Development and evaluation of a single-step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species

Objective To develop a single‐step multiplex PCR to differentiate the aquatic stages of Aedes aegypti, Aedes albopictus and Aedes vittatus collected from different breeding spots in arbovirus endemic/epidemic areas and to detect the most abundant species by the multiplex PCR. Methods Aquatic stages of different mosquito species were sampled by inspecting artificial and natural breeding sites in domestic and peridomestic areas. DNA was isolated from different stages of the three Aedes species. Using novel primers based on 18S rDNA sequence, a single‐step multiplex PCR was developed to clearly distinguish the three Aedes species. It was then evaluated in the aquatic stages of Aedes species collected from different areas. Results A total of 1150 aquatic stages were collected from 294 breeding spots, of which 156 contained Aedes species. Discarded tires were the major breeding spots of Aedes species. The aquatic stages were clustered into 230 pools; Ae. albopictus was detected in the largest number of pools, followed by Ae. aegypti and Ae. vittatus. Conclusions The Multiplex PCR clearly differentiated the aquatic stages of the three Aedes species and detected that Ae. albopictus was most profuse in different breeding spots surveyed, hence indicating to be the main vector in this region. So control measures can be designed against Ae. albopictus at an early stage to prevent any arboviral outbreak. This method is a convenient tool for precise identification of Aedes vectors during entomological surveys in arbovirus endemic/epidemic areas where several species coexist.     

First dengue virus detection in Aedes albopictus from Delhi, India: its breeding ecology and role in dengue transmission

Objective To report dengue virus and its disease transmission in Aedes albopictus in the National Capital Territory of Delhi, India. Methods Monthly Aedes surveys were carried out in 126 urban localities of Delhi in 2008 and 2009. Pools of all three species of Aedes mosquitoes were tested for Dengue virus (DENV) using an antigen‐capture enzyme‐linked immunosorbent assay. Results Aedes aegypti was the most prevalent species, breeding throughout the year. Aedes albopictus was found in 9.52% of surveyed localities including the central urban part of Delhi, in March and from August to October. Aedes albopictus and Aedes vittatus are adapting to breed in manmade containers in the urban areas of Delhi in addition to their natural habitats of bamboo bushes and rock pits. Of the 229 pools of Ae. aegypti and 34 pools of Ae. albopictus tested, 10.5% and 11.76% were positive for dengue virus, respectively. No dengue virus infection was recorded in Ae. vittatus. Conclusion This is the first report of dengue virus in Ae. albopictus from north India. Because DENV was detected in Ae. albopictus, which adapted to manmade containers, both its spread and transmission dynamics should be checked.     

Densovirus Oil Suspension Significantly Improves the Efficacy and Duration of Larvicidal Activity against Aedes albopictus

Aedes albopictus is the sole vector for various mosquito-borne viruses, including dengue, chikungunya, and Zika. Ecofriendly biological agents are required to reduce the spread of these mosquito-borne infections. Mosquito densoviruses (MDVs) are entomopathogenic mosquito-specific viruses, which can reduce the capacity of isolated vectors and decrease mosquito-borne viral disease transmission. However, their variable pathogenicity restricts their commercial use. In the present study, we developed a series of novel larvicide oil suspensions (denoted Bacillus thuringiensis (Bti) oil, Ae. albopictus densovirus (AalDV-5) oil, and a mixture of AalDV-5+Bti oil), which were tested against Ae. albopictus larvae under experimental semi-field and open-field conditions. The effect of AalDV-5 on non-target species was also evaluated. The combined effect of AalDV-5+Bti was greater than that of individual toxins and was longer lasting and more persistent compared with the laboratory AalDV-5 virus strain. The virus was quantified on a weekly basis by quantitative polymerase chain reaction (qPCR) and was persistently detected in rearing water as well as in dead larvae. Wildtype densovirus is not pathogenic to non-target organisms. The present findings confirm the improved effect of a mixed microbial suspension (AalDV-5+Bti oil) larvicide against Ae. albopictus. The development and testing of these products will enable better control of the vector mosquitoes.     

Wolbachia Do Not Induce Reactive Oxygen Species-Dependent Immune Pathway Activation in Aedes albopictus

Aedes albopictus is a major vector of dengue (DENV) and chikungunya (CHIKV) viruses, causing millions of infections annually. It naturally carries, at high frequency, the intracellular inherited bacterial endosymbiont Wolbachia strains wAlbA and wAlbB; transinfection with the higher-density Wolbachia strain wMel from Drosophila melanogaster led to transmission blocking of both arboviruses. The hypothesis that reactive oxygen species (ROS)-induced immune activation plays a role in arbovirus inhibition in this species was examined. In contrast to previous observations in Ae. aegypti, elevation of ROS levels was not observed in either cell lines or mosquito lines carrying the wild-type Wolbachia or higher-density Drosophila Wolbachia strains. There was also no upregulation of genes controlling innate immune pathways or with antioxidant/ROS-producing functions. These data suggest that ROS-mediated immune activation is not an important component of the viral transmission-blocking phenotype in this species.     

Insecticide-treated bednets to control dengue vectors: preliminary evidence from a controlled trial in Haiti

Objectives Insecticide‐treated bednets (ITNs) are effective in preventing nocturnally transmitted vector‐borne diseases, but their effect on diurnally active dengue vectors has never been studied. We investigated the efficacy of ITNs in reducing Aedes aegypti populations and dengue transmission. Methods A cluster‐randomized trial was carried out in Leogane, Haiti between July 2003 and July 2004. The study area (1017 houses) was divided into 18 sectors (clusters): nine received ITNs (Olyset® long‐lasting insecticidal bednets) and nine were untreated controls. Entomological surveys [measuring Breteau (BI), house (HI), container (CI) and pupae per person (PPI) indices and oviposition activity] were undertaken at baseline and at 1 and 5 months post‐intervention. All houses were georeferenced to enable spatial analysis. Control sectors received ITNs at 6 months, and a final entomological and attitudinal survey was undertaken at 12 months after baseline. Anti‐dengue IgM seropositivity rates were measured at baseline and after 12 months. Efficacy of ITNs was assessed by WHO cone bioassays. Results At 1‐month post‐intervention, entomological indices fell in all sectors, with HI and BI in the bednet sectors reduced by 6.7 (95% CI ?10.6, ?2.7; P < 0.01) and 8.4 (95% CI ?14.1, ?2.6; P < 0.01) respectively. Moreover at 1 month, ovitraps in control sectors were significantly more likely to be positive than in bednet sectors (P < 0.01). By 5 months, all indices remained low and HI, CI and BI were also significantly lower than that of baseline in the control arm. Curiously, at 5 months, HI, CI and BI were lower in the control arm than that in the bednet arm. A final survey, 12 months after the initial baseline study (5 months after bednets had been given to all households) indicated that all indices were significantly lower than that at baseline (P < 0.001). Control houses located within 50 m of a bednet house had significantly lower CI (Z = ?2.67, P = 0.008) and PPI (Z = ?2.19, P = 0.028) at 1 month, an effect that extended to 100 m by 5 months (Z = ?2.03, P = 0.042 and Z = ?2.37, P = 0.018 respectively), suggesting a spill‐over effect of the bednets. An IgM serosurvey showed a 15.3% decrease (95% CI 5.0–25.5%, P < 0.01) in the number of IgM‐positive individuals from baseline to12 months later. Conclusions Insecticide‐treated bednets had an immediate effect on dengue vector populations after their introduction, and over the next 5–12 months, the presence of ITNs may have continued to affect vector populations and dengue transmission.     

A Multidisciplinary Investigation of the First Chikungunya Virus Outbreak in Matadi in the Democratic Republic of the Congo

Early March 2019, health authorities of Matadi in the Democratic Republic of the Congo alerted a sudden increase in acute fever/arthralgia cases, prompting an outbreak investigation. We collected surveillance data, clinical data, and laboratory specimens from clinical suspects (for CHIKV-PCR/ELISA, malaria RDT), semi-structured interviews with patients/caregivers about perceptions and health seeking behavior, and mosquito sampling (adult/larvae) for CHIKV-PCR and estimation of infestation levels. The investigations confirmed a large CHIKV outbreak that lasted February–June 2019. The total caseload remained unknown due to a lack of systematic surveillance, but one of the two health zones of Matadi notified 2686 suspects. Of the clinical suspects we investigated (n = 220), 83.2% were CHIKV-PCR or IgM positive (acute infection). One patient had an isolated IgG-positive result (while PCR/IgM negative), suggestive of past infection. In total, 15% had acute CHIKV and malaria. Most adult mosquitoes and larvae (>95%) were Aedes albopictus. High infestation levels were noted. CHIKV was detected in 6/11 adult mosquito pools, and in 2/15 of the larvae pools. This latter and the fact that 2/6 of the CHIKV-positive adult pools contained only males suggests transovarial transmission. Interviews revealed that healthcare seeking shifted quickly toward the informal sector and self-medication. Caregivers reported difficulties to differentiate CHIKV, malaria, and other infectious diseases resulting in polypharmacy and high out-of-pocket expenditure. We confirmed a first major CHIKV outbreak in Matadi, with main vector Aedes albopictus. The health sector was ill-prepared for the information, surveillance, and treatment needs for such an explosive outbreak in a CHIKV-naïve population. Better surveillance systems (national level/sentinel sites) and point-of-care diagnostics for arboviruses are needed.     

Recently introduced Aedes albopictus in Corsica is competent to Chikungunya virus and in a lesser extent to dengue virus

Aedes albopictus has been established in Europe for some decades rendering temperate countries vulnerable to tropical diseases. The Italian chikungunya (CHIK) outbreak in the summer of 2007 demonstrated that indigenous transmission of CHIK was possible in Europe. To estimate the risk of a CHIK outbreak in Corsica, we assessed the vector competence of A. albopictus established in the island since 2006 towards a CHIK variant (E1-A226V). A dengue serotype 2 virus was also tested. Experimental infections showed that A. albopictus was highly competent to CHIK virus (disseminated infection rates ranging from 75% to 100%) and to a lesser extent, to dengue virus (12.5–68.8%). Moreover, A. albopictus ensured a high level of viral replication and was able to transmit the virus as early as 2 days after ingestion of infected blood with around 1 000 viral RNA available in salivary glands. The risk for a local transmission of CHIK is thus likely in Corsica, if other parameters determining the vector capacity of A. albopictus are suitable.     

Introduction of Aedes albopictus in Gabon: what consequences for dengue and chikungunya transmission?

The 2007 outbreak of chikungunya in Gabon has indicated the potential of this disease to spread beyond its usual range ensuing from the expansion of the mosquito Aedes albopictus . A few cases of dengue (DEN) infection were also detected. Because little is known about the potential for Gabonese mosquito species to transmit both chikungunya and DEN viruses (DENV), we conducted studies to determine the susceptibility of Ae. albopictus and Aedes aegypti collected in Libreville to both viruses by experimental infections. Disseminated infection rates were high for Ae. albopictus infected with chikungunya virus (CHIKV) (66.7–86%) and low with DENV (13–21.4%). Moreover, Ae. aegypti sp. formosus was a less efficient vector of CHIKV than Ae. albopictus . The recent introduction and dissemination of chikungunya associated with the invasion of Ae. albopictus in Africa illustrates the potential for CHIKV to spread to other parts of the world.     

Survival of Betta splendens fish (Regan, 1910) in domestic water containers and its effectiveness in controlling Aedes aegypti larvae (Linnaeus, 1762) in Northeast Brazil

Objective In Northeast Brazil, large domestic containers used to store water are important breeding sites of Aedes aegypti, the main vector of dengue fever. The objective of this study was to estimate the survival of Betta splendens (Perciformes: Osphronemidae) fish in domestic containers in Fortaleza (Ceará State), as well as its effectiveness in the control of premature A. aegypti stages. Methods The use of B. splendens was compared to Bacillus thuringiensis israelensis (Bti) in domestic containers. In a first home visit, B. splendens or Bti were applied to water containers. Two follow‐up visits were conducted after 3–4 and 5–6 months to assess the presence of viable fish in the containers and infestation by larvae. Betta splendens fish were still present in 97.6% of containers 45–60 days after application. When the fish was present, the infestation rate was significantly higher (P < 0.001) in the Bti group (IR ratio = 21.60; 95% CI: 6.46–72.28). In deposits where the fish remained, efficacy was 85% better than Bti. The permanence of fish was higher in concrete tanks (48.5%) located outside the house (47.5%) and at ground level (53.3%). We conclude that B. splendens may be suitable for biological control of A. aegypti larvae in large domestic water containers, but that appropriate measures should be taken to assure prolonged survival and the presence of fish in the containers.     

南昌市登革热传播媒介白纹伊蚊密度、抗药性及病原体携带监测研究

目的 掌握南昌地区白纹伊蚊密度、季节消长规律、登革热病毒携带以及对常用杀虫剂的抗药性程度,为合理选择和使用杀虫剂,科学防控登革热提供科学依据。方法 采用WHO推荐的幼虫浸渍法和成蚊接触筒法分别测定白纹伊蚊幼虫和成蚊的抗药性;密度监测:成蚊采用双层叠帐法,幼虫分别采用布雷图指数法(BI)、勺舀法和路径指数法;白纹伊蚊登革热病毒携带检测采用RT-PCR核酸检测。结果 2018年南昌地区白纹伊蚊成蚊呈单峰活动趋势,活动最高峰为6月份,全年平均帐诱指数为2.76只/(顶·h),平均布雷图指数(BI)为6.18,平均路径指数为4.67处/km,居民区帐诱指数、BI和路径指数远高于工地和公园生境。白纹伊蚊成蚊对溴氰菊酯和高效氯氰菊酯产生抗性,对马拉硫磷、氯菊酯、杀螟硫磷和残杀威表现敏感;白纹伊蚊幼虫除对溴氰菊酯表现为敏感外,对DDVP等其他6种杀虫剂分别表现出不同的抗性,抗性倍数处于9.09~76.19之间。南昌地区野外品系白纹伊蚊未检测到登革热病毒。结论 一旦有登革热病例的输入,南昌地区存在登革热传播风险, 应停用已产生高度抗药性的氯氰菊酯、残杀威等杀虫剂,重点清除居民区生境内白纹伊蚊孳生地,及时根据监测结果评估登革热发生或流行的风险,可为南昌地区登革热疫情的预测预警提供科学依据。     

Evaluation of Aedes aegypti,Aedes albopictus,and Culex quinquefasciatus Mosquitoes Competence to Oropouche virus Infection

The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.     

Association of human immune response to Aedes aegypti salivary proteins with dengue disease severity

Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.     

A Novel Anphevirus in Aedes albopictus Mosquitoes Is Distributed Worldwide and Interacts with the Host RNA Interference Pathway

The Asian tiger mosquito Aedes albopictus is a competent vector for several human arboviruses including dengue, chikungunya and Zika viruses. Mosquitoes also harbor insect-specific viruses (ISVs) that may modulate host physiology and potentially affect the transmission of viruses that are pathogenic to vertebrates, thus representing a potential tool for vector control strategies. In Ae. albopictus we identified a novel anphevirus (family Xinmoviridae; order Mononegavirales) provisionally designated here as Aedes albopictus anphevirus (AealbAV). AealbAV contains a ~12.4 kb genome that is highly divergent from currently known viruses but displays gene content and genomic organization typical of known anpheviruses. We identified AealbAV in several publicly available RNA-Seq datasets from different geographical regions both in laboratory colonies and field collected mosquitoes. Coding-complete genomes of AealbAV strains are highly similar worldwide (>96% nucleotide identity) and cluster according to the geographical origin of their hosts. AealbAV appears to be present in various body compartments and mosquito life stages, including eggs. We further detected AealbAV-derived vsiRNAs and vpiRNAs in publicly available miRNA-Seq libraries of Ae. albopictus and in samples experimentally coinfected with chikungunya virus. This suggests that AealbAV is targeted by the host RNA interference (RNAi) response, consistent with persistent virus replication. The discovery and characterization of AealbAV in Ae. albopictus will now allow us to identify its infection in mosquito populations and laboratory strains, and to assess its potential impact on Ae. albopictus physiology and ability to transmit arboviruses.     

Residual insecticidal activity of long‐lasting deltamethrin‐treated curtains after 1 year of household use for dengue control

Objective To evaluate the residual insecticidal activity of the PermaNet^® curtains on Aedes aegypti after 1 year of use in Thai households and to assess the influence of sun and dust exposure, washing practices and detergent use. Methods We sampled UV‐protected PermaNet^® curtains made of a long‐lasting deltamethrin‐[55 mg/m²] treated polyester netting, before (10 curtains) and after 8 (10 curtains) and 12 months (66 curtains) of household use in a field site in Chon Buri, Thailand. We assessed the residual insecticidal activity of the curtains by standard WHO bioassay, using a deltamethrin‐susceptible insectarium Aedes aegypti strain. Results Mosquito mortality was 100% before distribution, 100% at 8 months and 98.2% (95% CI 97.9–98.5) at 12 months of use. Sunlight, hand‐washing and detergent use had no effect on the residual insecticidal activity after 12 months. However, the mosquito survival rate increased by a factor of 6.4 (95% CI 3.5–11.8) on machine‐washed curtains and by a factor of 2.0 (95% CI 1.4–2.9) on curtains not covered by dust. Conclusion The residual insecticidal activity of PermaNet® curtains remains high after 12 months use under field conditions.     

Dengue vector prevalence and virus infection in a rural area in south India

We conducted a 2-year (1997-1999) longitudinal, entomological and virological study in three dengue endemic villages in Vellore district, Tamil Nadu, to understand the dynamics of dengue transmission. Aedes aegypti (Linn.), Ae. albopictus (Skuse) and Ae. vittatus (Bigot) were the prevalent vector species. Aedes aegypti was breeding throughout the year with a Breteau index ranging from 9.05 to 45.49. Aedes albopictus and Ae. vittatus were prevalent mainly in the rainy season. Small water holding containers (cemented tanks/cisterns) were the perennial breeding source of Ae. aegypti, and its abundance was significantly higher in semi-urbanized central areas than the peripheral areas of the villages. From 271 pools (4016 specimens) of adult females, eight dengue virus (DENV) isolates were obtained of which seven were from Ae. aegypti and one from Ae. albopictus. This is the first report of DENV isolation from Ae. albopictus in rural India. Infection rates in the two species were comparable. However, due to higher and perennial prevalence, Ae. aegypti is considered as primary vector with Ae. albopictus playing a secondary role. Despite circulation of all four serotypes (DENV 1-4) detected mainly during the transmission season, the high anthropophilic index of the vectors and their abundance, no human dengue case was reported, suggesting silent dengue transmission.     

Global Evolutionary History and Dynamics of Dengue Viruses Inferred from Whole Genome Sequences

Dengue is an arboviral disease caused by dengue virus (DENV), leading to approximately 25,000 deaths/year and with over 40% of the world’s population at risk. Increased international travel and trade, poorly regulated urban expansion, and warming global temperatures have expanded the geographic range and incidence of the virus in recent decades. This study used phylogenetic and selection pressure analyses to investigate trends in DENV evolution, using whole genome coding sequences from publicly available databases alongside newly sequenced isolates collected between 1963–1997 from Southeast Asia and the Pacific. Results revealed very similar phylogenetic relationships when using the envelope gene and the whole genome coding sequences. Although DENV evolution is predominantly driven by negative selection, a number of amino acid sites undergoing positive selection were found across the genome, with the majority located in the envelope and NS5 genes. Some genotypes appear to be diversifying faster than others within each serotype. The results from this research improve our understanding of DENV evolution, with implications for disease control efforts such as Wolbachia-based biocontrol and vaccine design.     

Differences in male–female ratios of Aedes albopictus (Diptera: Culicidae) following ultra-low volume adulticide applications

Suppression of Aedes albopictus populations is a substantial challenge for mosquito control programs globally because juveniles of this species are found in numerous kinds of domestic artificial containers that are difficult to detect, access, and eliminate. We conducted a multi-year assessment of the effect of different interventions to control Ae. albopictus near the northernmost geographic boundary of the species in temperate North America and deployed an array of BG-Sentinel traps for adult surveillance. Here we present the results of a comparative examination of adult sex ratios in urban and suburban areas, shifts in sex ratios after control interventions, and a discussion of the critical drivers of population dynamics of Ae. albopictus in our area. We collected significantly more male mosquitoes in urban as compared to suburban areas in June through September, but not in May (p < 0.001). The higher number of male mosquitoes in urban areas could be attributed to a higher number of larval habitats within a closer proximity of the surveillance traps and the lower flight dispersal of males. Following application of adulticides in urban areas, Ae. albopictus male populations were reduced by 88% on average, which was higher than the 69% reduction in female populations. The higher reduction of male mosquitoes could be attributed to the smaller body mass of the males and their higher susceptibility to adulticides. The results of this study are directly relevant to the development of suitable control strategies that depend on manipulation of males, such as the sterile insect technique. The results could also be used to refine mosquito abatement by providing more accurate methods to determine the need and timing of vector control.     

The development of predictive tools for pre‐emptive dengue vector control: a study of Aedes aegypti abundance and meteorological variables in North Queensland,Australia

Objectives To describe the meteorological influences on adult dengue vector abundance in Australia for the development of predictive models to trigger pre‐emptive control operation. Methods Multiple linear regression analyses were performed using meteorological data and female Aedes aegypti collection data from BG‐Sentinel Mosquito traps placed at 11 monitoring sites in Cairns, north Queensland. Results Considerable regression coefficients (R² = 0.64 and 0.61) for longer‐ and shorter‐term factor models respectively were derived. Longer‐term factors significantly associated with abundance of adult vectors were mean minimum temperature (lagged 6 month) and mean daily temperature (lagged 4 month), explaining the predictable increase in abundance during the wet season. Factors explaining fluctuation in abundance in the shorter term were mean relative humidity over the previous 2 week and current daily average temperature. Rainfall variables were not found to be strong predictors of A. aegypti abundance in either longer‐ or shorter‐term models. Conclusions The implications of these findings for the development of useful predictive models for vector abundance risks are discussed. Such models can be used to guide the application of pre‐emptive dengue vector control, and thereby enhance disease management.     

Distribution of Mosquito Larvae on Kosrae Island,Kosrae State,the Federated States of Micronesia

Surveys of mosquito larvae were carried out in six areas of Kosrae Island, Kosrae State, the Federated States of Micronesia in December 2009 and June 2012. A total of 962 larvae of six species were collected from 106 natural and artificial habitats. They were identified as Aedes aegypti, Ae. albopictus, Ae. marshallensis, Culex quinquefasciatus, Cx. annulirostris, and Cx. kusaiensis. This is the first report from Kosrae Island for three of these species—Ae. marshallensis, Cx. quinquefasciatus, and Cx. annulirostris. The most abundant species was Ae. albopictus, followed by Ae. marshallensis, and these two species were found in all areas. Relatively large numbers of Cx. quinquefasciatus and Cx. kusaiensis were found in five areas. Fewer Cx. annulirostris were found, and only in three areas. Aedes aegypti larvae were collected from a single habitat at Tafunsak in 2009. To prevent the outbreak of dengue fever, environmental management should focus on the destruction, alteration, disposal and recycling of containers that produce larger numbers of adult Aedes mosquitoes.