Insecticide resistance and genetic structure of Aedes aegypti populations from Rio de Janeiro State,Brazil

Vector control largely relies on neurotoxic chemicals, and insecticide resistance (IR) directly threatens their effectiveness. In some cases, specific alleles cause IR, and knowledge of the genetic diversity and gene flow among mosquito populations is crucial to track their arrival, rise, and spread. Here we evaluated Aedes aegypti populations’ susceptibility status, collected in 2016 from six different municipalities of Rio de Janeiro state (RJ), to temephos, pyriproxyfen, malathion, and deltamethrin. We collected eggs of Ae. aegypti in Campos dos Goytacazes (Cgy), Itaperuna (Ipn), Iguaba Grande (Igg), Itaboraí (Ibr), Mangaratiba (Mgr), and Vassouras (Vsr). We followed the World Health Organization (WHO) guidelines and investigated the degree of susceptibility/resistance of mosquitoes to these insecticides. We used the Rockefeller strain as a susceptible positive control. We genotyped the V1016I and F1534C knockdown resistance (kdr) alleles using qPCR TaqMan SNP genotyping assay. Besides, with the use of Ae. aegypti SNP-chip, we performed genomic population analyses by genotyping more than 15,000 biallelic SNPs in mosquitoes from each population. We added previous data from populations from other countries to evaluate the ancestry of RJ populations.All RJ Ae. aegypti populations were susceptible to pyriproxyfen and malathion and highly resistant to deltamethrin. The resistance ratios for temephos was below 3,0 in Cgy, Ibr, and Igg populations, representing the lowest rates since IR monitoring started in this Brazilian region. We found the kdr alleles in high frequencies in all populations, partially justifying the observed resistance to pyrethroid. Population genetics analysis showed that Ae. aegypti revealed potential higher migration among some RJ localities and low genetic structure for most of them. Future population genetic studies, together with IR data in Ae aegypti on a broader scale, can help us predict the gene flow within and among the Brazilian States, allowing us to track the dynamics of arrival and changes in the frequency of IR alleles, and providing critical information to improving vector control program.     

Pyrethroid resistance reduces the efficacy of space sprays for dengue control on the island of Martinique (Caribbean)

Background

Dengue fever is reemerging on the island of Martinique and is a serious threat for the human population. During dengue epidemics, adult Aedes aegypti control with pyrethroid space sprays is implemented in order to rapidly reduce transmission. Unfortunately, vector control programs are facing operational challenges with the emergence of pyrethroid resistant Ae. aegypti populations.

Methodology/Principal Findings

To assess the impact of pyrethroid resistance on the efficacy of treatments, applications of deltamethrin and natural pyrethrins were performed with vehicle-mounted thermal foggers in 9 localities of Martinique, where Ae. aegypti populations are strongly resistant to pyrethroids. Efficacy was assessed by monitoring mortality rates of naturally resistant and laboratory susceptible mosquitoes placed in sentinel cages. Before, during and after spraying, larval and adult densities were estimated. Results showed high mortality rates of susceptible sentinel mosquitoes treated with deltamethrin while resistant mosquitoes exhibited very low mortality. There was no reduction of either larval or adult Ae. aegypti population densities after treatments.

Conclusions/Significance

This is the first documented evidence that pyrethroid resistance impedes dengue vector control using pyrethroid-based treatments. These results emphasize the need for alternative tools and strategies for dengue control programs.     

Evaluation of boric acid as toxic sugar bait against resistant Aedes aegypti mosquitoes

Current methods of broad area application of contact insecticides used in mosquito control are becoming less effective, primarily due to resistance within mosquito populations. New methods that can deliver ingestible insecticides are being investigated as a means to mitigate resistance. This study evaluated insecticide delivery through toxic sugar baits (TSB) and resulting mortality of susceptible and resistant strains of Aedes aegypti. Two Ae. aegypti strains were evaluated using a 1% boric acid TSB: the susceptible Orlando 1952 (ORL) strain and the resistant Puerto Rican (PR) strain. The TSB resulted in high mortality for both ORL and PR strain of Ae. aegypti. Average mortality of female mosquitoes given TSB was 90.8% for PR and 99.3% for ORL. Our study suggests that targeting resistant mosquitoes with ingestible insecticides through TSBs could be a viable alternative to current mosquito control strategies and should be considered when developing an integrated vector management program.     

Filarial worms reduce Plasmodium infectivity in mosquitoes

Background

Co-occurrence of malaria and filarial worm parasites has been reported, but little is known about the interaction between filarial worm and malaria parasites with the same Anopheles vector. Herein, we present data evaluating the interaction between Wuchereria bancrofti and Anopheles punctulatus in Papua New Guinea (PNG). Our field studies in PNG demonstrated that An. punctulatus utilizes the melanization immune response as a natural mechanism of filarial worm resistance against invading W. bancrofti microfilariae. We then conducted laboratory studies utilizing the mosquitoes Armigeres subalbatus and Aedes aegypti and the parasites Brugia malayi, Brugia pahangi, Dirofilaria immitis, and Plasmodium gallinaceum to evaluate the hypothesis that immune activation and/or development by filarial worms negatively impact Plasmodium development in co-infected mosquitoes. Ar. subalbatus used in this study are natural vectors of P. gallinaceum and B. pahangi and they are naturally refractory to B. malayi (melanization-based refractoriness).

Methodology/Principal Findings

Mosquitoes were dissected and Plasmodium development was analyzed six days after blood feeding on either P. gallinaceum alone or after taking a bloodmeal containing both P. gallinaceum and B. malayi or a bloodmeal containing both P. gallinaceum and B. pahangi. There was a significant reduction in the prevalence and mean intensity of Plasmodium infections in two species of mosquito that had dual infections as compared to those mosquitoes that were infected with Plasmodium alone, and was independent of whether the mosquito had a melanization immune response to the filarial worm or not. However, there was no reduction in Plasmodium development when filarial worms were present in the bloodmeal (D. immitis) but midgut penetration was absent, suggesting that factors associated with penetration of the midgut by filarial worms likely are responsible for the observed reduction in malaria parasite infections.

Conclusions/Significance

These results could have an impact on vector infection and transmission dynamics in areas where Anopheles transmit both parasites, i.e., the elimination of filarial worms in a co-endemic locale could enhance malaria transmission.     

Insecticide resistance status and mechanisms in Aedes aegypti populations from Senegal

Aedes aegypti is the main epidemic vector of arboviruses in Africa. In Senegal, control activities are mainly limited to mitigation of epidemics, with limited information available for Ae. aegypti populations. A better understanding of the current Ae. aegypti susceptibility status to various insecticides and relevant resistance mechanisms involved is needed for the implementation of effective vector control strategies. The present study focuses on the detection of insecticide resistance and reveals the related mechanisms in Ae. aegypti populations from Senegal.Bioassays were performed on Ae. aegypti adults from nine Senegalese localities (Matam, Louga, Barkedji, Ziguinchor, Mbour, Fatick, Dakar, Kédougou and Touba). Mosquitoes were exposed to four classes of insecticides using the standard WHO protocols. Resistance mechanisms were investigated by genotyping for pyrethroid target site resistance mutations (V1016G, V1016I, F1534C and S989P) and measuring gene expression levels of key detoxification genes (CYP6BB2, CYP9J26, CYP9J28, CYP9J32, CYP9M6, CCEae3a and GSTD4).All collected populations were resistant to DDT and carbamates except for the ones in Matam (Northern region). Resistance to permethrin was uniformly detected in mosquitoes from all areas. Except for Barkédji and Touba, all populations were characterized by a susceptibility to 0.75% Permethrin. Susceptibility to type II pyrethroids was detected only in the Southern regions (Kédougou and Ziguinchor). All mosquito populations were susceptible to 5% Malathion, but only Kédougou and Matam mosquitoes were susceptible to 0.8% Malathion. All populations were resistant to 0.05% Pirimiphos-methyl, whereas those from Louga, Mbour and Barkédji, also exhibited resistance to 1% Fenitrothion. None of the known target site pyrethroid resistance mutations was present in the mosquito samples included in the genotyping analysis (performed in > 1500 samples). In contrast, a remarkably high (20-70-fold) overexpression of major detoxification genes was observed, suggesting that insecticide resistance is mostly mediated through metabolic mechanisms. These data provide important evidence to support dengue vector control in Senegal.     

Distribution of Voltage-Gated Sodium Channel (Nav) Alleles among the Aedes aegypti Populations In Central Java Province and Its Association with Resistance to Pyrethroid Insecticides

The emergence of insecticide resistant Aedes aegypti mosquitoes has hampered dengue control efforts. WHO susceptibility tests, using several pyrethroid compounds, were conducted on Ae. aegypti larvae that were collected and raised to adulthood from Semarang, Surakarta, Kudus and Jepara in Java. The AaNa_V gene fragment encompassing kdr polymorphic sites from both susceptible and resistant mosquitoes was amplified, and polymorphisms were associated with the resistant phenotype. The insecticide susceptibility tests demonstrated Ae, aegypti resistance to the pyrethroids, with mortality rates ranging from 1.6%–15.2%. Three non-synonymous polymorphisms (S989P, V1016G and F1534C) and one synonymous polymorphism (codon 982) were detected in the AaNa_V gene. Eight AaNa_V alleles were observed in specimens from Central Java. Allele 3 (SGF) and allele 7 (PGF) represent the most common alleles found and demonstrated strong associations with resistance to pyrethroids (OR = 2.75, CI: 0.97–7.8 and OR = 7.37, CI: 2.4–22.5, respectively). This is the first report of 8 Ae. aegypti AaNa_V alleles, and it indicates the development of resistance in Ae. aegypti in response to pyrethroid insecticide-based selective pressure. These findings strongly suggest the need for an appropriate integrated use of insecticides in the region. The 989P, 1016G and 1534C polymorphisms in the AaNa_V gene are potentially valuable molecular markers for pyrethroid insecticide resistance monitoring.     

The Impact of Selection with Diflubenzuron,a Chitin Synthesis Inhibitor,on the Fitness of Two Brazilian Aedes aegypti Field Populations

Several Aedes aegypti field populations are resistant to neurotoxic insecticides, mainly organophoshates and pyrethroids, which are extensively used as larvicides and adulticides, respectively. Diflubenzuron (DFB), a chitin synthesis inhibitor (CSI), was recently approved for use in drinking water, and is presently employed in Brazil for Ae. aegypti control, against populations resistant to the organophosphate temephos. However, tests of DFB efficacy against field Ae. aegypti populations are lacking. In addition, information regarding the dynamics of CSI resistance, and characterization of any potential fitness effects that may arise in conjunction with resistance are essential for new Ae. aegypti control strategies. Here, the efficacy of DFB was evaluated for two Brazilian Ae. aegypti populations known to be resistant to both temephos and the pyrethroid deltamethrin. Laboratory selection for DFB resistance was then performed over six or seven generations, using a fixed dose of insecticide that inhibited 80% of adult emergence in the first generation. The selection process was stopped when adult emergence in the diflubenzuron-treated groups was equivalent to that of the control groups, kept without insecticide. Diflubenzuron was effective against the two Ae. aegypti field populations evaluated, regardless of their resistance level to neurotoxic insecticides. However, only a few generations of DFB selection were sufficient to change the susceptible status of both populations to this compound. Several aspects of mosquito biology were affected in both selected populations, indicating that diflubenzuron resistance acquisition is associated with a fitness cost. We believe that these results can significantly contribute to the design of control strategies involving the use of insect growth regulators.     

Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance,recovery, and death

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site—the voltage-gated sodium channel (VGSC)—and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.     

Levels of cross-resistance to pyrethroids conferred by the Vssc knockdown resistance allele 410L+1016I+1534C in Aedes aegypti

Aedes aegypti is a primary vector of viral pathogens and is responsible for millions of human infections annually that represent critical public health and economic costs. Pyrethroids are one of the most commonly used classes of insecticides to control adult A. aegypti. The insecticidal activity of pyrethroids depends on their ability to bind and disrupt the voltage-sensitive sodium channel (VSSC). In mosquitoes, a common mechanism of resistance to pyrethroids is due to mutations in Vssc (hereafter referred as knockdown resistance, kdr). In this study, we found that a kdr (410L+V1016I+1534C) allele was the main mechanism of resistance in a pyrethroid-resistant strain of A. aegypti collected in Colombia. To characterize the level of resistance these mutations confer, we isolated a pyrethroid resistant strain (LMRKDR:RK, LKR) that was congenic to the susceptible Rockefeller (ROCK) strain. The full-length cDNA of Vssc was cloned from LKR and no additional resistance mutations were present. The levels of resistance to different pyrethroids varied from 3.9- to 56-fold. We compared the levels of resistance to pyrethroids, DCJW and DDT between LKR and what was previously reported in two other congenic strains that share the same pyrethroid-susceptible background (the ROCK strain), but carry different kdr alleles (F1534C or S989P + V1016G). The resistance conferred by kdr alleles can vary depending on the stereochemistry of the pyrethroid. The 410L+1016I+1534C kdr allele does not confer higher levels of resistance to six of ten pyrethroids, relative to the 1534C allele. The importance of these results to understand the evolution of insecticide resistance and mosquito control are discussed.     

Assembly of the Genome of the Disease Vector Aedes aegypti onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

The mosquito Aedes aegypti transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled — there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of Ae. aegypti, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes Ae. aegypti resistant to the human parasite Brugia malayi, and generated a list of candidate genes that could be affecting the trait.     

Organophosphate Resistance in Aedes aegypti: Study from Dengue Hemorrhagic Fever Endemic Subdistrict in Riau,Indonesia

Background:Dengue hemorrhagic fever (DHF) is a significant health problem. The high number of cases requires preventions, including controlling the dengue vector, Aedes aegypti mosquito. One of the control methods is the use of insecticides containing organophosphate. This study aims to detect organophosphate resistance in Aedes aegypti from DHF endemic subdistrict, Riau, Indonesia by a sensitivity test of temephos and 5% malathion and measuring the activity of non-specific alpha and beta esterase enzymes.Methods:This observational study determined Aedes aegypti resistance from larvae to adult in one DHF endemic subdistrict in Riau, Indonesia. The bioassay was used for temephos sensitivity of Aedes aegypti larvae. The LC99 value was analyzed using probit and compared with the diagnostic value from WHO. The WHO susceptibility test was conducted to determine 5% malathion resistance from adult mosquitoes. The mortality of less than 90% was declared as resistant. Measurement of alpha and beta esterase levels used Lee''s microplate assay technique based on visual identification and absorbance value (AV).Results:The results showed that Aedes aegypti were resistant to temephos. It also showed that adult mosquitoes were resistant to 5% malathion. Based on the alpha esterase activity test, it was found that most of the mosquitoes showed very sensitive meanwhile, based on the beta esterase activity test, most of the mosquitoes were moderate resistance.Conclusion:This study suggests that Aedes aegypti population from DHF endemic subdistrict in Riau, Indonesia are indicated to develop resistance to organophosphate.Key Words: Aedes aegypti, Dengue Hemorrhagic fever, Organophosphate, Resistance     

Mosquito metallomics reveal copper and iron as critical factors for Plasmodium infection

Iron and copper chelation restricts Plasmodium growth in vitro and in mammalian hosts. The parasite alters metal homeostasis in red blood cells to its favor, for example metabolizing hemoglobin to hemozoin. Metal interactions with the mosquito have not, however, been studied. Here, we describe the metallomes of Anopheles albimanus and Aedes aegypti throughout their life cycle and following a blood meal. Consistent with previous reports, we found evidence of maternal iron deposition in embryos of Ae. aegypti, but less so in An. albimanus. Sodium, potassium, iron, and copper are present at higher concentrations during larval developmental stages. Two An. albimanus phenotypes that differ in their susceptibility to Plasmodium berghei infection were studied. The susceptible white stripe (ws) phenotype was named after a dorsal white stripe apparent during larval stages 3, 4, and pupae. During larval stage 3, ws larvae accumulate more iron and copper than the resistant brown stripe (bs) phenotype counterparts. A similar increase in copper and iron accumulation was also observed in the susceptible ws, but not in the resistant bs phenotype following P. berghei infection. Feeding ws mosquitoes with extracellular iron and copper chelators before and after receiving Plasmodium-infected blood protected from infection and simultaneously affected follicular development in the case of iron chelation. Unexpectedly, the application of the iron chelator to the bs strain reverted resistance to infection. Besides a drop in iron, iron-chelated bs mosquitoes experienced a concomitant loss of copper. Thus, the effect of metal chelation on P. berghei infectivity was strain-specific.     

Kdr genotyping (V1016I,F1534C) of the Nav channel of Aedes aegypti (L.) mosquito populations in Harris County (Houston), Texas,USA, after Permanone 31–66 field tests and its influence on probability of survival

Aedes aegypti (L.) is an important mosquito vector of emerging arboviruses such as Zika, dengue, yellow fever, and chikungunya. To quell potential disease outbreaks, its populations are controlled by applying pyrethroid insecticides, which selection pressure may lead to the development of insecticide resistance. Target site insensitivity to pyrethroids caused by non-synonymous knockdown resistance (kdr) mutations in the voltage-gated sodium (Na_V) channel is a predominant mechanism of resistance in mosquitoes. To evaluate the potential impact of pyrethroid resistance on vector control, Ae. aegypti eggs were collected from eight mosquito control operational areas in Harris County, Texas, and emerged females were treated in field tests at four different distances from the pyrethroid Permanone 31–66 source. The females were genotyped by melting curve analyses to detect two kdr mutations (V1016I and F1534C) in the Na_V channel. Harris County females had higher survivorship rates at each distance than the pyrethroid-susceptible Orlando strain females. Survivorship increased with distance from the pyrethroid source, with 39% of field-collected mosquitoes surviving at 7.62 m and 82.3% at 22.86 m from the treatment source. Both the V1016I and F1534C pyrethroid resistant genotypes were widely distributed and at high frequency, with 77% of the females being double homozygous resistant (II/CC), this being the first report of kdr mutations in Ae. aegypti in Harris County. Analysis of the probability of survival for each mutation site independently indicated that the CC genotype had similar probability of survival as the FC heterozygous, while the II genotype had higher survival than both the VI and VV, that did not differ. The double homozygous resistant genotype (II/CC) had the highest probability of survival. A linear model estimated probability of survival for areas and genotypes. The high frequency and widespread distribution of double-homozygote pyrethroid-resistant Ae. aegypti may jeopardize disease vector control efforts in Harris County.     

Changes in enzyme titres with age in four geographical strains of Aedes aegypti and their association with insecticide resistance

Abstract. The enzymes acetylcholinesterase, glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PD), and general esterases were assayed in four strains of Aedes aegypti mosquitoes aged between 1 and 30 days. Microtitre plate methods were used to assay activity in the homogenates of individual mosquitoes. The levels of GST and G6PD declined with the age of the mosquitoes, while the activity for the other enzymes remained constant. Soluble protein content was also found to decline with mosquito age in all the strains. Insecticide bioassays showed that two strains (Trinidad and Virtudes) of Ae. aegypti were resistant to DDT, deltamethrin and malathion, whereas two other strains (Bangkok and Indian) were susceptible to all four classes of insecticides tested. Higher esterase activity levels in the resistant compared to the susceptible strains were assumed to be the cause of organophosphate resistance. The combination of DDT and deltamethrin resistance in two strains with normal GST and G6PD characteristics suggests that a kdr-type nerve insensitivity mechanism may be involved.     

Identifying insecticide resistance genes in mosquito by combining AFLP genome scans and 454 pyrosequencing

AFLP‐based genome scans are widely used to study the genetics of adaptation and to identify genomic regions potentially under selection. However, this approach usually fails to detect the actual genes or mutations targeted by selection owing to the difficulties of obtaining DNA sequences from AFLP fragments. Here, we combine classical AFLP outlier detection with 454 sequencing of AFLP fragments to obtain sequences from outlier loci. We applied this approach to the study of resistance to Bacillus thuringiensis israelensis (Bti) toxins in the dengue vector Aedes aegypti. A genome scan of Bti‐resistant and Bti‐susceptible A. aegypti laboratory strains was performed based on 432 AFLP markers. Fourteen outliers were detected using two different population genetic algorithms. Out of these, 11 were successfully sequenced. Three contained transposable elements (TEs) sequences, and the 10 outliers that could be mapped at a unique location in the reference genome were located on different supercontigs. One outlier was in the vicinity of a gene coding for an aminopeptidase potentially involved in Bti toxin‐binding. Patterns of sequence variability of this gene showed significant deviation from neutrality in the resistant strain but not in the susceptible strain, even after taking into account the known demographic history of the selected strain. This gene is a promising candidate for future functional analysis.     

Natural arbovirus infection rate and detectability of indoor female Aedes aegypti from Mérida,Yucatán,Mexico

Arbovirus infection in Aedes aegypti has historically been quantified from a sample of the adult population by pooling collected mosquitoes to increase detectability. However, there is a significant knowledge gap about the magnitude of natural arbovirus infection within areas of active transmission, as well as the sensitivity of detection of such an approach. We used indoor Ae. aegypti sequential sampling with Prokopack aspirators to collect all mosquitoes inside 200 houses with suspected active ABV transmission from the city of Mérida, Mexico, and tested all collected specimens by RT-PCR to quantify: a) the absolute arbovirus infection rate in individually tested Ae. aegypti females; b) the sensitivity of using Prokopack aspirators in detecting ABV-infected mosquitoes; and c) the sensitivity of entomological inoculation rate (EIR) and vectorial capacity (VC), two measures ABV transmission potential, to different estimates of indoor Ae. aegypti abundance. The total number of Ae. aegypti (total catch, the sum of all Ae. aegypti across all collection intervals) as well as the number on the first 10-min of collection (sample, equivalent to a routine adult aspiration session) were calculated. We individually tested by RT-PCR 2,161 Aedes aegypti females and found that 7.7% of them were positive to any ABV. Most infections were CHIKV (77.7%), followed by DENV (11.4%) and ZIKV (9.0%). The distribution of infected Aedes aegypti was overdispersed; 33% houses contributed 81% of the infected mosquitoes. A significant association between ABV infection and Ae. aegypti total catch indoors was found (binomial GLMM, Odds Ratio > 1). A 10-min indoor Prokopack collection led to a low sensitivity of detecting ABV infection (16.3% for detecting infected mosquitoes and 23.4% for detecting infected houses). When averaged across all infested houses, mean EIR ranged between 0.04 and 0.06 infective bites per person per day, and mean VC was 0.6 infectious vectors generated from a population feeding on a single infected host per house/day. Both measures were significantly and positively associated with Ae. aegypti total catch indoors. Our findings provide evidence that the accurate estimation and quantification of arbovirus infection rate and transmission risk is a function of the sampling effort, the local abundance of Aedes aegypti and the intensity of arbovirus circulation.