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This work reviews and discusses how a deeper knowledge of mosquito time‐allocation, particularly resting periods, can enhance the effectiveness and deployment of classical vector control tools. As more high‐resolution data are gathered by novel tracking devices, follow‐up questions investigating mosquito behaviour are likely to arise, requiring the development of comprehensive frameworks to properly collect, summarize and analyse them. We demonstrate how novel stereoscopic 3D mapping tools can generate unique data and discuss available metrics to adequately quantify mosquito behaviour particularly under (semi‐) field conditions. Incorporation of mosquito 3D tracking with ecological, evolutionary and quantitative genetics approaches would enable a deeper understanding of the physiological and genetic basis driving resting behaviours. The ability to better determine when, where and how mosquitoes are allocating resting periods might constitute a major input to the advancement of control strategies. o Este trabajo repasa y discute como un conociemiento más profundo de la repartición temporal de los mosquitos, en particular sus periodos de reposo, puede mejorar la eficacia y despliegue de herraminetas clásicas de control de vectores. A medida que se recopilan más datos de alta resolución mediante nuevos dispositivos de rastreo, es probable que nuevas preguntas de seguimiento acerca del comportamiento de los mosquitos surjan, lo que requerirá el desarollo de marcos integrales para recopilarlos, resumirlos, y analizarlos adecuadamente. Demonstramos cómo nuevas herramientas de mapeo estereoscópico en 3D pueden generar datos únicos y discutimos las diferentes métricas disponibles para cuantificar adecuadamente el comportamiento de los mosquitos, particularmente bajo condiciones de (semi)campo. La incorporación del rastreo 3D de mosquitos a enfoques ecológicos, evolutivos, y de genética cuantitativa permitíra una comprensión más profunda de las bases fisiológicas y genéticas que regulan los comportamientos de reposo. La capacidad de determinar con mayor precisión cuándo, dónde, y cómo los mosquitos reparten sus periodos de reposo podría constituir un aporte clave para el desarollo de estragegias de control.

Aedes-borne viruses (ABVs) such as dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV) contribute significantly to the global burden of infectious diseases, disproportionately affecting disadvantaged populations from tropical and subtropical urban areas. ABVs can be transmitted from female mosquitoes to their progeny by vertical transmission via transovarial and/or trans-egg vertical transmission and contribute to the maintenance of infected-mosquito populations year-round in endemic regions. This study describes the natural infection rate of DENV, CHIKV, and ZIKV in field-caught male Aedes (Sergentomyia) aegypti (Linnaeus) mosquitoes from Mérida, Yucatán, México, as a proxy for the occurrence of vertical virus transmission. We used indoor sequential sampling with Prokopack aspirators to collect all mosquitoes inside houses from ABV hotspots areas. Collections were performed in a DENV and CHIKV post-epidemic phase and during a period of active ZIKV transmission. We individually RT-qPCR tested all indoor collected Ae. aegypti males (1,278) followed by Sanger sequencing analysis for final confirmation. A total of 6.7% male mosquitoes were positive for ABV (CHIKV = 5.7%; DENV = 0.9%; ZIKV = 0.1%) and came from 21.0% (30/143) houses infested with males. Most ABV-positive male mosquitoes were positive for CHIKV (84.8%). The distribution of ABV-positive Ae. aegypti males was aggregated in a few households, with two houses having 11 ABV-positive males each. We found a positive association between ABV-positive males and females per house. These findings suggested the occurrence of vertical arbovirus transmission within the mosquito populations in an ABV-endemic area and, a mechanism contributing to viral maintenance and virus re-emergence among humans in post-epidemic periods. Graphical Abstract

There is an increased need to mitigate the emergence of insecticide resistance and incorporate new formulations and modes of application to control the urban vector Aedes aegypti. Most research and development of insecticide formulations for the control of Ae. aegypti has focused on their peridomestic use as truck-mounted ULV-sprays or thermal fogs despite the widespread knowledge that most resting Ae. aegypti are found indoors. A recent modification of indoor residual spraying (IRS), termed targeted IRS (TIRS) works by restricting applications to 1.5 m down to the floor and on key Ae. aegypti resting sites (under furniture). TIRS also opens the possibility of evaluating novel residual insecticide formulations currently being developed for malaria IRS. We evaluated the residual efficacy of chlorfenapyr, formulated as Sylando 240SC, for 12 months on free-flying field-derived pyrethroid-resistant Ae. aegypti using a novel experimental house design in Merida, Mexico. On a monthly basis, 600 female Ae. aegypti were released into the houses and left indoors with access to sugar solution for 24 hours. After the exposure period, dead and alive mosquitoes were counted in houses treated with chlorfenapyr as well as untreated control houses to calculate 24-h mortality. An evaluation for these exposed cohorts of surviving mosquitoes was extended up to seven days under laboratory conditions to quantify \"delayed mortality\". Mean acute (24-h) mortality of pyrethroid-resistant Ae. aegypti ranged 80-97% over 5 months, dropping below 30% after 7 months post-TIRS. If delayed mortality was considered (quantifying mosquito mortality up to 7 days after exposure), residual efficacy was above 90% for up to 7 months post-TIRS application. Generalized Additive Mixed Models quantified a residual efficacy of chlorfenapyr of 225 days (ca. 7.5 months). Chlorfenapyr represents a new option for TIRS control of Ae. aegypti in urban areas, providing a highly-effective time of protection against indoor Ae. aegypti females of up to 7 months.

Aedes (Stegomyia) albopictus (Skuse), a mosquito vector with increasing capacity to transmit human arboviral infections worldwide, naturally harbors the intracellular bacterium Wolbachia spp. This relationship has been observed in native mosquito populations of Ae. albopictus around the world and, more recently, in Mérida, Yucatán, México (abbreviated hereafter as MID). This study provides additional evidence confirming the presence of Wolbachia A (wAlbA) and B (wAlbB) strains in field-collected Ae. albopictus of Mérida, based on wsp gene DNA sequencing analyses of 14 positive PCR samples. Maximum likelihood (ML) analyses of the wsp gene showed high nucleotide sequence homology between Wolbachia from Merida and the globally reported strains A and B, suggesting that these two Wolbachia strains have remained stable in Ae. albopictus over time, regardless of the geographical area. To the best of our knowledge, this is the first report of Wolbachia gene sequencing in native populations of Ae. albopictus in Yucatán, Mexico. Currently many Aedes mosquitoes (e.g., Ae. aegypti) harboring intracellular Wolbachia B bacteria are being released as part of arbovirus and mosquito control programs in Yucatan and globally. Understanding the presence and stability of Wolbachia strains across different Aedes host backgrounds is crucial to ensuring the long-term effectiveness of biological interventions aimed at controlling mosquito populations and arbovirus transmission in endemic areas.

Insecticide-based approaches remain a key pillar for Aedes -borne virus (ABV, dengue, chikungunya, Zika) control, yet they are challenged by the limited effect of traditional outdoor insecticide campaigns responding to reported arboviral cases and by the emergence of insecticide resistance in mosquitoes. A three-arm Phase II unblinded entomological cluster randomized trial was conducted in Merida, Yucatan State, Mexico, to quantify the entomological impact of targeted indoor residual spraying (TIRS, application of residual insecticides in Ae. aegypti indoor resting sites) applied preventively 2 months before the beginning of the arbovirus transmission season. Trial arms involved the use of two insecticides with unrelated modes of action (Actellic 300CS, pirimiphos-methyl, and SumiShield 50WG, clothianidin) and a control arm where TIRS was not applied. Entomological impact was quantified by Prokopack adult collections performed indoors during 10 min per house. Regardless of the insecticide, conducting a preventive TIRS application led to significant reductions in indoor Ae. aegypti densities, which were maintained at the same levels as in the low arbovirus transmission period (Actellic 300CS reduced Ae. aegypti density up to 8 months, whereas SumiShield 50WG up to 6 months). The proportional reduction in Ae. aegypti abundance in treatment houses compared to control houses was 50–70% for Actellic 300CS and 43–63% for SumiShield 50WG. Total operational costs including insecticide ranged from US$4.2 to US$10.5 per house, depending on the insecticide cost. Conducting preventive residual insecticide applications can maintain Ae. aegypti densities at low levels year-round with important implications for preventing ABVs in the Americas and beyond.

Background Dengue is a major public health problem in Mexico, where the use of chemical insecticides to control the principal dengue vector, Aedes aegypti , is widespread. Resistance to insecticides has been reported in multiple sites, and the frequency of kdr mutations associated with pyrethroid resistance has increased rapidly in recent years. In the present study, we characterized patterns of insecticide resistance in Ae. aegypti populations in five small towns surrounding the city of Merida, Mexico. Methods A cross-sectional, entomological survey was performed between June and August 2013 in 250 houses in each of the five towns. Indoor resting adult mosquitoes were collected in all houses and four ovitraps were placed in each study block. CDC bottle bioassays were conducted using F 0 -F 2 individuals reared from the ovitraps and kdr allele (Ile1016 and Cys1534) frequencies were determined. Results High, but varying, levels of resistance to chorpyrifos-ethyl was detected in all study towns, complete susceptibility to bendiocarb in all except one town, and variations in resistance to deltamethrin between towns, ranging from 63–88 % mortality. Significant associations were detected between deltamethrin resistance and the presence of both kdr alleles. Phenotypic resistance was highly predictive of the presence of both alleles, however, not all mosquitoes containing a mutant allele were phenotypically resistant. An analysis of genotypic differentiation (exact G test) between the five towns based on the adult female Ae. aegypti collected from inside houses showed highly significant differences ( p  < 0.0001) between genotypes for both loci. When this was further analyzed to look for fine scale differences at the block level within towns, genotypic differentiation was significant for both loci in San Lorenzo (Ile1016, p  = 0.018 and Cys1534, p  = 0.007) and for Ile1016 in Acanceh ( p  = 0.013) and Conkal ( p  = 0.031). Conclusions The results from this study suggest that 3 years after switching chemical groups, deltamethrin resistance and a high frequency of kdr alleles persisted in Ae. aegypti populations. The spatial variation that was detected in both resistance phenotypes and genotypes has practical implications, both for vector control operations as well as insecticide resistance management strategies.

Spatial emanators (SE) are innovative tools for controlling indoor Aedes aegypti due to their relatively easy use and high efficacy. Large-scale implementation challenges include community adoption, particularly ensuring proper installation and timely replacement as SE efficacy wanes. We conducted a three-arm, open-label entomological cluster randomized controlled trial with a crossover design, involving 588 households, to assess the entomological effect of the community use of metofluthrin emanators. Arms were: \"no treatment\"; \"community-led deployment\" (CD), where the households were responsible for installing and replacing SE with minimal guidance; and \"managed deployment\" (MD), where the research team handled SE installation and replacement. Emanators were replaced every 3 weeks across four deployment cycles, followed by a crossover between the CD and MD arms. Indoor resting mosquitoes were collected using Prokopack aspirators, and human landing counts (HLCs) were conducted in a subset of 12 houses (4 by arm) at the first, fourth, fifth, and eighth SE replacement rounds. Values of each endpoint during all sampling periods were compared using generalized linear mixed effects models (GLMM), the coefficients of the best-fitting model estimated that SE intervention reduced the number of Ae. aegypti per house by 32.7% (95%CI = 16.2-46.0%) in the CD arm and 36.8% (21.1-49.3%) in the MD arm. HLCs accounted 74-94% efficacy (MD) and 35-79% (CD). The crossover analysis found no significant difference between periods and arms, demonstrating the community's ability to manage SE as effectively as research team, even without prior training. This trial suggests that safe, portable SE are suited to deployment by householders as a rapid response to local Aedes-borne disease outbreaks even in the presence of high pyrethroid resistance in the local Aedes population. In urban areas where effective coverage and resourcing is a challenge to control campaigns, community \"ownership\" of SE products may enhance the impact of insecticidal interventions.

With the recent occurrence of locally transmitted Aedes-borne viruses in the continental United States and Europe, and a lack of effective vaccines, new approaches to control Aedes aegypti and Aedes albopictus are needed. In sub-tropical urban settings in the US, Ae. albopictus is a dominant nuisance and arbovirus vector species. Unfortunately, the vector control toolbox against Ae. albopictus is not as well developed as for Ae. aegypti. Here, we evaluate the efficacy, longevity, and range of protectiveness of a novel passive metofluthrin emanator (10% active ingredient in a polyethylene mesh) against Ae. albopictus indoors and outdoors. Four studies were conducted comparing the presence of the metofluthrin emanator to a control lacking emanator with interest in quantifying efficacy by human landing counts. Studies evaluated the effect of an emanator at varying distances from one or more human volunteers indoors and outdoors. Efficacy of emanators over time since activation was also evaluated. Mixed-effects models determined that sitting in close proximity to an emanator reduced landings by 89.5% outdoors and by 74.6% indoors. The emanator was determined protective when located immediately next to a human volunteer outdoors but not uniformly protective when located further away. The emanator was protective at all tested distances from the device indoors. Mortality of mosquitoes exposed to metofluthrin emanators was ~2x higher than those who were not exposed in indoor conditions. Finally, a Generalized Additive Model determined that emanators used continuously outdoors lost their effect after 2.5 weeks and stopped inducing paralysis in mosquitoes after 3.8 weeks of use. We show strong and lasting efficacy of 10% metofluthrin emanators against field Ae. albopictus both in indoor and outdoor conditions. Metofluthrin emanators can protect people from Ae. albopictus bites, representing a viable option for reducing human-mosquito contacts at home and beyond.

Triatoma dimidiata, the main vector of Trypanosoma cruzi throughout South Mexico and Central America, infest domiciles and peridomestic ecotopes of rural and semi-rural communities. This study reports the effect of the residual application of the organophosphate pirimiphos-methyl in a microencapsulated formulation (Actellic 300CS) for the control of intradomiciliary and peridomestic T. dimidiata in the community of Tekik de Regil (hereafter Tekik) in Yucatan, Southeast Mexico. From March to October 2022, a two-arm, unblinded entomological trial was performed in Tekik. Timed Manual Collections (TMC) characterized house and peridomicile infestation by T. dimidiata prior (baseline) and after the residual spraying (RS) (post-intervention) of a microencapsulated formulation. A total of 120 premises were surveyed (60 positive and 60 negative for T. dimidiata), randomly allocated 1:1 to treatment (RS with Actellic 300CS) and control (no RS) arms. Monthly post-spraying entomological surveys (May-October) with TMC were carried out in a random sample of ten houses from each arm. We analyzed the association between the treatment and post-intervention infestations using chi-square contingency tables. The estimated efficacy of the intervention with the 95% Confidence Intervals (CI) was calculated with the efficacy formula, using the Odds Ratio (OD) calculated from a binomial Generalized Linear Mixed Model (GLMM) from the positive premises in the baseline survey and post-intervention, using time as a random effect. Domestic infestations post-intervention were only detected in the control group (2/60 houses, 3.3%). Cumulative peridomestic infestation was significantly higher in the control arm (31.7%; 19/60) compared to the treatment arm (11.7%; 7/60) (X2 = 0.007, p < 0.01). The cumulative 6-month estimated efficacy of the intervention (% reduction in treatment versus control arm) was 65% (95% CI: 14%-79%). A single application of Actellic 300CS reduced T. dimidiata infestations by more than 60% for up to 6 months and provides evidence of an alternative formulation suitable for triatomine control in 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.