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

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

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.     

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.     

Aedes aegypti lines for combined sterile insect technique and incompatible insect technique applications: the importance of host genomic background

Aedes aegypti L. (Diptera: Culicidae), being the primary vector of pathogenic arboviruses, is a target for the development of novel genetic approaches to complement current conventional vector control strategies such as the combined sterile insect and incompatible insect technique (SIT/IIT). A transinfected line of Ae. aegypti carrying the wAlbB Wolbachia strain (WB2) was introgressed into two genomic backgrounds, Brazil and Mexico, producing two new Ae. aegypti strains (WB2-BRA and WB2-MEX). These strains were evaluated with respect to several life-history traits such as fecundity, fertility, longevity, pupa size, pupation curve, and male mating competitiveness, as well as their response to irradiation. Our results show that the impact of Wolbachia infection depends on the genomic background and that the Brazilian one had no significant effect, whereas the Mexican one negatively affected fertility, longevity, and pupal size. Interestingly, Wolbachia-infected Ae. aegypti lines required a lower irradiation dose to achieve complete female sterility than the uninfected ones. The present findings are discussed given the potential use of Wolbachia-infected Ae. aegypti lines in combined SIT/IIT population suppression programs.     

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.     

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 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.     

Costs and benefits of Wolbachia infection in immature Aedes albopictus depend upon sex and competition level

Bacterial endosymbionts induce various effects on hosts and can dramatically impact host fitness and development. An example is provided by obligate, maternally-inherited Wolbachia, which infect a broad range of invertebrates. Wolbachia are capable of altering host reproduction, thereby promoting infection spread. Wolbachia also pose direct physiological costs and benefits to hosts, complicating their categorization as parasites or mutualists. This study examines for an effect of Wolbachia infection in intra-specific larval competition by Aedes albopictus mosquitoes, with the goal of examining for an impact of Wolbachia infection in mixed populations. Similar to prior work examining for an influence of Wolbachia infection on the fitness of A. albopictus in adults, the results presented here support the hypothesized impact of Wolbachia across all life stages, including immatures. The differential competitiveness of infected larvae detected in our experiments indicates that Wolbachia infected A. albopictus females are less competitive relative to uninfected females when competing under highly competitive conditions. In contrast, under low competitive pressures, infected females experience higher survivorship. Thus, Wolbachia infection shifts from parasitism to mutualism as a function of developmental conditions. Results are discussed in relation to the invasion and persistence of Wolbachia in A. albopictus populations.The results are important to the evolution of stable Wolbachia symbioses, including Wolbachia invasion of an uninfected population. The resulting infection dynamics that occur in an infected population are discussed.     

The effects of simulated rainfall on immature population dynamics of Aedes albopictus and female oviposition

Larvae of Aedes albopictus Skuse typically inhabit natural and artificial containers. Since these larval habitats are replenished by rainfall, Ae. albopictus may experience increased loss of immature stages in areas with high levels of rainfall. In this study, we investigated the effects of rainfall and container water level on population density, and oviposition activity of Ae. albopictus. In field and laboratory experiments, we found that rainfall resulted in the flushing of breeding habitats. Excess rain negatively impacted larval and pupal retention, especially in small habitats. When filled with water to overflowing, container habitats were significantly repellent to ovipositing females. Taken together, these data suggest that rainfall triggers population loss of Ae. albopictus and related species through a direct detrimental effect (flushing out) and an indirect effect (ovipositional repellency).     

Artificial Triple Wolbachia Infection in Aedes albopictus Yields a New Pattern of Unidirectional Cytoplasmic Incompatibility

Obligately intracellular Wolbachia bacteria infect numerous invertebrates and often manipulate host reproduction to facilitate the spread of infection. An example of reproductive manipulation is Wolbachia-induced cytoplasmic incompatibility (CI), which occurs commonly in insects. This CI has been the focus both of basic scientific studies of naturally occurring invasion events and of applied investigations on the use of Wolbachia as a vehicle to drive desired genotypes into insect populations (“gene drive” or “population replacement” strategies). The latter application requires an ability to generate artificial infections that cause a pattern of unidirectional incompatibility with the targeted host population. A suggested target of population replacement strategies is the mosquito Aedes albopictus (Asian tiger mosquito), an important invasive pest and disease vector. Aedes albopictus individuals are naturally “superinfected” with two Wolbachia types: wAlbA and wAlbB. Thus, generating a strain that is unidirectionally incompatible with field populations requires the introduction of an additional infection into the preexisting superinfection. Although prior reports demonstrate an ability to transfer Wolbachia infections to A. albopictus artificially, including both intra- and interspecific Wolbachia transfers, previous efforts have not generated a strain capable of invading natural populations. Here we describe the generation of a stable triple infection by introducing Wolbachia wRi from Drosophila simulans into a naturally superinfected A. albopictus strain. The triple-infected strain displays a pattern of unidirectional incompatibility with the naturally infected strain. This unidirectional CI, combined with a high fidelity of maternal inheritance and low fecundity effects, suggests that the artificial cytotype could serve as an appropriate vehicle for gene drive.Wolbachia spp. are maternally inherited, obligately intracellular bacteria that commonly infect invertebrates, including ∼20% of insect species (2). A hypothesized explanation for the evolutionary success of Wolbachia is its ability to affect host reproduction; cytoplasmic incompatibility (CI) is one of the most widely reported effects (25). Unidirectional CI can occur when the Wolbachia infection type present in a male differs from that in his mate. Although the precise mechanism is unknown, a lock/key model has been proposed in which the Wolbachia infection modifies the sperm during spermatogenesis (27). If the male inseminates a female lacking a compatible Wolbachia type, the modified sperm fail to achieve karyogamy. In contrast, “rescue” of the modified sperm occurs in embryos from females infected with compatible Wolbachia types. Thus, in populations that include both infected and uninfected individuals, Wolbachia-infected females can mate successfully with all males in the population. In contrast, uninfected females can reproduce successfully only with uninfected males. This pattern of unidirectional CI allows Wolbachia to spread rapidly through host populations.Previous studies of insects with multiple Wolbachia types have demonstrated that unidirectional CI can be additive (4, 5). Multiple Wolbachia infection types within an individual male may independently modify sperm, requiring a similar combination of infection types in female mates for compatibility. Additive unidirectional CI can result in repeated population replacement events, in which single- or double-infection cytotypes are replaced by a Wolbachia “superinfection” (i.e., individuals harboring two or more infections).The concept of population replacement has attracted attention for its potential applications. A frequently referenced strategy is based on the replacement of natural populations with modified populations that are refractory to disease transmission (1, 4, 8, 12, 22). A Wolbachia-based population replacement strategy requires the generation of artificial infection types that differ from those of the targeted populations.Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tiger mosquito, is native to Asia and is a globally invasive insect. Examples of introduction and establishment include North and South America (11), and recent invasions have extended to Africa, Australia, and Europe (9). In addition to being an invasive pest, this mosquito is an aggressive daytime human biter and has been implicated as a vector of animal (20) and human (11) disease. Recent reports have highlighted its role as a primary vector during recent chikungunya virus epidemics (17, 21).Aedes albopictus populations are naturally infected with two Wolbachia types: wAlbA and wAlbB (13, 24). Therefore, to employ Wolbachia as a vehicle for population replacement, an additional, incompatible infection must be introduced into the natural infection types. Previously, Wolbachia strain wRi was successfully established in A. albopictus by microinjecting the cytoplasm of Drosophila simulans (Riverside) into the embryos of aposymbiotic (i.e., without Wolbachia) A. albopictus mosquitoes (28). As hypothesized, the resulting artificial infection displayed a pattern of bidirectional CI when these mosquitoes were crossed with the naturally double infected strain. Thus, the modification/rescue mechanism(s) of the wRi infection is known to differ from those of the naturally occurring infection types. Therefore, we hypothesized that individuals harboring the combined wRi, wAlbA, and wAlbB infections would be unidirectionally incompatible with the naturally infected mosquitoes.To develop a strain appropriate for an applied population replacement strategy, we have performed experiments to generate an artificial triple infection. Following embryonic microinjection, experiments were designed to examine individuals across generations for the hypothesized unidirectional CI pattern, to determine the stability and segregation of the different infection types, and to characterize the relative fitness of triple-infected individuals.     

Wolbachia modulates Chikungunya replication in Aedes albopictus

The Aedes albopictus mosquito has been involved as the principal vector of recent major outbreaks due to the chikungunya virus (CHIKV). The species is naturally infected by two strains of Wolbachia (wAlbA and wAlbB). Wolbachia infections are thought to have spread by manipulating the reproduction of their hosts; cytoplasmic incompatibility is the mechanism used by Wolbachia to invade natural populations of many insects including Ae. albopictus. Here, we report a study on the effects of removing Wolbachia from Ae. albopictus on CHIKV replication and examine the consequences of CHIKV infection on some life‐history traits (survival and reproduction) of Wolbachia‐free Ae. albopictus. We found that Wolbachia‐free mosquitoes maintained a highly heterogeneous CHIKV replication compared to Wolbachia‐infected individuals. In Wolbachia‐infected Ae. albopictus, the regular increase of CHIKV followed by a steady viral load from day 4 post‐infection onwards was concomitant with a decline in Wolbachia density. This profile was also detected when examining the two key organs for viral transmission, the midgut and the salivary glands. Moreover, Wolbachia‐free Ae. albopictus was not altered in life‐history traits such as survival, oviposition and hatching characteristics whether infected or not with CHIKV. We found that Wolbachia is not essential for viral replication, its presence could lead to optimize replication from day 4 post‐infection onwards, coinciding with a decrease in Wolbachia density. Wolbachia may regulate viral replication in Ae. albopictus, with consequences on survival and reproduction.     

Evaluation of partial sterility in mating performance and reproduction of the West Indian sweetpotato weevil,Euscepes postfasciatus

The sterile insect technique (SIT) is based on population and behavioral ecology and is widely used to suppress or eradicate target pest insect populations. The effectiveness of SIT depends on the ability of the released sterile males to mate with and inseminate wild females. The use of gamma‐radiation to induce sterility is, however, associated with negative impacts not only on reproductive cells but also on somatic cells. Consequently, irradiation for sterilization diminishes mating performance over time. In this study, we evaluated the balance between the irradiation dose and both fertility and mating propensity in Euscepes postfasciatus (Fairmaire) (Coleoptera: Curculionidae) for 22 days following irradiation. The mating propensity of males irradiated with a 150‐Gy dose, as currently used to induce complete sterility of E. postfasciatus in the SIT program in Okinawa Prefecture, was equal to that of non‐irradiated weevils for up to 6 days, and the mating propensity of males irradiated with a dose of 125 Gy was equal to that of non‐irradiated weevils for twice this period (12 days). The fertilization ability of weevils irradiated with a dose of 125 Gy was reduced by 4.6% in males and 0.6% in females, compared to the potential fertilization ability. We also discuss the possibility of the application of partially sterilized insects in eradication programs.     

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.     

Radiation biology of mosquitoes

There is currently renewed interest in assessing the feasibility of the sterile insect technique (SIT) to control African malaria vectors in designated areas. The SIT relies on the sterilization of males before mass release, with sterilization currently being achieved through the use of ionizing radiation. This paper reviews previous work on radiation sterilization of Anopheles mosquitoes. In general, the pupal stage was irradiated due to ease of handling compared to the adult stage. The dose-response curve between the induced sterility and log (dose) was shown to be sigmoid, and there was a marked species difference in radiation sensitivity. Mating competitiveness studies have generally been performed under laboratory conditions. The competitiveness of males irradiated at high doses was relatively poor, but with increasing ratios of sterile males, egg hatch could be lowered effectively. Males irradiated as pupae had a lower competitiveness compared to males irradiated as adults, but the use of partially-sterilizing doses has not been studied extensively. Methods to reduce somatic damage during the irradiation process as well as the use of other agents or techniques to induce sterility are discussed. It is concluded that the optimal radiation dose chosen for insects that are to be released during an SIT programme should ensure a balance between induced sterility of males and their field competitiveness, with competitiveness being determined under (semi-) field conditions. Self-contained ⁶⁰Co research irradiators remain the most practical irradiators but these are likely to be replaced in the future by a new generation of high output X ray irradiators.     

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.     

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.     

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

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

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.     

Indoor-Breeding of Aedes albopictus in Northern Peninsular Malaysia and Its Potential Epidemiological Implications

Background

The mosquito Ae. albopictus is usually adapted to the peri-domestic environment and typically breeds outdoors. However, we observed its larvae in most containers within homes in northern peninsular Malaysia. To anticipate the epidemiological implications of this indoor-breeding, we assessed some fitness traits affecting vectorial capacity during colonization process. Specifically, we examined whether Ae. albopictus exhibits increased survival, gonotrophic activity and fecundity due to the potential increase in blood feeding opportunities.

Methodology/Principal Findings

In a series of experiments involving outdoors and indoors breeding populations, we found that Ae. albopictus lives longer in the indoor environment. We also observed increased nighttime biting activity and lifetime fecundity in indoor/domestic adapted females, although they were similar to recently colonized females in body size.

Conclusion/Significance

Taken together these data suggest that accommodation of Ae. albopictus to indoor/domestic environment may increase its lifespan, blood feeding success, nuisance and thus vectorial capacity (both in terms of increased vector-host contacts and vector population density). These changes in the breeding behavior of Ae. albopictus, a potential vector of several human pathogens including dengue viruses, require special attention.     

The Effect of Pyriproxyfen as a “Population Growth Regulator” against Aedes albopictus under Semi-Field Conditions

An insect growth regulator, pyriproxyfen, has been used for the control of a range of pest insects, including mosquitoes. Pyriproxyfen is effective in inhibiting adult emergence and sterilizing adult females. The Asian tiger mosquito, Aedes albopictus (Skuse), is an important vector of dengue and chikungunya, and is expanding its distribution throughout Europe and the Americas. In the present study, we evaluated the impact of pyriproxyfen-treated bed nets on population growth of Ae. albopictus under semi-field conditions, using 6 small microcosms. We created microcosms containing breeding sites to simulate the natural ecosystem of vector mosquito and installing miniature bed net treated with 350 mg/m² pyriproxyfen in Experiment I and 35 mg/m² in Experiment II. For each experiment, we also established microcosms installing untreated polyethylene net (untreated control). The installing nets were provided with artificially torn holes, to simulate damage and allow mosquitoes to penetrate. We released 100 pairs of Ae. albopictus into each microcosm, and allowed them to feed on a mouse under the bed nets at approximately 1-week intervals. In comparison with the untreated control microcosms, the number of eggs laid by the released adults in the pyriproxyfen-treated microcosms was significantly lower in both Experiment I and II. Moreover, egg hatchability was significantly suppressed and pupal mortality was increased. Our results indicate that tarsal contact with pyriproxyfen has been shown to suppress egg production and hatchability in adult females and the auto-dissemination of pyriproxyfen into larval breeding sites by adult mosquitoes, through contact with pyriproxyfen-treated polyethylene bed nets, may suppress the mosquito population density.