Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.     

Comparison of Vector Competence of Aedes mediovittatus and Aedes aegypti for Dengue Virus: Implications for Dengue Control in the Caribbean

Background

Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes.

Methods

To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1–4 per os at viral titers of 5–6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution.

Results

Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus.

Conclusions

This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs.     

The Neovolcanic Axis Is a Barrier to Gene Flow among Aedes aegypti Populations in Mexico That Differ in Vector Competence for Dengue 2 Virus

Background

Aedes aegypti is the main mosquito vector of the four serotypes of dengue virus (DENV). Previous population genetic and vector competence studies have demonstrated substantial genetic structure and major differences in the ability to transmit dengue viruses in Ae. aegypti populations in Mexico.

Methodology/Principal Findings

Population genetic studies revealed that the intersection of the Neovolcanic axis (NVA) with the Gulf of Mexico coast in the state of Veracruz acts as a discrete barrier to gene flow among Ae. aegypti populations north and south of the NVA. The mosquito populations north and south of the NVA also differed in their vector competence (VC) for dengue serotype 2 virus (DENV2). The average VC rate for Ae. aegypti mosquitoes from populations from north of the NVA was 0.55; in contrast the average VC rate for mosquitoes from populations from south of the NVA was 0.20. Most of this variation was attributable to a midgut infection and escape barriers. In Ae. aegypti north of the NVA 21.5% failed to develop midgut infections and 30.3% of those with an infected midgut failed to develop a disseminated infection. In contrast, south of the NVA 45.2% failed to develop midgut infections and 62.8% of those with an infected midgut failed to develop a disseminated infection.

Conclusions

Barriers to gene flow in vector populations may also impact the frequency of genes that condition continuous and epidemiologically relevant traits such as vector competence. Further studies are warranted to determine why the NVA is a barrier to gene flow and to determine whether the differences in vector competence seen north and south of the NVA are stable and epidemiologically significant.     

Complement-Related Proteins Control the Flavivirus Infection of Aedes aegypti by Inducing Antimicrobial Peptides

The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry in vertebrates and invertebrates as an immune defense mechanism. Thioester (TE)-containing proteins (TEPs), which show high similarity to mammalian complement C3, are thought to play a key role in innate immunity in arthropods. Herein, we report that a viral recognition cascade composed of two complement-related proteins limits the flaviviral infection of Aedes aegypti. An A. aegypti macroglobulin complement-related factor (AaMCR), belonging to the insect TEP family, is a crucial effector in opposing the flaviviral infection of A. aegypti. However, AaMCR does not directly interact with DENV, and its antiviral effect requires an A. aegypti homologue of scavenger receptor-C (AaSR-C), which interacts with DENV and AaMCR simultaneously in vitro and in vivo. Furthermore, recognition of DENV by the AaSR-C/AaMCR axis regulates the expression of antimicrobial peptides (AMPs), which exerts potent anti-DENV activity. Our results both demonstrate the existence of a viral recognition pathway that controls the flaviviral infection by inducing AMPs and offer insights into a previously unappreciated antiviral function of the complement-like system in arthropods.     

Visual-Olfactory Integration in the Human Disease Vector Mosquito Aedes aegypti

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Temephos Resistance in Aedes aegypti in Colombia Compromises Dengue Vector Control

Background

Control and prevention of dengue relies heavily on the application of insecticides to control dengue vector mosquitoes. In Colombia, application of the larvicide temephos to the aquatic breeding sites of Aedes aegypti is a key part of the dengue control strategy. Resistance to temephos was recently detected in the dengue-endemic city of Cucuta, leading to questions about its efficacy as a control tool. Here, we characterize the underlying mechanisms and estimate the operational impact of this resistance.

Methodology/Principal Findings

Larval bioassays of Ae. aegypti larvae from Cucuta determined the temephos LC₅₀ to be 0.066 ppm (95% CI 0.06–0.074), approximately 15× higher than the value obtained from a susceptible laboratory colony. The efficacy of the field dose of temephos at killing this resistant Cucuta population was greatly reduced, with mortality rates <80% two weeks after application and <50% after 4 weeks. Neither biochemical assays nor partial sequencing of the ace-1 gene implicated target site resistance as the primary resistance mechanism. Synergism assays and microarray analysis suggested that metabolic mechanisms were most likely responsible for the temephos resistance. Interestingly, although the greatest synergism was observed with the carboxylesterase inhibitor, DEF, the primary candidate genes from the microarray analysis, and confirmed by quantitative PCR, were cytochrome P450 oxidases, notably CYP6N12, CYP6F3 and CYP6M11.

Conclusions/Significance

In Colombia, resistance to temephos in Ae. aegypti compromises the duration of its effect as a vector control tool. Several candidate genes potentially responsible for metabolic resistance to temephos were identified. Given the limited number of insecticides that are approved for vector control, future chemical-based control strategies should take into account the mechanisms underlying the resistance to discern which insecticides would likely lead to the greatest control efficacy while minimizing further selection of resistant phenotypes.     

Co-occurrence Patterns of the Dengue Vector Aedes aegypti and Aedes mediovitattus, a Dengue Competent Mosquito in Puerto Rico

Aedes aegypti is implicated in dengue transmission in tropical and subtropical urban areas around the world. Ae. aegypti populations are controlled through integrative vector management. However, the efficacy of vector control may be undermined by the presence of alternative, competent species. In Puerto Rico, a native mosquito, Ae. mediovittatus, is a competent dengue vector in laboratory settings and spatially overlaps with Ae. aegypti. It has been proposed that Ae. mediovittatus may act as a dengue reservoir during inter-epidemic periods, perpetuating endemic dengue transmission in rural Puerto Rico. Dengue transmission dynamics may therefore be influenced by the spatial overlap of Ae. mediovittatus, Ae. aegypti, dengue viruses, and humans. We take a landscape epidemiology approach to examine the association between landscape composition and configuration and the distribution of each of these Aedes species and their co-occurrence. We used remotely sensed imagery from a newly launched satellite to map landscape features at very high spatial resolution. We found that the distribution of Ae. aegypti is positively predicted by urban density and by the number of tree patches, Ae. mediovittatus is positively predicted by the number of tree patches, but negatively predicted by large contiguous urban areas, and both species are predicted by urban density and the number of tree patches. This analysis provides evidence that landscape composition and configuration is a surrogate for mosquito community composition, and suggests that mapping landscape structure can be used to inform vector control efforts as well as to inform urban planning.     

Efficacy of sticky and standard ovitraps for Aedes aegypti in Trinidad,West Indies

The double sticky trap (DST) is described for the first time and is evaluated along with standard ovitraps and sticky traps (STs) to determine population densities of Ae. aegypti in the urban township of St. Augustine and the rural community of Tamana, Trinidad, West Indies. Ten houses were selected at each study site. At each of the ten houses, one ovitrap, one ST, and one DST were placed using the criteria established for placement of ovitraps. The results showed the three trapping methods successfully collected Ae. aegypti mosquitoes. All three traps collected significantly more adults or eggs in St. Augustine than in Tamana. DSTs collected 2,286 adults from St. Augustine vs 316 adults from Tamana (p<0.002), STs collected 1,480 and 220 adults, respectively (p<0.01), and the ovitraps collected 2,735 and 517 eggs, respectively from St. Augustine and Tamana (p<0.002). Based on these results, the DSTs collected significantly (P<0.02) more adults than the STs. The DSTs and STs collected both adult and immature stages which can be used for toxicology, virology, and PCR studies and are suitable alternative Ae. aegypti surveillance tools for the Caribbean and Latin American region.     

Ammonia metabolism in Aedes aegypti

We investigated the mechanisms by which Aedes aegypti mosquitoes are able to metabolize ammonia. When females were given access to solutions containing NH(4)Cl or to a blood meal, hemolymph glutamine and proline concentrations increased markedly, indicating that ammonium/ammonia can be removed from the body through the synthesis of these two amino acids. The importance of glutamine synthetase was shown when an inhibitor of the enzyme was added to the meal causing the glutamine concentration in hemolymph to decrease significantly, while the proline concentration increased dramatically. Unexpectedly, we found an important role for glutamate synthase. When mosquitoes were fed azaserine, an inhibitor of glutamate synthase, the glutamine concentration increased and the proline concentration decreased significantly. This confirms the presence of glutamate synthase in mosquitoes and suggests that this enzyme contributes to the production of glutamate for proline synthesis. Several key enzymes related to ammonium/ammonia metabolism showed activity in homogenates of mosquito fat body and midgut. The mosquito genes encoding glutamate dehydrogenase, glutamine synthetase, glutamate synthase, pyrroline-5-carboxylate synthase were cloned and sequenced. The mRNA expression patterns of these genes were examined by a real-time RT-PCR in fat body and midgut. The results show that female mosquitoes have evolved efficient mechanisms to detoxify large loads of ammonium/ammonia.     

Trisomy in Aedes aegypti.

A trisomic (2n=6+1) pupa of the yellow fever mosquito Aedes aegypti has been found. The trisomy involved chromosome 3 which is intermediate in size between 1 and 2. The extra chromosome formed a univalent or a trivalent during meiosis.     

Insecticide-Driven Patterns of Genetic Variation in the Dengue Vector Aedes aegypti in Martinique Island

Effective vector control is currently challenged worldwide by the evolution of resistance to all classes of chemical insecticides in mosquitoes. In Martinique, populations of the dengue vector Aedes aegypti have been intensively treated with temephos and deltamethrin insecticides over the last fifty years, resulting in heterogeneous levels of resistance across the island. Resistance spreading depends on standing genetic variation, selection intensity and gene flow among populations. To determine gene flow intensity, we first investigated neutral patterns of genetic variability in sixteen populations representative of the many environments found in Martinique and experiencing various levels of insecticide pressure, using 6 microsatellites. Allelic richness was lower in populations resistant to deltamethrin, and consanguinity was higher in populations resistant to temephos, consistent with a negative effect of insecticide pressure on neutral genetic diversity. The global genetic differentiation was low, suggesting high gene flow among populations, but significant structure was found, with a pattern of isolation-by-distance at the global scale. Then, we investigated adaptive patterns of divergence in six out of the 16 populations using 319 single nucleotide polymorphisms (SNPs). Five SNP outliers displaying levels of genetic differentiation out of neutral expectations were detected, including the kdr-V1016I mutation in the voltage-gated sodium channel gene. Association tests revealed a total of seven SNPs associated with deltamethrin resistance. Six other SNPs were associated with temephos resistance, including two non-synonymous substitutions in an alkaline phosphatase and in a sulfotransferase respectively. Altogether, both neutral and adaptive patterns of genetic variation in mosquito populations appear to be largely driven by insecticide pressure in Martinique.     

Genetic Diversity and Phylogeny of Aedes aegypti,the Main Arbovirus Vector in the Pacific

Background

The Pacific region is an area unique in the world, composed of thousands of islands with differing climates and environments. The spreading and establishment of the mosquito Aedes aegypti in these islands might be linked to human migration. Ae. aegypti is the major vector of arboviruses (dengue, chikungunya and Zika viruses) in the region. The intense circulation of these viruses in the Pacific during the last decade led to an increase of vector control measures by local health authorities. The aim of this study is to analyze the genetic relationships among Ae. aegypti populations in this region.

Methodology/Principal Finding

We studied the genetic variability and population genetics of 270 Ae. aegypti, sampled from 9 locations in New Caledonia, Fiji, Tonga and French Polynesia by analyzing nine microsatellites and two mitochondrial DNA regions (CO1 and ND4). Microsatellite markers revealed heterogeneity in the genetic structure between the western, central and eastern Pacific island countries. The microsatellite markers indicate a statistically moderate differentiation (F_ST = 0.136; P < = 0.001) in relation to island isolation. A high degree of mixed ancestry can be observed in the most important towns (e.g. Noumea, Suva and Papeete) compared with the most isolated islands (e.g. Ouvea and Vaitahu). Phylogenetic analysis indicated that most of samples are related to Asian and American specimens.

Conclusions/Significance

Our results suggest a link between human migrations in the Pacific region and the origin of Ae. aegypti populations. The genetic pattern observed might be linked to the island isolation and to the different environmental conditions or ecosystems.     

Modeling Dengue Vector Dynamics under Imperfect Detection: Three Years of Site-Occupancy by Aedes aegypti and Aedes albopictus in Urban Amazonia

Aedes aegypti and Ae. albopictus are the vectors of dengue, the most important arboviral disease of humans. To date, Aedes ecology studies have assumed that the vectors are truly absent from sites where they are not detected; since no perfect detection method exists, this assumption is questionable. Imperfect detection may bias estimates of key vector surveillance/control parameters, including site-occupancy (infestation) rates and control intervention effects. We used a modeling approach that explicitly accounts for imperfect detection and a 38-month, 55-site detection/non-detection dataset to quantify the effects of municipality/state control interventions on Aedes site-occupancy dynamics, considering meteorological and dwelling-level covariates. Ae. aegypti site-occupancy estimates (mean 0.91; range 0.79–0.97) were much higher than reported by routine surveillance based on ‘rapid larval surveys’ (0.03; 0.02–0.11) and moderately higher than directly ascertained with oviposition traps (0.68; 0.50–0.91). Regular control campaigns based on breeding-site elimination had no measurable effects on the probabilities of dwelling infestation by dengue vectors. Site-occupancy fluctuated seasonally, mainly due to the negative effects of high maximum (Ae. aegypti) and minimum (Ae. albopictus) summer temperatures (June-September). Rainfall and dwelling-level covariates were poor predictors of occupancy. The marked contrast between our estimates of adult vector presence and the results from ‘rapid larval surveys’ suggests, together with the lack of effect of local control campaigns on infestation, that many Aedes breeding sites were overlooked by vector control agents in our study setting. Better sampling strategies are urgently needed, particularly for the reliable assessment of infestation rates in the context of control program management. The approach we present here, combining oviposition traps and site-occupancy models, could greatly contribute to that crucial aim.     

Midgut bacterial dynamics in Aedes aegypti

In vector mosquitoes, the presence of midgut bacteria may affect the ability to transmit pathogens. We have used a laboratory colony of Aedes aegypti as a model for bacterial interspecies competition and show that after a blood meal, the number of species (culturable on Luria-Bertani agar) that coexist in the midgut is low and that about 40% of the females do not harbor any cultivable bacteria. We isolated species belonging to the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas, and we also determined their growth rates, antibiotic resistance, and ex vivo inhibition of each other. To investigate the possible existence of coadaptation between midgut bacteria and their host, we fed Ae.?aegypti cohorts with gut bacteria from human, a frog, and two mosquito species and followed the bacterial population growth over time. The dynamics of the different species suggests coadaptation between host and bacteria, and interestingly, we found that Pantoea stewartii isolated from Ae.?aegypti survive better in Ae.?aegypti as compared to P.?stewartii isolated from the malaria mosquito Anopheles gambiae.     

Feedback regulation of vitellogenin synthesis in Aedes aegypti and Aedes atropalpus

Blood-fed hemiovariectomized Aedes aegypti synthesized vitellogenin at the same rate as intact females. Removal of both developing ovaries from blood-fed females diminished vitellogenin synthesis gradually. To determine the concentration of circulating vitellogenin, vitellin was purified using hydrophobic affinity chromatography. A titration curve of antibody-antigen complex was developed and the concentration of vitellogenin circulating in ovariectomized females was found to be five-fold higher than that in intact females.Ovaries removed from vitellogenic donors did not induce vitellogenin synthesis in non-vitellogenic (blood-fed, decapitated) recipients. Implanting a pinocytotic ovary into blood-fed females in which vitellogenesis was arrested by ovariectomy, restored vitellogenin synthesis.These results indicate that vitellogenin synthesis in Aedes aegypti is controlled by a feedback-inhibition mechanism, rather than an ovarian factor, and the ovary acts as a sink.