Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Individual, Household, and Community Drivers of Dengue Virus Infection Risk in Kamphaeng Phet Province, Thailand.

BACKGROUND: Dengue virus (DENV) often circulates endemically. In such settings with high levels of transmission, it remains unclear whether there are risk factors that alter individual infection risk. METHODS: We tested blood taken from individuals living in multigenerational households in Kamphaeng Phet province, Thailand for DENV antibodies (N = 2364, mean age 31 years). Seropositivity ranged from 45.4% among those 1-5 years old to 99.5% for those >30 years. Using spatially explicit catalytic models, we estimated that 11.8% of the susceptible population gets infected annually. RESULTS: We found that 37.5% of the variance in seropositivity was explained by unmeasured household-level effects with only 4.2% explained by spatial differences between households. The serostatus of individuals from the same household remained significantly correlated even when separated by up to 15 years in age. CONCLUSIONS: These findings show that despite highly endemic transmission, persistent differences in infection risk exist across households, the reasons for which remain unclear.

Genetic mapping of specific interactions between Aedes aegypti mosquitoes and dengue viruses.

Specific interactions between host genotypes and pathogen genotypes (G×G interactions) are commonly observed in invertebrate systems. Such specificity challenges our current understanding of invertebrate defenses against pathogens because it contrasts the limited discriminatory power of known invertebrate immune responses. Lack of a mechanistic explanation, however, has questioned the nature of host factors underlying G×G interactions. In this study, we aimed to determine whether G×G interactions observed between dengue viruses and their Aedes aegypti vectors in nature can be mapped to discrete loci in the mosquito genome and to document their genetic architecture. We developed an innovative genetic mapping strategy to survey G×G interactions using outbred mosquito families that were experimentally exposed to genetically distinct isolates of two dengue virus serotypes derived from human patients. Genetic loci associated with vector competence indices were detected in multiple regions of the mosquito genome. Importantly, correlation between genotype and phenotype was virus isolate-specific at several of these loci, indicating G×G interactions. The relatively high percentage of phenotypic variation explained by the markers associated with G×G interactions (ranging from 7.8% to 16.5%) is consistent with large-effect host genetic factors. Our data demonstrate that G×G interactions between dengue viruses and mosquito vectors can be assigned to physical regions of the mosquito genome, some of which have a large effect on the phenotype. This finding establishes the existence of tangible host genetic factors underlying specific interactions between invertebrates and their pathogens in a natural system. Fine mapping of the uncovered genetic loci will elucidate the molecular mechanisms of mosquito-virus specificity.

Impact of daily temperature fluctuations on dengue virus transmission by Aedes aegypti.

Most studies on the ability of insect populations to transmit pathogens consider only constant temperatures and do not account for realistic daily temperature fluctuations that can impact vector-pathogen interactions. Here, we show that diurnal temperature range (DTR) affects two important parameters underlying dengue virus (DENV) transmission by Aedes aegypti. In two independent experiments using different DENV serotypes, mosquitoes were less susceptible to virus infection and died faster under larger DTR around the same mean temperature. Large DTR (20 °C) decreased the probability of midgut infection, but not duration of the virus extrinsic incubation period (EIP), compared with moderate DTR (10 °C) or constant temperature. A thermodynamic model predicted that at mean temperatures <18 °C, DENV transmission increases as DTR increases, whereas at mean temperatures >18 °C, larger DTR reduces DENV transmission. The negative impact of DTR on Ae. aegypti survival indicates that large temperature fluctuations will reduce the probability of vector survival through EIP and expectation of infectious life. Seasonal variation in the amplitude of daily temperature fluctuations helps to explain seasonal forcing of DENV transmission at locations where average temperature does not vary seasonally and mosquito abundance is not associated with dengue incidence. Mosquitoes lived longer and were more likely to become infected under moderate temperature fluctuations, which is typical of the high DENV transmission season than under large temperature fluctuations, which is typical of the low DENV transmission season. Our findings reveal the importance of considering short-term temperature variations when studying DENV transmission dynamics.

Species abundance and density of malaria vectors in Western Thailand and implications for disease transmission.

Understanding the dynamics of malaria vectors and their interactions with environmental factors is crucial for effective malaria control. This study investigated the abundance, species composition, seasonal variations, and malaria infection status of female mosquitoes in malaria transmission and non-transmission areas in Western Thailand. Additionally, the susceptibility of malaria vectors to pyrethroid insecticides was assessed. Entomological field surveys were conducted during the hot, wet, and cold seasons in both malaria transmission areas (TA) and non-transmission areas (NTA). The abundance and species composition of malaria vectors were compared between TA and NTA. The availability of larval habitats and the impact of seasonality on vector abundance were analyzed. Infection with Plasmodium spp. in primary malaria vectors was determined using molecular techniques. Furthermore, the susceptibility of malaria vectors to pyrethroids was evaluated using the World Health Organization (WHO) susceptibility test. A total of 9799 female mosquitoes belonging to 54 species and 11 genera were collected using various trapping methods. The number of malaria vectors was significantly higher in TA compared to NTA (P < 0.001). Anopheles minimus and An. aconitus were the predominant species in TA, comprising over 50% and 30% of the total mosquitoes collected, respectively. Seasonality had a significant effect on the availability of larval habitats in both areas (P < 0.05) but did not impact the abundance of adult vectors (P > 0.05). The primary malaria vectors tested were not infected with Plasmodium spp. The WHO susceptibility test revealed high susceptibility of malaria vectors to pyrethroids, with mortality rates of 99-100% at discriminating concentrations. The higher abundance of malaria vectors in the transmission areas underscores the need for targeted control measures in these regions. The susceptibility of malaria vectors to pyrethroids suggests the continued effectiveness of this class of insecticides for vector control interventions. Other factors influencing malaria transmission risk in the study areas are discussed. These findings contribute to our understanding of malaria vectors and can inform evidence-based strategies for malaria control and elimination efforts in Western Thailand.

Dengue-1 virus clade replacement in Thailand associated with enhanced mosquito transmission.

Dengue viruses (DENV) are characterized by extensive genetic diversity and can be organized in multiple, genetically distinct lineages that arise and die out on a regular basis in regions where dengue is endemic. A fundamental question for understanding DENV evolution is the relative extent to which stochastic processes (genetic drift) and natural selection acting on fitness differences among lineages contribute to lineage diversity and turnover. Here, we used a set of recently collected and archived low-passage DENV-1 isolates from Thailand to examine the role of mosquito vector-virus interactions in DENV evolution. By comparing the ability of 23 viruses isolated on different dates between 1985 and 2009 to be transmitted by a present-day Aedes aegypti population from Thailand, we found that a major clade replacement event in the mid-1990s was associated with virus isolates exhibiting increased titers in the vector's hemocoel, which is predicted to result in a higher probability of transmission. This finding is consistent with the hypothesis that selection for enhanced transmission by mosquitoes is a possible mechanism underlying major DENV clade replacement events. There was significant variation in transmission potential among isolates within each clade, indicating that in addition to vector-driven selection, other evolutionary forces act to maintain viral genetic diversity. We conclude that occasional adaptive processes involving the mosquito vector can drive major DENV lineage replacement events.

Development and evaluation of a pyriproxyfen-treated device to control the dengue vector, Aedes aegypti (L.) (Diptera:Culicidae).

The resurgence of dengue fever and the chikungunya epidemic make the control of Aedes aegypti mosquitoes, the vectors of these diseases, critically important. We developed and evaluated an Ae. aegypti control device that is visually-attractive to mosquitoes. This pyriproxyfen-treated device was evaluated for its impact on Ae. aegypti egg production and population dynamics in dengue-endemic areas in Thailand. The device consists of a "high rise" shaped ovitrap/ resting station covered with black cotton cloth. The device is easily collapsible and transportable. Ae. aegypti are generally drawn towards darker, shadier areas making this device physically attractive as a resting station to mosquitoes of all physiological stages. The results show this device suppressed Ae. aegypti populations after it was introduced into a village. The observed effect was primarily the result of the Ae. aegypti exposure to pyriproxyfen shortly after adult emergence or after taking a blood meal resulting in decreased egg production. We believe the device may be further improved physically and the formulation should be replaced to provide even better efficacy for controlling Ae. aegypti mosquito, populations.

Underrecognized mildly symptomatic viremic dengue virus infections in rural Thai schools and villages.

BACKGROUND: The understanding of dengue virus (DENV) transmission dynamics and the clinical spectrum of infection are critical to informing surveillance and control measures. Geographic cluster studies can elucidate these features in greater detail than cohort studies alone. METHODS: A 4-year longitudinal cohort and geographic cluster study was undertaken in rural Thailand. Cohort children underwent pre-/postseason serology and active school absence-based surveillance to detect inapparent and symptomatic dengue. Cluster investigations were triggered by cohort dengue and non-dengue febrile illnesses (positive and negative clusters, respectively). RESULTS: The annual cohort incidence of symptomatic dengue ranged from 1.3% to 4.4%. DENV-4 predominated in the first 2 years, DENV-1 in the second 2 years. The inapparent-to-symptomatic infection ratio ranged from 1.1:1 to 2.9:1. Positive clusters had a 16.0% infection rate, negative clusters 1.1%. Of 119 infections in positive clusters, 59.7% were febrile, 20.2% were afebrile with other symptoms, and 20.2% were asymptomatic. Of 16 febrile children detected during cluster investigations who continued to attend school, 9 had detectable viremia. CONCLUSIONS: Dengue transmission risk was high near viremic children in both high- and low-incidence years. Inapparent infections in the cohort overestimated the rate of asymptomatic infections. Ambulatory children with mild febrile viremic infections could represent an important component of dengue transmission.

The impact of insect growth regulators on adult emergence inhibition and the fitness of Aedes aegypti field populations in Thailand.

BACKGROUND: The frequent use of insecticides in vector control causes the development of insecticide resistance. Insect growth regulators (IGRs), which effect insect development, are used as a promising alternative to control resistant insect vectors. This study aimed to develop novel effective tools for Aedes aegypti control by evaluating the efficacy of different IGRs on larval development, blood feeding capacity, fecundity, and fertility in females and sperm productivity in males across geographical regions of Thailand. METHODS: The efficacy of 16 technical grade IGRs were evaluated against laboratory strain Ae. aegypti larvae in order to determine their emergence inhibition (EI) at 50% and 95% under laboratory conditions. Six IGRs were selected for fecundity, fertility, and sperm productivity studies using feed-through treatments at EI95 concentration levels against adult Ae. aegypti field strains. RESULTS: The results from larval bioassay tests indicate that juvenile hormone mimics (EI50 = 0.010-0.229 ppb; EI95 = 0.066-1.118 ppb) and chitin synthesis inhibitors affecting CHS1 (EI50 = 0.240-2.412 ppb; EI95 = 0.444-4.040 ppb) groups effectively inhibited adult Ae. aegypti emergence. Methoprene and fenoxycarb significantly reduced blood feeding capacity. Egg production was comparable among strains while methoprene, pyriproxyfen and diflubenzuron induced egg production. Egg retention was detected in females fed on diflubenzuron. Methoprene, fenoxycarb, diflubenzuron, and teflubenzuron reduced egg hatching rates in mosquito field strains compared to laboratory strain. Male mosquitoes fed on fenoxycarb showed significantly lower sperm production compared to other treatments. CONCLUSION: Juvenile hormone analogues and chitin synthesis inhibitors affecting CHS1 groups showed excellent results in adult emergence inhibition in this study. They also disrupted reproductive systems in both adult males and females. This study suggested that they can be used as an alternative larvicide in mosquito control programs.

Entomological Risk Assessment for Dengue Virus Transmission during 2016-2020 in Kamphaeng Phet, Thailand.

Individual houses with high risks of dengue virus (DENV) transmission might be a source of virus transmission within the neighborhood. We conducted an entomological risk assessment for DENV transmission at the household level, comprising family cohort members residing in the same location, to assess the risk for dengue virus transmitted by mosquito vectors. The studies were conducted in Kamphaeng Phet Province, Thailand, during 2016-2020. Entomological investigations were performed in 35 cohort families on day 1 and day 14 after receiving dengue case reports. DENV was found in 22 Aedes samples (4.9%) out of 451 tested samples. A significantly higher DENV infection rate was detected in vectors collected on day 1 (6.64%) compared to those collected on day 14 (1.82%). Annual vector surveillance was carried out in 732 houses, with 1002 traps catching 3653 Aedes females. The majority of the 13,228 water containers examined were made from plastic and clay, with used tires serving as a primary container, with 59.55% larval abundance. Larval indices, as indicators of dengue epidemics and to evaluate disease and vector control approaches, were calculated. As a result, high values of larval indices indicated the considerably high risk of dengue transmission in these communities.

A Comparative Study of Mosquito and Sand Fly (Diptera: Psychodidae: Phlebotominae) Sampling Using Dry Ice and Chemically Generated Carbon Dioxide From Three Different Prototype CO2 Generators.

A comparative study was conducted to test the efficiency of Centers for Disease Control and Prevention (CDC) light traps baited with either dry ice or carbon dioxide (CO2) produced from one of three different sources in collecting mosquitoes (Diptera: Culicidae) in Thailand. Treatments consisted of dry ice pellets, CO2 gas produced from one of three prototype CO2 generator systems (TDA, CUBE, Moustiq-Air Med-e-Cell - MEC), and a CDC light trap without a CO2 source. The best performing prototype from Thailand was then tested in collecting sand flies (Diptera: Psychodidae: Phlebotominae) in Greece. A total of 12,798 mosquitoes and 8,329 sand flies were sampled during the experimentation. The most prevalent mosquito species collected in Thailand were: Culex vishnui Theobald > Anopheles minimus Theobald > Culex tritaeniorhynchus Giles > Anopheles sawadwongporni Rattanarithikul & Green. By far the most prevalent sand fly species collected in Thessaloniki was Phlebotomus perfiliewi Parrot followed by Phlebotomus tobbi Adler and Theodor and Phlebotomus simici Nitzulescu. In general, the TDA treatment was the only treatment with no significant difference from the dry ice-treatment in mean trap catches. Although dry ice-baited traps caught higher numbers of mosquitoes and sand flies than the TDA-baited traps, there was no difference in the number of species collected. Results indicate that the traps baited with the TDA CO2 generator were as attractive as traps supplied with dry ice and, therefore, the TDA CO2 generator is a suitable alternative to dry ice as a source of carbon dioxide for use with adult mosquito and sand fly traps.

Too "sexy" for the field? Paired measures of laboratory and semi-field performance highlight variability in the apparent mating fitness of Aedes aegypti transgenic strains.

BACKGROUND: Evaluating and improving mating success and competitive ability of laboratory-reared transgenic mosquito strains will enhance the effectiveness of proposed disease-control strategies that involve deployment of transgenic strains. Two components of the mosquito rearing process, larval diet quantity and aquatic environment - which are linked to physiological and behavioural differences in adults - are both relatively easy to manipulate. In mosquitoes, as for many other arthropod species, the quality of the juvenile habitat is strongly associated with adult fitness characteristics, such as longevity and fecundity. However, the influence of larval conditioning on mating performance is poorly understood. Here, we investigated the combined effects of larval diet amount and environmental water source on adult male mating success in a genetically modified strain of Aedes aegypti mosquitoes in competition with wild-type conspecifics. Importantly, this research was conducted in a field setting using low generation laboratory and wild-type lines. RESULTS: By controlling larval diet (high and low) and rearing water source (field-collected and laboratory water), we generated four treatment lines of a genetically modified strain of Ae. aegypti tagged with fluorescent sperm. Laboratory reared mosquitoes were then competed against a low generation wild-type colony in a series of laboratory and semi-field mating experiments. While neither food quantity nor larval aquatic environment were found to affect male mating fitness, the transgenic lines consistently outperformed wild-types in laboratory competition assays, an advantage that was not conferred to semi-field tests. CONCLUSIONS: Using a model transgenic system, our results indicate that differences in the experimental conditions of laboratory- and field-based measures of mating success can lead to variation in the perceived performance ability of modified strains if they are only tested in certain environments. While there are many potential sources of variation between laboratory and field lines, laboratory adaptation - which may occur over relatively few generations in this species - may directly impact mating ability depending on the context in which it is measured. We suggest that colony-hybridization with field material can potentially be used to mitigate these effects in a field setting. Release programs utilising mass-produced modified laboratory strains should incorporate comparative assessments of quality in candidate lines.

Extensive Genetic Differentiation between Homomorphic Sex Chromosomes in the Mosquito Vector, Aedes aegypti.

Mechanisms and evolutionary dynamics of sex-determination systems are of particular interest in insect vectors of human pathogens like mosquitoes because novel control strategies aim to convert pathogen-transmitting females into nonbiting males, or rely on accurate sexing for the release of sterile males. In Aedes aegypti, the main vector of dengue and Zika viruses, sex determination is governed by a dominant male-determining locus, previously thought to reside within a small, nonrecombining, sex-determining region (SDR) of an otherwise homomorphic sex chromosome. Here, we provide evidence that sex chromosomes in Ae. aegypti are genetically differentiated between males and females over a region much larger than the SDR. Our linkage mapping intercrosses failed to detect recombination between X and Y chromosomes over a 123-Mbp region (40% of their physical length) containing the SDR. This region of reduced male recombination overlapped with a smaller 63-Mbp region (20% of the physical length of the sex chromosomes) displaying high male-female genetic differentiation in unrelated wild populations from Brazil and Australia and in a reference laboratory strain originating from Africa. In addition, the sex-differentiated genomic region was associated with a significant excess of male-to-female heterozygosity and contained a small cluster of loci consistent with Y-specific null alleles. We demonstrate that genetic differentiation between sex chromosomes is sufficient to assign individuals to their correct sex with high accuracy. We also show how data on allele frequency differences between sexes can be used to estimate linkage disequilibrium between loci and the sex-determining locus. Our discovery of large-scale genetic differentiation between sex chromosomes in Ae. aegypti lays a new foundation for mapping and population genomic studies, as well as for mosquito control strategies targeting the sex-determination pathway.

Laboratory and semi-field evaluation of Mosquito Dunks against Aedes aegypti and Aedes albopictus larvae (Diptera: Culicidae).

Laboratory bioassays and semi-field studies were conducted on the efficacy and longevity of Mosquito Dunks (7,000 ITU/mg Bti) in order to determine the concentration-response relationship and the effectiveness on the potency of the Bti product against Aedes mosquito species based on the WHO protocol standard methods and to determine the longevity of release for this product against Ae. aegypti mosquito larvae in water storage containers. This bio-potency study with the late 3rd instar larvae of Ae. aegypti and Ae. albopictus was carried out according to WHO standard protocols. The six concentrations of the Bti product used in each test were replicated 4 times with 25 mosquito larvae. Probit analysis was then used to determine the LC50 and LC95 which was equated with dosages of 1.02 and 1.86 ppm for Ae. aegypti; and 0.39 and 0.84 ppm for Ae. albopictus, which reveals a potency of 382.95 and 303.74 ITU/mg, respectively. The semi-field evaluation of this product in 200-liter earthen jars against 3rd instar larvae of Ae. aegypti showed satisfactory control of greater than 80% at 11 weeks post-treatment.

No evidence for local adaptation of dengue viruses to mosquito vector populations in Thailand.

Despite their epidemiological importance, the evolutionary forces that shape the spatial structure of dengue virus genetic diversity are not fully understood. Fine-scale genetic structure of mosquito vector populations and evidence for genotype × genotype interactions between dengue viruses and their mosquito vectors are consistent with the hypothesis that the geographical distribution of dengue virus genetic diversity may reflect viral adaptation to local mosquito populations. To test this hypothesis, we measured vector competence in all sympatric and allopatric combinations of 14 low-passage dengue virus isolates and two wild-type populations of Aedes aegypti mosquitoes sampled in Bangkok and Kamphaeng Phet, two sites located about 300 km apart in Thailand. Despite significant genotype × genotype interactions, we found no evidence for superior vector competence in sympatric versus allopatric vector-virus combinations. Viral phylogenetic analysis revealed no geographical clustering of the 14 isolates, suggesting that high levels of viral migration (gene flow) in Thailand may counteract spatially heterogeneous natural selection. We conclude that it is unlikely that vector-mediated selection is a major driver of dengue virus adaptive evolution at the regional scale that we examined. Dengue virus local adaptation to mosquito vector populations could happen, however, in places or times that we did not test, or at a different geographical scale.

Frequent in-migration and highly focal transmission of dengue viruses among children in Kamphaeng Phet, Thailand.

Revealing the patterns and determinants of the spread of dengue virus (DENV) at local scales is central to understanding the epidemiology and evolution of this major human pathogen. We performed a phylogenetic analysis of the envelope (E) genes of DENV-1, -2, -3, and -4 isolates (involving 97, 23, 5, and 74 newly collected sequences, respectively) sampled from school-based cohort and village-based cluster studies in Kamphaeng Phet, Thailand, between 2004 and 2007. With these data, we sought to describe the spatial and temporal patterns of DENV spread within a rural population where a future vaccine efficacy trial is planned. Our analysis revealed considerable genetic diversity within the study population, with multiple lineages within each serotype circulating for various lengths of time during the study period. These results suggest that DENV is frequently introduced into both semi-urban and rural areas in Kamphaeng Phet from other populations. In contrast, the persistence of viral lineages across sampling years was observed less frequently. Analysis of phylogenetic clustering indicated that DENV transmission was highly spatially and temporally focal, and that it occurred in homes rather than at school. Overall, the strength of temporal clustering suggests that seasonal bottlenecks in local DENV populations facilitate the invasion and establishment of viruses from outside of the study area, in turn reducing the extent of lineage persistence.

Fine scale spatiotemporal clustering of dengue virus transmission in children and Aedes aegypti in rural Thai villages.

BACKGROUND: Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages. METHODOLOGY/PRINCIPAL FINDINGS: Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile "index" cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80-100 meters away (p<0.001). Significantly more Ae. aegypti were DENV-infectious (i.e., DENV-positive in head/thorax) in positive clusters (23/1755; 1.3%) than negative clusters (1/1548; 0.1%). In positive clusters, 8.2% of mosquitoes were DENV-infectious in index houses, 4.2% in other houses with DENV-infected children, and 0.4% in houses without infected children (p<0.001). The DENV infection rate in contacts was 47.4% in houses with infectious mosquitoes, 28.7% in other houses in the same cluster, and 10.8% in positive clusters without infectious mosquitoes (p<0.001). Ae. aegypti pupae and adult females were more numerous only in houses containing infectious mosquitoes. CONCLUSIONS/SIGNIFICANCE: Human and mosquito infections are positively associated at the level of individual houses and neighboring residences. Certain houses with high transmission risk contribute disproportionately to DENV spread to neighboring houses. Small groups of houses with elevated transmission risk are consistent with over-dispersion of transmission (i.e., at a given point in time, people/mosquitoes from a small portion of houses are responsible for the majority of transmission).

Ecological modeling of Aedes aegypti (L.) pupal production in rural Kamphaeng Phet, Thailand.

BACKGROUND: Aedes aegypti (L.) is the primary vector of dengue, the most important arboviral infection globally. Until an effective vaccine is licensed and rigorously administered, Ae. aegypti control remains the principal tool in preventing and curtailing dengue transmission. Accurate predictions of vector populations are required to assess control methods and develop effective population reduction strategies. Ae. aegypti develops primarily in artificial water holding containers. Release recapture studies indicate that most adult Ae. aegypti do not disperse over long distances. We expect, therefore, that containers in an area of high development site density are more likely to be oviposition sites and to be more frequently used as oviposition sites than containers that are relatively isolated from other development sites. After accounting for individual container characteristics, containers more frequently used as oviposition sites are likely to produce adult mosquitoes consistently and at a higher rate. To this point, most studies of Ae. aegypti populations ignore the spatial density of larval development sites. METHODOLOGY: Pupal surveys were carried out from 2004 to 2007 in rural Kamphaeng Phet, Thailand. In total, 84,840 samples of water holding containers were used to estimate model parameters. Regression modeling was used to assess the effect of larval development site density, access to piped water, and seasonal variation on container productivity. A varying-coefficients model was employed to account for the large differences in productivity between container types. A two-part modeling structure, called a hurdle model, accounts for the large number of zeroes and overdispersion present in pupal population counts. FINDINGS: The number of suitable larval development sites and their density in the environment were the primary determinants of the distribution and abundance of Ae. aegypti pupae. The productivity of most container types increased significantly as habitat density increased. An ecological approach, accounting for development site density, is appropriate for predicting Ae. aegypti population levels and developing efficient vector control programs.