations of Ae. aegypti are basal to continental African populations; (c) after diverging 7 MYA (95% HPD: 4–15 MYA) from its nearest formally described relative (Ae. mascarensis), Ae. aegypti moved to continental Africa less than 85,000 years ago, where it recently (<1,000 years ago) split into two recognized subspecies Ae. aegypti formosus and a human commensal, Ae. aegypti aegypti; (d) the Madagascar samples form a clade more distant from all other Ae. aegypti than the named species Ae. mascarensis, implying that Madagascar may harbour a new cryptic species; and (e) there is evidence of introgression between Ae. mascarensis and Ae. aegypti on Réunion, and between the two subspecies elsewhere in the SWIO, a likely consequence of recent introductions of domestic Ae. aegypti aegypti from Asia.     

Morphogenesis of Sindbis virus in three subclones of Aedes albopictus (mosquito) cells.

The morphogenesis of Sindbis virus in three Aedes albopictus subcloned cell lines was examined. Each line was distinguishable with respect to morphology, cytopathic response to infection, and progeny yield. C7-10 cells, which produced the highest titers of virus and exhibited the most severe cytopathic response, were characterized ultrastructurally by the presence of budding particles at the cell surface and at the membranes of internal vesicles. C6/36 cells, which displayed a moderate cytotoxic response, manifested similar features in response to Sindbis virus infection. Both cell types also produced a structure composed of an electron-dense matrix in which nucleocapsids were embedded. Internally matured virions were released by exocytosis from these cells. In addition to a lack of cytopathic effect, u4.4 cells also failed to exhibit obvious morphogenetic changes upon infection. Virus particles were occasionally seen within vesicles, but budding at the cell surface was not detected. The mechanism of release of internally matured virions was not apparent. These studies provide further evidence that these three subcloned mosquito cell lines represent different tissues in the larval or adult insect.     

Genetic characters of the globally spread tiger mosquito,Aedes albopictus (Diptera,Culicidae): implications from mitochondrial gene COI

There has been a rapid global expansion of Aedes albopictus , with varying biological characters and vector competence according to geographic and evolutionary origins of the invading populations. Based on mitochondrial gene cytochrome c oxidase I (COI) sequences from both native and invasive regions, genetic characters of Ae. albopictus were examined. Phylogenetic analyses indicate lineage differentiation in the original areas, and only one of the lineages was involved in the worldwide expansion. Multiple invasions were detected in populations in Africa and the Americas, whereas no obvious genetic structure was found in European populations. Asian populations showed high genetic diversity, with 42 private haplotypes being detected in this region. In addition, other genetic divergence has occurred, as in Japan and Pakistan, where populations showed significant differences from all other Asian populations. Altogether, populations in China displayed the highest genetic diversity (Hd =0.946, π=1.609%) and significantly negative Tajima's D (?1.88475) and Fu's FS (?24.43873). This result may be attributable to the insecticide interventions used to control dengue epidemics.     

First record and establishment of the mosquito Aedes albopictus in Spain

The invasive mosquito Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) was detected for the first time in Spain, in Sant Cugat del Vallès, a city in the north-east of the country (41 degrees 28' N, 2 degrees 4' E, altitude 120 m), during August 2004. A male and one larva were collected in the backyard of a house and in a tree hole, respectively. Dense populations of adults and larvae were found in subsequent surveys, confirming the establishment of the species in the area. This is the first report of the establishment of this species in the Iberian Peninsula.     

Bird pollination in an angraecoid orchid on Reunion Island (Mascarene Archipelago, Indian Ocean)

BACKGROUND AND AIMS: Although numerous angraecoid orchids in Madagascar display typical sphingophilous syndrome (i.e. white, nectariferous, long-spurred flowers, producing a strong scent at the crepuscule that is attractive to moths), three species of Angraecum in Reunion, belonging to the endemic section Hadrangis, have atypical unscented and short-spurred flowers. The aim of the study was to investigate the implication of plant-pollinator interaction on the evolution of floral morphology of these peculiar island floral forms. METHODS: The flower morphology of A. striatum (one of the three section Hadrangis species) was investigated by performing a set of floral measures, and the reproductive biology was investigated by a set of hand pollination experiments. Natural pollinators were observed by means of a digital video camera. Pollinator efficiency (pollen removal and deposition) and reproductive success (fruit set) were quantified once a week in natural field conditions during the 2005 flowering season (i.e. from January to March). KEY RESULTS: The orchid is self-compatible but requires a pollinator to achieve fruit set. Only one pollinator was observed, the endemic white-eye Zosterops borbonicus (Zosteropidae). These birds perched on inflorescences, and probed most fresh-looking flowers on each plant for nectar. Nectar was both abundant (averaging 7.7 microL) and dilute (averaging 9.7 % sugar in sucrose equivalents). Birds were mostly active between 0830 and 0930 h. Visits to plants were extremely short, lasting from 9 to 27 s. At the study site, 60.9 % of flowers had pollen removed, and 46.4 % had pollinia deposited on stigmas. The proportion of flowers that initiated a fruit averaged 20.6 % in natural conditions. CONCLUSIONS: For the first time, a bird-pollinated orchid is described from a sub-tribe that is mainly specialized for moth pollination. This study documents a morphological shift in flowers in response to pollinator adaptations in the insular context of the Mascarene Archipelago.     

Effects on male fitness of removing Wolbachia infections from the mosquito Aedes albopictus

Abstract.  Cytoplasmic incompatibility (CI) induced by maternally inherited Wolbachia bacteria is a potential tool for the suppression of insect pest species with appropriate patterns of infection. The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) is known to be infected by two strains of Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae), w Alb A and w Alb B, throughout its geographical distribution. This infection pattern theoretically restricts the application of CI-based control strategies. However, Wolbachia can be horizontally transferred using embryonic microinjection to generate incompatible transfected lines harbouring a single new strain of Wolbachia. In order to assess the feasibility of this approach, the effects of Wolbachia removal on mosquito fitness need to be clearly evaluated as the removal of natural superinfection is an inescapable step of this approach. Previous research has shown that uninfected females, produced by antibiotic treatment, showed a decrease in fitness compared with those infected with Wolbachia. In this study, the effect of Wolbachia removal on male fitness was investigated. Longevity and reproductive potential (mating competitiveness and sperm capacity) were assessed in both laboratory cages and greenhouses. No differences were observed between uninfected and infected males with respect to longevity, mating rate, sperm capacity and mating competitiveness in either laboratory conditions or greenhouses. The preservation of fitness in males of Ae. albopictus deprived of natural Wolbachia infection is discussed in relation to the development of incompatible insect technique suppression strategies. Finally, the potential application of aposymbiotic males in mark–release–recapture studies is suggested.     

Intraspecific variation in nuclear DNA content among world populations of a mosquito,Aedes albopictus (Skuse)

Summary Aedes albopictus is commonly distributed in most parts of the Oriental region and on many islands in the Indian and the Pacific Oceans. The species was recently introduced into the United States and Brazil. Feulgen cytophotometric quantitation of haploid nuclear DNA content was carried out for 37 populations of Ae. albopictus to determine the extent of intraspecific variation in nuclear DNA content and whether the range expansion of the species has coincided with an increase in DNA content. The haploid nuclear DNA content varied nearly three-fold. The minimum DNA content was 0.62 pg in Koh Samui from Thailand, and the maximum DNA content was 1.66 pg in Houston-61 from the United States. Statistical comparisons of populations revealed significant differences in DNA contents. No geographic clustering of populations was noted with respect to DNA content. In general, populations from the United States and Brazil had higher DNA contents, but there was no indication that the range expansion had occurred hand in hand with an increase in DNA content. Each population had a specific amount of DNA that is probably imposed by the microenvironment.     

A linkage map of the Asian tiger mosquito (Aedes albopictus) based on cDNA markers

The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse), is an important vector of a number of arboviruses, and populations exhibit extreme variation in adaptive traits such as egg diapause, cold hardiness, and autogeny (ability to mature a batch of eggs without blood feeding). The genetic basis of some of these traits has been established, but lack of a high-resolution linkage map has prevented in-depth genetic analyses of the genes underlying these complex traits. We report here on the breeding of 4 F(1) intercross mapping families and the use of these to locate 35 cDNA markers to the A. albopictus linkage map. The present study increases the number of markers on the A. albopictus cDNA linkage map from 38 to 73 and the density of markers from 1 marker/5.7 cM to 1 marker/2.9 cM and adds 9, 16, and 10 markers to the 3 linkage groups, respectively. The overall lengths of the 3 linkage groups are 64.5, 76.5, and 71.6 cM, respectively, for a combined length of 212.6 cM. Despite conservation in the order of most genes among the 4 families and a previous mapping family, we found substantial heterogeneity in the amount of recombination among markers. This was most marked in linkage group I, which varied between 16.7 and 69.3 cM. A map integrating the results from these 4 families with an earlier cDNA linkage map is presented.     

Role of extracellular virus on the maintenance of the persistent infection induced in Aedes albopictus (mosquito) cells by Sindbis virus.

Sindbis virus infection of cultured mosquito cells was found to have no effect on the growth of these cells; instead, a persistent infection of the culture followed an initial acute phase of rapid virus synthesis. Nearly all of the cells in the acute stage of infection were found to actively release virus in an infectious-center assay and to contain significant amounts of virus antigen as determined by immunofluorescence. Cells in the persistent phase of infection released few virions into the media, and only a small percentage of the cultured cells could be demonstrated to contain detectable amounts of virus antigen by immunofluorescence assay. In spite of the fact that nearly 100% of the cells in the persistent phase of infection were found to be virus negative by the two assays described above, the culture as a whole totally excluded the expression of superinfecting virus, as did cells in the acute phase, suggesting that most of the persistently infected cells did, indeed, contain virus information. Prevention of reinfection of the cells in the persistent phase by eliminating extracellular virus resulted in a curing of the culture such that it responded to infection by added virus much as would an uninfected culture.     

Exclusion of superinfecting homologous virus by Sindbis virus-infected Aedes albopictus (mosquito) cells.

The infection of tissue-cultured Aedes albopictus (mosquito) cells by an alphavirus ultimately results in a persistently infected cell population which can be maintained in the laboratory for years. One characteristic of this culture is that it will not support the replication of superinfecting homologous virus. We have previously shown that mosquito cells persistently infected with Sindbis virus produce an antiviral agent which when applied to uninfected mosquito cells suppresses Sindbis virus replication. The exclusion of virus replication in the antiviral-agent-treated cells is similar to the phenomenon of homologous interference described in alphavirus-infected vertebrate cells. In this study we examined the expression of homologous interference in three lines of mosquito cells and compared the expression of homologous interference to the effects of the antiviral activity. The cell lines were found to differ in their ability to express homologous interference, and evidence suggests that the mosquito cells may suppress replication by homologous interference or by the action of the antiviral agent.     

Quantitative trait loci determining autogeny and body size in the Asian tiger mosquito (Aedes albopictus)

The majority of mosquito species require a blood meal to stimulate vitellogenesis and subsequent oviposition (anautogeny), but some autogenous individuals complete their first ovarian cycle without a blood meal. Autogeny may be facultative or obligatory. In this study, we selected for an autogenous strain in the Asian tiger mosquito Aedes albopictus and examined an F(1) intercross population for quantitative trait loci (QTL) determining the autogeny trait as well as wing length as a proxy for body size. Using composite interval mapping, we identified four QTL for each trait and observed considerable overlap in genome positions between each QTL for autogeny (follicle size) and wing length. Most QTL were minor in magnitude, individually explaining <10% of the phenotypic variation. Alleles from the autogenous parent generally showed a dominance or overdominance effect on both phenotypes. Strong genetic and phenotypic correlations indicate that autogeny and wing length are determined by up to four clusters of tightly linked genes or the potential pleiotropic effects of single genes. Although females from the autogenous strain produced approximately fivefold more eggs following a blood meal than through autogeny, we suggest that the maintenance of alleles for autogeny in natural populations is likely due to balancing selection. Autogeny should be favored under conditions of limited host availability for blood feeding or increased defensive behavior by the host and adequate larval nutrition. Correlation between autogeny and body size may reflect an increased ability for larger females to accumulate sufficient nutrient reserves to support oogenesis without the requirement for a blood meal.