We report the first use of near-infrared spectroscopy for rapid, low-cost, noninvasive detection of Zika virus in Aedes aegypti .

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. 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 (Na_V), 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. 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. 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.

(1) Background: The deployment of the bacterium Wolbachia to reduce arbovirus transmission is ongoing in several countries worldwide. When Wolbachia-carrying Aedes aegypti are released and established in the field, females may feed on dengue-infected hosts. The effects of simultaneous exposure on life-history traits of Ae. aegypti to Wolbachia wMel strain and dengue-1 virus DENV-1 remain unclear. (2) Methods: We monitored 4 groups (mosquitoes with either DENV-1 or Wolbachia, coinfected with DENV-1 and Wolbachia, as well as negative controls) to estimate Ae. aegypti survival, oviposition success, fecundity, collapsing and fertility of quiescent eggs for 12 weeks. (3) Results: Neither DENV-1 nor Wolbachia had a significant impact on mosquito survival nor on mosquito fecundity, although the last parameter showed a tendency to decrease with ageing. There was a significant decrease in oviposition success in individuals carrying Wolbachia. Wolbachia infection and storage time significantly increased egg collapse parameter on the egg viability assay, while DENV-1 had a slight protective effect on the first four weeks of storage. (4) Conclusions: Despite limitations, our results contribute to better understanding of the tripartite interaction of virus, bacteria and mosquito that may take place in field conditions and aid in guaranteeing the Wolbachia strategy success.

The transmission of dengue (DENV) and Zika (ZIKV) has been continuously increasing worldwide. An efficient arbovirus surveillance system is critical to designing early-warning systems to increase preparedness of future outbreaks in endemic countries. The Near Infrared Spectroscopy (NIRS) is a promising high throughput technique to detect arbovirus infection in Ae. aegypti with remarkable advantages such as cost and time effectiveness, reagent-free, and non-invasive nature over existing molecular tools for similar purposes, enabling timely decision making through rapid detection of potential disease. Our aim was to determine whether NIRS can differentiate Ae. aegypti females infected with either ZIKV or DENV single infection, and those coinfected with ZIKV/DENV from uninfected ones. Using 200 Ae. aegypti females reared and infected in laboratory conditions, the training model differentiated mosquitoes into the four treatments with 100% accuracy. DENV-, ZIKV-, and ZIKV/DENV-coinfected mosquitoes that were used to validate the model could be correctly classified into their actual infection group with a predictive accuracy of 100%, 84%, and 80%, respectively. When compared with mosquitoes from the uninfected group, the three infected groups were predicted as belonging to the infected group with 100%, 97%, and 100% accuracy for DENV-infected, ZIKV-infected, and the co-infected group, respectively. Preliminary lab-based results are encouraging and indicate that NIRS should be tested in field settings to evaluate its potential role to monitor natural infection in field-caught mosquitoes.

Several countries have been using Wolbachia deployments to replace highly competent native Aedes aegypti populations with Wolbachia-carrying mosquitoes with lower susceptibility to arboviruses such as dengue, Zika, and chikungunya. In Rio de Janeiro, Wolbachia deployments started in 2015 and still present a moderate introgression with a modest reduction in dengue cases in humans (38%). Here, we evaluated the vector competence of wild-type and wMel-infected Ae. aegypti with a Brazilian genetic background to investigate whether virus leakage could contribute to the observed outcomes in Brazil. We collected the specimens in three areas of Rio de Janeiro with distinct frequencies of mosquitoes with wMel strain and two areas with wild Ae. aegypti. The mosquitoes were orally exposed to two titers of DENV-1 and the saliva of DENV-1-infected Ae. aegypti was microinjected into wMel-free mosquitoes to check their infectivity. When infected with the high DENV-1 titer, the presence of wMel did not avoid viral infection in mosquitoes’ bodies and saliva but DENV-1-infected wMel mosquitoes produced lower viral loads than wMel-free mosquitoes. On the other hand, wMel mosquitoes infected with the low DENV-1 titer were less susceptible to virus infection than wMel-free mosquitoes, although once infected, wMel and wMel-free mosquitoes exhibited similar viral loads in the body and the saliva. Our results showed viral leakage in 60% of the saliva of wMel mosquitoes with Brazilian background; thus, sustained surveillance is imperative to monitor the presence of other circulating DENV-1 strains capable of overcoming the Wolbachia blocking phenotype, enabling timely implementation of action plans.

The deployment of the Wolbachia w Mel strain is currently underway in multiple dengue-endemic municipalities across Brazil. The efficacy of this strategy in Rio de Janeiro remains uncertain, primarily due to the difficulty in sustaining high w Mel prevalence in regions previously subjected to large-scale releases. A key contributing factor was the routine rotation of insecticides within the framework of Integrated Vector Management (IVM), which led to the use of the larvicide Spinosad for Aedes control in urban areas. This compound was associated with a precipitous decline in Aedes aegypti mosquitoes, regardless the w Mel infection status. While w Mel-uninfected population recovered within weeks, w Mel-infected population remained at low levels likely due to the fitness costs imposed by w Mel on egg viability. To assess the long-term persistence of wMel following this demographic collapse, we conducted mosquito sampling across 12 neighborhoods in Rio de Janeiro, 30 months after mosquito populations crashed. Our findings reveal that w Mel introgression remains suboptimal, with a mean frequency of 9.87% across the sampled areas. Only two neighborhoods exhibited wMel frequencies exceeding 15%, likely reflecting ongoing localized releases. The reduced prevalence underscores the challenges of achieving self-sustaining w Mel establishment in complex urban environments and highlight critical considerations for the implementation of Wolbachia -based dengue control programs in endemic regions. Sponsorship: This study was supported by grants from the CNPq – Conselho Nacional de Desenvolvimento Científico e Tecnológico (312282/2022-2, 307209/2023-7) and by FAPERJ – Fundação Carlos Chagas Filho de Amparo a Pesquisa no Estado do Rio de Janeiro (E-26/211.159/2019, E-26/204.108/2024, E26/2001.844/2017, E-26/210.335/2022, E-26/210.537/2024).