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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: Aedes albopictus, the Asian tiger mosquito, is an exotic invasive species in Europe. It has substantial public health relevance due to its potential role in transmitting several human pathogens. Out of the European coun‑ tries, Spain has one of the highest risk levels of autochthonous arbovirus transmission due to both the high density of Ae. albopictus and the extensive tourist infux from vector-endemic areas. This study aims to investigate the suscep‑ tibility of fve Ae. albopictus populations from mainland Spain and the Balearic Islands to a Brazilian Zika virus (ZIKV) strain. Methods: The F1 generation of each Ae. albopictus population was orally challenged with a ZIKV-infected blood meal (1.8×106 PFU/ml). At 7 and 14 days post-infection (dpi), mosquito bodies (thorax and abdomen) and heads were individually analysed through RT-qPCR to determine the infection rate (IR) and dissemination rate (DR), respectively. The saliva of infected mosquitoes was inoculated in Vero cells and the transmission rate was assessed by plaque assay or RT-qPCR on ~33 individuals per population. Results: The IR and DR ranged between 12–88%, and 0–60%, respectively, suggesting that ZIKV is capable of cross‑ ing the midgut barrier. Remarkably, no infectious viral particle was found in saliva samples, indicating a low ability of ZIKV to overcome the salivary gland barrier. A subsequent assay revealed that a second non-infective blood meal 48 h after ZIKV exposure did not infuence Ae. albopictus vector competence. Conclusions: The oral experimental ZIKV infections performed here indicate that Ae. albopictus from Spain become infected and disseminate the virus through the body but has a limited ability to transmit the Brazilian ZIKV strain through biting. Therefore, the results suggest a limited risk of autochthonous ZIKV transmission in Spain by Ae. albopictus info:eu-repo/semantics/publishedVersion

A Zika virus (ZIKV) pandemic started soon after the first autochthonous cases in Latin America. Although Aedes aegypti is pointed as the primary vector in Latin America, little is known about the fitness cost due to ZIKV infection. We investigated the effects of ZIKV infection on the life-history traits of Ae. aegypti females collected in three districts of Rio de Janeiro, Brazil (Barra, Deodoro, and Porto), equidistant ~25 km each other. Aedes aegypti mosquitoes were classified into infected (a single oral challenge with ZIKV) and superinfected (two ZIKV-infected blood meals spaced by 7 days each other). ZIKV infection reduced Ae. aegypti survival in two of the three populations tested, and superinfection produced a sharper increase in mortality in one of those populations. We hypothesized higher mortality with the presence of more ZIKV copies in Ae. aegypti females from Porto. The number of eggs laid per clutch was statistically similar between vector populations and infected and uninfected mosquitoes. Infection by ZIKV not affected female oviposition success. ZIKV infection impacted Ae. aegypti vectorial capacity by reducing its lifespan, although female fecundity remained unaltered. The outcome of these findings to disease transmission intensity still needs further evaluation.

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.

Malaria transmission in South America is overwhelmingly located in the Amazon region with limited cases outside that biome. A key factor in the mitigation of malaria transmission is the determination of vector diversity and bionomics in endemic areas. Anopheles mosquitoes were collected in four different landscapes of Cruzeiro do Sul-Acre, the current area with highest malaria transmission in Brazil. We performed adult mosquito collections every three months over two years and associated vector occurrence with local abiotic factors. A total of 1,754 Anopheles belonging to nine species were collected, but only four of them (An. albitarsis s.l. Lynch-Arribalzaga, An. braziliensis Chagas, An. peryassui Dyar and Knab, and An. triannulatus Neiva and Pinto) represented 77.1% of the total. Vector density and diversity was uneven across field sites and collection periods. Higher Anopheles abundance (54.8%) and richness were observed in a deforested palm tree area (IFC), with An. braziliensis the most frequent mosquito (40.5%). Only 7.3% of mosquitoes were collected in the SAB village, but 66.4% of them were An. darlingi and An. oswaldoi, species often regarded as primary and secondary vectors of malaria in the Amazon region. A distinct biting preference was observed between 18:00-19:40. The distance from the nearest breeding site and minimum temperature explained 41.6% of the Anopheles community composition. Our data show that the Anopheles species composition may present great variation on a microgeographic scale.

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.

Background Field ecologists often rely on mark-release-recapture (MRR) experiments to estimate population dynamics parameters for a given species. In the case of a medically important taxon, i.e., a disease vector, inferences on species survival and dispersal rates are particularly important as they have the potential to provide insights into disease transmission dynamics in endemic areas. Medical entomologists have traditionally used fluorescent dusts to externally mark the cuticle of insects. However, dust marking is usually restricted to the adult life stage because immature insects lose the mark when they molt. Methodology/Principal Findings We evaluated the efficacy of 13 trace elements in marking nymphs of three native Brazilian Chagas disease vectors: Triatoma brasiliensis, Triatoma pseudomaculata, and Rhodnius nasutus. Cr and Cu were detected in over 97% of T. brasiliensis (34/35 31/31 for Cr and Cu), while Cu and Mn were detected in more than 95% of T. pseudomaculata (29/29 for Cu and 28/29 for Mn) tested 120 days after marking. Only Mn marked over 90% of R. nasutus nymphs (38/41). Overall, trace elements had no negative effects on T. pseudomaculata longevity, but As-marked T. brasiliensis nymphs (p, Author Summary Mark, release and recapture (MRR) is one of the most powerful techniques employed by field ecologists, and is used to provide relevant estimates of natural populations such as dispersal, survival rates, and population densities. A reliable, persistent and effective marking method is a sine qua non condition to conduct a MRR experiment. Despite their relevance as Chagas disease vectors in the Americas, triatomine bugs have seldom been subjected to MRR studies. So far, estimates on triatomine dispersal have largely relied on molecular/genetic approaches, which are not direct measurements of the distance between the release and recapture point. We evaluated the persistence and efficiency of trace elements, chemicals present in very low quantities in the organism of a targeted species, to mark the nymphs of three Chagas disease vectors of the Brazilian caatinga biome, Triatoma brasiliensis, T. pseudomaculata, and Rhodnius nasutus. At least one trace element was shown to be a good marker for each triatomine species: more than 97% of T. brasiliensis were still marked with Cr and Cu after 120 days; more than 95% of T. pseudomaculata were marked with Cu and Mn and more than 90% of R. nasutus were marked with Mn 120 days after marking. Only As-marked T. brasiliensis and Cd-marked R. nasutus nymphs had significantly shorter lifespan. We believe these findings show that trace elements are a reliable and persistent marking method for triatomine nymphs and hope this is the first step towards the development of meaningful ecological estimates through the application of the MRR methodology.

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.

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.