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Background Botanical substances such as essential oils (EOs) have demonstrated insecticidal properties and are a valid option for vector control. However, free EOs are unreliable as mosquito larvicides due their easy degradation by environmental exposure to ultraviolet light and higher temperatures. Here, we assessed the efficacy of a mosquito larvicide based on orange oil in a yeast-based delivery system against Aedes aegypti strains with different resistance status towards chemical neurotoxic insecticides. This larvicide preparation was physicochemically characterized in a previous report. Methods Larvae of four Ae. aegypti strains from different regions of Brazil and different resistance profiles for deltamethrin (pyrethroid) and temephos (organophosphate) were tested against yeast-encapsulated orange oil (YEOO) in laboratory conditions for measurement of LC50 and LC90 values. The same assays were performed with the Belo Horizonte strain under environmental conditions (natural light and temperature). The resistance profiles of these strains were compared to the Rockefeller reference strain in all conditions. Results YEOO was found to be a highly active larvicide (LC50 Ae. aegypti strains tested in both laboratory conditions (LC50 = 8.1–24.7 mg/L) and environmental conditions with natural light and temperature fluctuation (LC50 = 20.0–49.9 mg/L). Moreover, all strains were considered susceptible (RR 90–95 of Belo Horizonte in the laboratory, probably due the higher heterogeneity associated with older egg papers (> 5 months). Conclusion YEOO demonstrates high larvicidal activity against Ae. aegypti strains with resistant phenotypes for deltamethrin (PY) and temephos (OP). This larvicidal activity suggests the potential for the development of YEOO as an alternative intervention to synthetic insecticides in integrated vector management programs, for populations with resistance to commonly used insecticides. Graphical Abstract

Deployment of Wolbachia to mitigate dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) transmission is ongoing in 12 countries. One way to assess the efficacy of Wolbachia releases is to determine invasion rates within the wild population of Aedes aegypti following their release. Herein we evaluated the accuracy, sensitivity and specificity of the Near Infrared Spectroscopy (NIRS) in estimating the time post death, ZIKV-, CHIKV-, and Wolbachia-infection in trapped dead female Ae. aegypti mosquitoes over a period of 7 days. Regardless of the infection type, time post-death of mosquitoes was accurately predicted into four categories (fresh, 1 day old, 2–4 days old and 5–7 days old). Overall accuracies of 93.2, 97 and 90.3% were observed when NIRS was used to detect ZIKV, CHIKV and Wolbachia in dead Ae. aegypti female mosquitoes indicating NIRS could be potentially applied as a rapid and cost-effective arbovirus surveillance tool. However, field data is required to demonstrate the full capacity of NIRS for detecting these infections under field conditions., Santos et al. demonstrate that the Near Infrared Spectroscopy (NIRS) can accurately estimate the death time of trapped female Aedes aegypti and vector infection with Zika virus, Chikungunya virus, or Wolbachia in a 7-day trapping period. This study suggests that NIRS may provide an accurate and inexpensive tool that improves arbovirus surveillance systems.

Aedes aegypti is adapted to live in close association with human dwellings, where it lays eggs in several man-made container types with a broad range of size, shape, and material. Biotic and abiotic conditions of larval habitats determine the abundance and body size of emerging adult mosquitoes. Here, we estimated the predictive potential of physicochemical water variables for Culicidae immature abundance and Ae. aegypti adult body size in four neighborhoods with distinct urban landscapes in Rio de Janeiro, Brazil. Domestic water holding containers (N = 240) were inspected for the presence of Culicidae immatures and had several physiochemical parameters measured. Larvae and pupae were counted, and pupae were reared to the adult stage for taxonomic identification. Dry weight and wing size were measured for Ae. aegypti adult mosquitoes (N = 981). The association between larval habitat parameters with Culicidae abundance and Ae. aegypti body size data was estimated through linear mixed models and generalized linear mixed models, respectively, with the neighborhood as random effect. The abundance of immature Culicidae in larval habitats (from which >90% of adults emerging from field collected pupae were Ae. aegypti) was positively associated with container volume and the dissolved organic carbon concentration (DOC). Female average dry weight and male and female wing lengths were positively associated with larval habitat temperature whereas male average dry weight was positively related to water conductivity. Aedes aegypti originating from larval habitats with Ae. albopictus exhibited no differences in median wing length and dry body weight when compared with specimens collected in containers exclusively colonized by Ae. aegypti. These results demonstrate that container water volume (characteristic easily observed in the field) and DOC (often higher in unmanaged water holding recipients) is related to higher Ae. aegypti immature density. Estimating the effects of physicochemical water variables on immature abundance and adult body size can provide valuable information for predicting arbovirus transmission risk in endemic settings. Funding Agencies|US National Institutes of Health Fogarty International Center FIRCA [R03TW00744601A1]; Conselho Nacional de Desenvolvimento Cientifico e TecnologicoNational Council for Scientific and Technological Development (CNPq) [12446/2018]; Preventing and Combating the Zika Virus MCTIC/FNDCT-CNPq/MEC-CAPES/MS-Decit. [440929/2016-4]; Fundacao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro [E-26/203.064/2016, E-26/201.335/2016]; Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior-CAPESCAPES [001]; Coordination for the Improvement of Higher Education Personnel-CAPESCAPES; Fiocruz (Programa Institucional de Internacionalizacao da Fiocruz PrInt FiocruzCAPES)

Despite worldwide efforts to understand the transmission dynamics of Zika virus (ZIKV), scanty evaluation has been made on the vector competence of Aedes aegypti fed directly on viremic human and non-human primates (NHPs). We blood-fed Ae. aegypti from two districts in Rio de Janeiro on six ZIKV infected pregnant rhesus macaques at several time points, half of which were treated with Sofosbuvir (SOF). Mosquitoes were analyzed for vector competence after 3, 7 and 14 days of incubation. Although viremia extended up to eight days post monkey inoculation, only mosquitoes fed on the day of the peak of viremia, recorded on day two, became infected. The influence of SOF treatment could not be assessed because the drug was administered just after mosquito feeding on day two. The global infection, dissemination and transmission rates were quite low (4.09%, 1.91% and 0.54%, respectively), no mosquito was infected when viremia was below 1.26 &times, 105 RNA copies/mL. In conclusion, Ae. aegypti vector competence for ZIKV from macaques is low, likely to be due to low viral load and the short duration of ZIKV viremia in primates suitable for infecting susceptible mosquitoes. If ZIKV infection in human and macaques behaves similarly, transmission of the Zika virus in nature is most strongly affected by vector density.

Background Effective mosquito control approaches incorporate both adult and larval stages. For the latter, physical, biological, and chemical control have been used with varying results. Successful control of larvae has been demonstrated using larvicides including insect growth regulators, e.g. the organophosphate temephos, as well as various entomopathogenic microbial species. However, a variety of health and environmental issues are associated with some of these. Laboratory trials of essential oils (EO) have established the larvicidal activity of these substances, but there are currently no commercially available EO-based larvicides. Here we report on the development of a new approach to mosquito larval control using a novel, yeast-based delivery system for EO. Methods Food-grade orange oil (OO) was encapsulated into yeast cells following an established protocol. To prevent environmental contamination, a proprietary washing strategy was developed to remove excess EO that is adsorbed to the cell exterior during the encapsulation process. The OO-loaded yeast particles were then characterized for OO loading, and tested for efficacy against Aedes aegypti larvae. Results The composition of encapsulated OO extracted from the yeast microparticles was demonstrated not to differ from that of un-encapsulated EO when analyzed by high performance liquid chromatography. After lyophilization, the oil in the larvicide comprised 26–30 percentage weight (wt%), and is consistent with the 60–65% reduction in weight observed after the drying process. Quantitative bioassays carried with Liverpool and Rockefeller Ae. aegypti strains in three different laboratories presented LD50 of 5.1 (95% CI: 4.6–5.6) to 27.6 (95% CI: 26.4–28.8) mg/l, for L1 and L3/L4 mosquito larvae, respectively. LD90 ranged between 18.9 (95% CI: 16.4–21.7) mg/l (L1 larvae) to 76.7 (95% CI: 69.7–84.3) mg/l (L3/L4 larvae). Conclusions The larvicide based on OO encapsulated in yeast was shown to be highly active (LD50 Ae. aegypti. These results demonstrate its potential for incorporation in an integrated approach to larval source management of Ae. aegypti. This novel approach can enable development of affordable control strategies that may have significant impact on global health.

Mosquito midgut microbiota is a key component of vector competence, as gut bacteria can disturb pathogen development. In this study, we addressed the microbiota composition of Aedes aegypti during its lifespan, under field conditions. We also investigated the possible effects of environment, dietary regime and ageing on the gut community composition. We employed culture independent and dependent approaches to characterise vector microbiota. There was evidence of a lifelong stable core microbiota after mosquitoes were released into an urban settlement, where they presumably fed on a range of vertebrate hosts and carbohydrate sources. This core was formed mainly of bacteria belonging to the genera Pseudomonas, Acinetobacter, Aeromonas and Stenotrophomonas and to the families Oxalobacteraceae, Enterobacteriaceae and Comamonadaceae. We showed that both dietary regime and age were associated with the abundance of some bacterial groups in the Ae. aegypti microbiota. The majority of the bacterial groups we identified have been detected in the midgut of Ae. aegypti from laboratory and wild populations, indicating a possible core microbiota associated with this mosquito species. Our findings suggest that Ae. aegypti harbours a stable bacterial community during its adult life, similar to mosquito populations from distinct geographic areas, which may be further explored for arbovirus biocontrol strategies.

We report the first use of near-infrared spectroscopy for rapid, low-cost, noninvasive detection of Zika virus in Aedes aegypti., The accelerating global spread of arboviruses, such as Zika virus (ZIKV), highlights the need for more proactive mosquito surveillance. However, a major challenge during arbovirus outbreaks has been the lack of rapid and affordable tests for pathogen detection in mosquitoes. We show for the first time that near-infrared spectroscopy (NIRS) is a rapid, reagent-free, and cost-effective tool that can be used to noninvasively detect ZIKV in heads and thoraces of intact Aedes aegypti mosquitoes with prediction accuracies of 94.2 to 99.3% relative to quantitative reverse transcription polymerase chain reaction (RT-qPCR). NIRS involves simply shining a beam of light on a mosquito to collect a diagnostic spectrum. We estimated in this study that NIRS is 18 times faster and 110 times cheaper than RT-qPCR. We anticipate that NIRS will be expanded upon for identifying potential arbovirus hotspots to guide the spatial prioritization of vector control.

Background. Chemical control is still a major strategy to constrain vector density and mitigate pathogen transmission. However, insecticide overuse poses a high selective pressure, favouring the spread of resistance alleles in natural populations. In an insecticide-free environment, a fitness cost is expected in resistant insects when compared to susceptible counterparts. This study investigates whether insecticide resistance to an organophosphate (temephos) and a pyrethroid (deltamethrin) is associated with fitness traits in four Aedes aegypti wild populations sampled every three months over one year. Findings. We measured development time from larvae to adult, female survival, wing length, fecundity, and adult resistance to starvation in field insecticide resistant Ae. aegypti populations four times over a year. These results were confronted with resistance levels to temephos and deltamethrin and with potentially related mechanisms, including a kdr mutation in the pyrethroid target site. No differences in fitness cost were found after contrasting mosquitoes from the same population collected throughout a year, irrespective of differences in insecticide resistance levels. Additionally, significant differences were not observed among field populations. However, compared to the reference strain Rockefeller, field females survived significantly less. Moreover, larval development was equal or slower in three out of four field populations. In no case differences were evidenced in starvation tolerance, wing length, and fecundity. Conclusions. Overall, field resistant mosquitoes seemed to have a slight fitness disadvantage when compared with the Rockefeller susceptible strain which might represent a potential fitness cost of insecticide resistance. However, after comparing Ae. aegypti from the same population but sampled at different moments, or from different field populations, mosquito life-history traits varied independently of resistance ratios. The metabolic deviations necessary to overcome the adverse effects of insecticides may cause an energy trade-off that affects energy allocation and ultimately basic demands of insect biology. The extent of fitness cost due to insecticide resistance is critical information to delay the evolution of resistance in wild vector populations.

The impact of senescence and pathogen infection on Aedes aegypti life-history traits remains poorly understood. This laboratory study focused on the impact of Zika virus (ZIKV) infection and the age of first blood intake on blood meal and clutch sizes, and more importantly on the egg production ratio per μL of blood. Three groups of ZIKV-infected and uninfected Ae. aegypti females that received their first blood meal at 7 (young feeders), 14 (mature feeders) and 21 days old (old feeders) were monitored daily for survival and received a blood meal free of ZIKV once a week. The number of eggs laid per female were registered 3±4 days after blood feeding. Infection by ZIKV and age of feeding produced a strong negative impact on survival and oviposition success (e.g. likelihood of laying at least one egg per gonotrophic cycle). Interestingly, clutch size presented a dramatic reduction on uninfected mosquitoes, but raised from 36.5 in clutch1 to 55.1 eggs in clutch 3. Blood meal size remained stable in uninfected females, while a slight increase was observed for the infected counterparts. In uninfected Ae. aegypti, egg production was strongly affected by the age of feeding with younger females laying three times more eggs than when older. On the other hand, ZIKV-infected mosquitoes had a constant but low egg production. Overall, mosquito senescence and ZIKV infection had an impact on mosquito egg production by causing a sharp decrease in the number of eggs along the clutches for uninfected mosquitoes and a slight increase for infected mosquitoes. Despite some study limitations, our results contribute to a better understanding of the effects of mosquito aging and pathogen infection on the vectorial capacity of Ae. aegypti.

Background In the tropics, the utilization of insecticides is still an important strategy for controlling Aedes aegypti, the principle vector of dengue, chikungunya and Zika viruses. However, increasing insecticide resistance in Ae. aegypti populations might hinder insecticide efficacy on a long-term basis. It will be important to understand the dynamics and evolution of insecticide resistance by assessing its frequency and the mechanisms by which it occurs. Methodology/Principal findings The insecticide resistance status of four Brazilian Ae. aegypti populations was monitored. Quantitative bioassays with the major insecticides employed in the country was performed: the adulticide deltamethrin (a pyrethroid—PY) and the larvicides, temephos (an organophosphate) and diflubenzuron (a chitin synthesis inhibitor). Temephos resistance was detected in all populations although exhibiting a slight decrease over time probably due to the interruption of field use. All vector populations were susceptible to diflubenzuron, recently introduced in the country to control Ae. aegypti. Resistance against deltamethrin was extremely high in three populations. Molecular assays investigated substitutions in the voltage gated sodium channel (NaV), the PY target site, at positions 1011, 1016 and 1534. Elevated frequencies of substitutions Val1016Ile and Phe1534Cys related to high PY resistance levels were identified. Biochemical assays detected alterations in the activities of two detoxifying enzyme classes related to metabolic resistance, glutathion-S-transferases and esterases. The results obtained were evaluated in the context of both recent insecticide use and the records of dengue incidence in each locality. Conclusions/Significance The four Ae. aegypti populations evaluated were resistant to the neurotoxic insecticides, temephos and deltamethrin. However, they were still susceptible to diflubenzuron. A probable correlation between adult insect resistance to PY and the domestic application of insecticides is discussed, pointing to the need for awareness measures regarding the correct utilization by citizens. This work aims to contribute to the efficient and rational management of Ae. aegypti control of both larvae and adults., Author summary Among the pathogens transmitted by Aedes aegypti, dengue virus is the most important due to the number of people affected or at risk and the high rate of mortality worldwide. The confirmation that Ae. aegypti is also the vector of Zika, chikungunya and urban yellow fever poses serious consequences for public health, pointing to the need of reevaluating current vector control strategies. Although there is growing recognition of the importance of social participation and community engagement to prevent high levels of infestation, insecticides are considered important vector control tools. Nevertheless, the massive and indiscriminate adoption of insecticides to control larvae and adults contributes to resistance spread. In particular, the domestic use of adulticides, especially in epidemic seasons, is assumed to induce high levels of resistance in Ae. aegypti populations. However, the consequences of insecticide interruption upon the resistance of field populations has been less investigated. We evaluated, in four Brazilian regions over one year, the dynamics of dengue vector population resistance to the principal insecticides used in the country. The main resistance mechanisms were also investigated. Data are discussed taking into account the potential relationship among dengue outbreaks, public and private chemical control and insecticide resistance.

Different urban structures might affect the life history parameters of Aedes aegypti and, consequently, dengue transmission. Container productivity, probability of daily survival (PDS) and dispersal rates were estimated for mosquito populations in a high income neighbourhood of Rio de Janeiro. Results were contrasted with those previously found in a suburban district, as well as those recorded in a slum. After inspecting 1,041 premises, domestic drains and discarded plastic pots were identified as the most productive containers, collectively holding up to 80% of the total pupae. In addition, three cohorts of dust-marked Ae. aegypti females were released and recaptured daily using BGS-Traps, sticky ovitraps and backpack aspirators in 50 randomly selected houses; recapture rate ranged from 5-12.2% within cohorts. PDS was determined by two models and ranged from 0.607-0.704 (exponential model) and 0.659-0.721 (non-linear model), respectively. Mean distance travelled varied from 57-122 m, with a maximum dispersal of 263 m. Overall, lower infestation indexes and adult female survival were observed in the high income neighbourhood, suggesting a lower dengue transmission risk in comparison to the suburban area and the slum. Since results show that urban structure can influence mosquito biology, specific control strategies might be used in order to achieve cost-effective Ae. aegypti control.

Background Zika virus (ZIKV) emerged in the Pacific Ocean and subsequently caused a dramatic Pan‐American epidemic after its first appearance in the Northeast region of Brazil in 2015. The virus is transmitted by Aedes mosquitoes. We evaluated the role of temperature and infectious doses of ZIKV in vector competence of Brazilian populations of Ae. aegypti and Ae. albopictus. Methodology/Principal findings Two Ae. aegypti (Rio de Janeiro and Natal) and two Ae. albopictus (Rio de Janeiro and Manaus) populations were orally challenged with five viral doses (102 to 106 PFU / ml) of a ZIKV strain (Asian genotype) isolated in Northeastern Brazil, and incubated for 14 and 21 days in temperatures mimicking the spring-summer (28°C) and winter-autumn (22°C) mean values in Brazil. Detection of viral particles in the body, head and saliva samples was done by plaque assays in cell culture for determining the infection, dissemination and transmission rates, respectively. Compared with 28°C, at 22°C, transmission rates were significantly lower for both Ae. aegypti populations, and Ae. albopictus were not able to transmit the virus. Ae. albopictus showed low transmission rates even when challenged with the highest viral dose, while both Ae. aegypti populations presented higher of infection, dissemination and transmission rates than Ae. albopictus. Ae. aegypti showed higher transmission efficiency when taking virus doses of 105 and 106 PFU/mL following incubation at 28°C; both Ae. aegypti and Ae. albopictus were unable to transmit ZIKV with virus doses of 102 and 103 PFU/mL, regardless the incubation temperature. Conclusions/Significance The ingested viral dose and incubation temperature were significant predictors of the proportion of mosquito’s biting becoming infectious. Ae. aegypti and Ae. albopictus have the ability to transmit ZIKV when incubated at 28°C. However Brazilian populations of Ae. aegypti exhibit a much higher transmission potential for ZIKV than Ae. albopictus regardless the combination of infection dose and incubation temperature., Author summary Zika virus is an arbovirus that has become endemic in Brazilian territory and in tropical and subtropical countries of the Americas since 2015. The virus is transmitted by Aedes mosquitoes. Aedes aegypti and Aedes albopictus are widespread in Brazil. To evaluated the influence of temperature and the effect of the infectious dose of ZIKV in vector competence, Brazilian populations of Ae. aegypti and Ae. albopictus were orally exposed to different infectious doses, distributed from 102 to 106 PFU / ml and incubated at 22°C and 28°C. We experimentally demonstrated that both populations of Ae. aegypti and Ae. albopictus have the ability to transmit ZIKV when incubated at 28°C, however the infectious dose strongly influenced the proportion of mosquitoes that were able to transmit the virus. Ae. albopictus populations showed low transmission rates when challenged with the highest viral dose, while Ae. aegypti populations are more susceptible, presenting high rates of infection, dissemination and transmission. When incubated at 22°C, Ae. albopictus populations were not able to transmit the virus. Combined, the results indicate that Brazilian populations of Ae. aegypti exhibit a much higher transmission potential for ZIKV than Ae. albopictus.