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Over half the world's population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito-human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = -9.11) and 12.4% (95% CI 4.2%, ∞); z = -2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Vector-borne diseases are among the most burdensome infectious diseases worldwide with high burden to health systems in developing regions in the tropics. For many of these diseases, vector control to reduce human biting rates or arthropod populations remains the primary strategy for prevention. New vector control interventions intended to be marketed through public health channels must be assessed by the World Health Organization for public health value using data generated from large-scale trials integrating epidemiological endpoints of human health impact. Such phase III trials typically follow large numbers of study subjects to meet necessary power requirements for detecting significant differences between treatment arms, thereby generating substantive and complex datasets. Data is often gathered directly in the field, in resource-poor settings, leading to challenges in efficient data reporting and/or quality assurance. With advancing technology, mobile data collection (MDC) systems have been implemented in many studies to overcome these challenges. Here we describe the development and implementation of a MDC system during a randomized-cluster, placebo-controlled clinical trial evaluating the protective efficacy of a spatial repellent intervention in reducing human infection with Aedes-borne viruses (ABV) in the urban setting of Iquitos, Peru, as well as the data management system that supported it. We discuss the benefits, remaining capacity gaps and the key lessons learned from using a MDC system in this context in detail.

BACKGROUND:Insecticides used against Aedes aegypti and other disease vectors can elicit a multitude of dose-dependent effects on behavioral and bionomic traits. Estimating the potential epidemiological impact of a product requires thorough understanding of these effects and their interplay at different dosages. Volatile spatial repellent (SR) products come with an additional layer of complexity due to the potential for altered movement of affected mosquitoes and diffusion of volatile particles of the product beyond the treated house. Here, we propose a paired experimental design and statistical inference framework for estimating these nuanced effects of volatile SRs. METHOD:We fitted a continuous-time Markov chain model in a Bayesian framework to data on marked mosquitoes released in interconnected experimental huts conducted in Iquitos, Peru. We estimated the effects of two dosages of transfluthrin on Ae. aegypti behaviors associated with human-vector contact: repellency, exiting and knockdown in the treated space and in connected, adjacent huts. We validated the framework using simulated data. RESULTS:The odds of a female Ae. aegypti being repelled, and thus prevented from entering a treated hut (HT), increased at both dosages (low dosage: odds = 1.64, 95% highest density interval (HDI) = 1.30-2.09; high dosage: odds = 1.35, HDI = 1.04-1.67). The relative risk of exiting from the treated hut was reduced (low: RR = 0.70, HDI = 0.62-1.09; high: RR = 0.70, HDI = 0.40-1.06), with this effect carrying over to untreated spaces two huts away from the treated hut (H2) (low: RR = 0.79, HDI = 0.59-1.01; high: RR = 0.66, HDI = 0.50-0.87). Knockdown rates were increased in both treated and downstream huts, particularly under high dosage (HT: RR = 8.37, HDI = 2.11-17.35; H1: RR = 1.39, HDI = 0.52-2.69; H2: RR = 2.22, HDI = 0.96-3.86). CONCLUSIONS:Our framework is effective at elucidating multiple effects of volatile chemicals used in SR products, as well as their downstream effects. For the examined formulations of transfluthrin, we found notable dose-dependent effects on repellency, movement and knockdown that carry over to adjacent, untreated spaces.

Vismia gracilis extracts were tested against Aedes aegypti to assess mortality and behavioural effects. The leaf hexanic extract (L-Hex) presented increased larvicidal activity with exposure period: LC50 46.48 µg/mL (24 h) and LC50 20.57 µg/mL (48 h). Eight compounds were annotated/isolated from the L-Hex active extract, 4 benzophenones and 4 anthraquinones. Considering chemometric findings, the benzophenone moiety, tested as the commercial benzophenone, promoted larval mortality (LC50 16.35 µg/mL). Both the L-Hex extract and benzophenone induced intestinal damage in larvae. Benzophenone also promoted toxicity and behavioural effects in female adults. These findings highlighted the potential use of this class of compounds for developing vector-control products. [ABSTRACT FROM AUTHOR]

Mosquitoes are important vectors that transmit pathogens to human and other vertebrates. Each mosquito species has specific ecological requirements and bionomic traits that impact human exposure to mosquito bites, and hence disease transmission and vector control. A study of human biting mosquitoes and their bionomic characteristics was conducted in West Sumba and Southwest Sumba Districts, Nusa Tenggara Timur Province, Indonesia from May 2015 to April 2018. Biweekly human landing catches (HLC) of night biting mosquitoes both indoors and outdoors caught a total of 73,507 mosquito specimens (59.7% non-Anopheles, 40.3% Anopheles). A minimum of 22 Culicinae species belonging to four genera (Aedes, Armigeres, Culex, Mansonia), and 13 Anophelinae species were identified. Culex quinquefasciatus was the dominant Culicinae species, Anopheles aconitus was the principal Anopheles species inland, while An. sundaicus was dominant closer to the coast. The overall human biting rate (HBR) was 10.548 bites per person per night (bpn) indoors and 10.551 bpn outdoors. Mosquitoes biting rates were slightly higher indoors for all genera with the exception of Anopheles, where biting rates were slightly higher outdoors. Diurnal and crepuscular Aedes and Armigeres demonstrated declining biting rates throughout the night while Culex and Anopheles biting rates peaked before midnight and then declined. Both anopheline and non-anopheline populations did not have a significant association with temperature (p = 0.3 and 0.88 respectively), or rainfall (p = 0.13 and 0.57 respectively). The point distribution of HBR and seasonal variables did not have a linear correlation. Data demonstrated similar mosquito–human interactions occurring outdoors and indoors and during early parts of the night implying both indoor and outdoor disease transmission potential in the area–pointing to the need for interventions in both spaces. Integrated vector analysis frameworks may enable better surveillance, monitoring and evaluation strategies for multiple diseases. Author summary: This study outlines the array of mosquitoes that bite humans at night on the island of Sumba, Indonesia, with data on behavioural traits that impact when and where disease transmission may occur. Biweekly human landing catches (HLCs) were performed in four selected houses in 12 clusters (villages) from sunset to sunrise over a three years period (May 2015 to April 2018). The collection and analysis of 73,507 mosquito specimens revealed the presence of various species of Anopheles, Aedes, Culex, Armigeres and Mansonia, that potentially transmit several diseases including malaria, filaria, dengue and other mosquito borne viral diseases. Even though these data represent only night-time collections, this represents a comprehensive geographic description and inventory of species, bionomics and temporal distribution of mosquitoes on the island of Sumba. Data demonstrate that the high diversity of species with associated diversity in behaviours results in mosquito-human contact occurring throughout the night and both indoors and outdoors–relevant to both disease transmission and intervention applicability. Vector specific behaviours are specifically relevant to intervention strategies for specific diseases. The use of molecular methods to determine and validate morphological identification of specimens resulted in the characterization of multiple novel sequences–indicating the presence of undescribed species, members of cryptic species complexes or species without molecular data. Species identification using molecular methods are essential towards determine vector species compositions–especially in areas where data is absent. Though the correlation between temperature, rainfall and HBR was not statistically significant, the presence of mosquito populations throughout the year allow for perennial transmission of mosquito-borne diseases. Overall, these findings represent baseline and novel data for Sumba and may be utilized to develop disease and vector-specific or integrated strategies that mitigate the transmission of mosquito borne diseases in Indonesia. [ABSTRACT FROM AUTHOR]

Aedes aegypti and Aedes albopictus vector dengue, chikungunya and Zika viruses. With both species expanding their global distributions at alarming rates, developing effective surveillance equipment is a continuing priority for public health researchers. Sound traps have been shown, in limited testing, to be highly species-specific when emitting a frequency corresponding to a female mosquito wingbeat. Determining male mosquito capture rates in sound traps based on lure frequencies in endemic settings is the next step for informed deployment of these surveillance tools. We field-evaluated Male Aedes Sound Traps (MASTs) set to either 450 Hz, 500 Hz, 550 Hz or 600 Hz for sampling Aedes aegypti and/or Aedes albopictus and compared catch rates to BG-Sentinel traps within Pacific (Madang, Papua New Guinea) and Latin American (Molas, Mexico and Orange Walk Town, Belize) locations. MASTs set to 450–550 Hz consistently caught male Ae. aegypti at rates comparable to BG-Sentinel traps in all locations. A peak in male Ae. albopictus captures in MASTs set at 550 Hz was observed, with the lowest mean abundance recorded in MASTs set to 450 Hz. While significantly higher abundances of male Culex were sampled in MASTs emitting lower relative frequencies in Molas, overall male Culex were captured in significantly lower abundances in the MASTs, relative to BG-Sentinel traps within all locations. Finally, significant differences in rates at which male Aedes and Culex were positively detected in trap-types per weekly collections were broadly consistent with trends in abundance data per trap-type. MASTs at 550 Hz effectively captured both male Ae. aegypti and Ae. albopictus while greatly reducing bycatch, especially male Culex, in locations where dengue transmission has occurred. This high species-specificity of the MAST not only reduces staff-time required to sort samples, but can also be exploited to develop an accurate smart-trap system—both outcomes potentially reducing public health program expenses. Author summary: The continued global expansion of Aedes aegypti and Aedes albopictus, which transmit viruses causing dengue and Zika, necessitates the further development of effective mosquito traps for monitoring and predicting transmission risk. Mosquito sound traps attract male mosquitoes by producing a frequency which mimics the wingbeat sound of female mosquitoes. As few insects will respond to such sounds, these traps can be highly species-specific, saving significant person-time to sort samples, potentially paving the way for more accurate smart-trap systems and reduced surveillance costs. To date, limited research has occurred within field-settings investigating the capture rates of male mosquitoes in sound traps set to different frequencies. To this point, we performed trials in dengue endemic environments to quantify capture rates of mosquitoes in Male Aedes Sound Traps set to different frequencies in Papua New Guinea, Mexico and Belize. We found that both male Ae. aegypti and Ae. albopictus responded positively to frequencies between 450 and 600 Hz. Additionally, male Culex mosquitoes were also caught, but at lower abundances in traps set to higher sound frequencies. These traps, when set to 550 Hz, effectively capture male Ae. aegypti or Ae. albopictus with limited bycatch. [ABSTRACT FROM AUTHOR]

Spatial repellents (SRs) reduce human-mosquito contact by preventing mosquito entrance into human-occupied spaces and interfering with host-seeking and blood-feeding. A new model to synthesize experimental data on the effects of transfluthrin on Aedes aegypti explores how SR effects interact to impact the epidemiology of diseases vectored by these mosquitoes. Our results indicate that the greatest impact on force of infection is expected to derive from the chemical's lethal effect but delayed biting and the negative effect this may have on the mosquito population could elicit substantial impact in the absence of lethality. The relative contributions of these effects depend on coverage, chemical dose, and housing density. We also demonstrate that, through an increase in the number of potentially mosquito bites, increased partial blood-feeding and reduced exiting may elicit adverse impacts, which could offset gains achieved by other effects. Our analysis demonstrates how small-scale experimental data can be leveraged to derive expectations of epidemiological impact of SRs deployed at larger scales. Author summary: Mosquito control strategies that reduce bites to humans through multiple, non-lethal modes of action may be important in controlling mosquito-borne diseases where insecticidal strategies are ineffective. Assessing how effective such tools are in reducing infections is not clear-cut due to the multiple ways these products affect mosquitoes' behavior and life cycle. We introduce a paired experimental and mathematical framework to analyze and combine data from experiments on the several effects of a transfluthrin formulation and assess its public health impact. We show that, while product-induced lethality accounts for the majority of the product's impact, delayed blood feeding can, through its negative impact on mosquito population sizes, elicit its own substantial impact. Adverse effects of increased partial blood-feeding and reduced exiting could offset gains achieved by other effects such delayed blood feeding and lethality. Our model offers a way of synthesizing the results of feasible experiments at small scales to assess public health impact at large scales. [ABSTRACT FROM AUTHOR]

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Background: Mosquito-borne viruses—such as Zika, chikungunya, dengue fever, and yellow fever, among others—are of global importance. Although vaccine development for prevention of mosquito-borne arbovirus infections has been a focus, mitigation strategies continue to rely on vector control. However, vector control has failed to prevent recent epidemics and arrest expanding geographic distribution of key arboviruses, such as dengue. As a consequence, there has been increasing necessity to further optimize current strategies within integrated approaches and advance development of alternative, innovative strategies for the control of mosquito-borne arboviruses. Methods and findings: This review, intended as a general overview, is one of a series being generated by the Worldwide Insecticide resistance Network (WIN). The alternative strategies discussed reflect those that are currently under evaluation for public health value by the World Health Organization (WHO) and represent strategies of focus by globally recognized public health stakeholders as potential insecticide resistance (IR)-mitigating strategies. Conditions where these alternative strategies could offer greatest public health value in consideration of mitigating IR will be dependent on the anticipated mechanism of action. Arguably, the most pressing need for endorsement of the strategies described here will be the epidemiological evidence of a public health impact. Conclusions: As the burden of mosquito-borne arboviruses, predominately those transmitted by Aedes aegypti and A. albopictus, continues to grow at a global scale, new vector-control tools and integrated strategies will be required to meet public health demands. Decisions regarding implementation of alternative strategies will depend on key ecoepidemiological parameters that each is intended to optimally impact toward driving down arbovirus transmission. [ABSTRACT FROM AUTHOR]

A mark–release–recapture study was conducted to refine the "push–pull" strategy for controlling the dengue, chikungunya, and Zika virus vector Aedes aegypti in a peridomestic environment by determining optimal locations and distances from human-occupied experimental huts for placement of the "pull" component (Biogents Sentinel™ [BGS] traps) to maximize the capture of mosquitoes. The BGS traps were placed at portals of entry (windows or doors) or corners of the experimental huts and at varying distances from the huts (0, 3, and 10 m). The location optimization trials revealed higher trap capture rates and reduction in entry of mosquitoes when the BGS traps were positioned nearer the experimental hut portals of entry than those placed in the corner of the huts. The trap capture rate at huts' portals of entry was 38.7% (116/300), while the corners recorded 23.7% (71/300). The percentage reduction in entry of mosquitoes into the huts was 69% and 31% at portals of entry and corners or vertices, respectively. In the distance optimization trials, the highest captures were recorded at 0 m (18.5%; 111/600) and 10 m (14.2%; 128/900) distances from the hut. Moreover, the highest percentage reduction in entry of mosquitoes into the huts occurred for traps set at 0 m (65.6%) compared with 3 m (17.2%) or 10 m (14.6%) distances from the huts. [ABSTRACT FROM AUTHOR]

The efficacy of the BG-Sentinel™ (BGS) trap as a 'pull' component of a 'push-pull' system (PPS) for management of the dengue vector, Aedes aegypti, was evaluated using local households in Pu Tuey, Kanchanaburi, Thailand. The pull component was the concluding phase of a 3-part investigation using a PPS combination spatial repellent (SR) and BGS trap to capture adult vector mosquitoes. Two sentinel households were selected for evaluation of BGS trap efficacy based on the highest pretrial indoor resting densities of Ae. aegypti using Centers for Disease Control and Prevention (CDC) mechanical backpack collections. Potential Ae. aegypti resting sites around the selected houses were identified as possible competing sites that might influence the BGS trap capture efficiency. Results showed that BGS traps were productive in capturing Ae. aegypti females (93.4% of all Aedes collected) in the presence of competing man-made, artificial resting sites. The CDC backpack aspirator collections provided an indirect measure of local Aedes population, although technically not comparable for supporting productivity of BGS traps due to different collection days and households sampled. The predominant competing resting sites were water containers found within 3 m around the outside of sentinel households. The most productive BGS collections between houses differed by location. The most productive period of operation for Ae. aegypti BGS trapping was between 1330 and 1730 h. The BGS trap appears an effective 'pull' device in the PPS strategy in natural settings. [ABSTRACT FROM AUTHOR]

Background: Aedes aegypti is a primary vector of dengue virus (DENV), the causative agent of dengue fever, an arthropod-borne disease of global importance. Although a vaccine has been recommended for prevention, current dengue prevention strategies rely on vector control. Recently, volatile pyrethroids—spatial repellents—have received interest as a novel delivery system for adult Ae. aegypti control. Understanding the full range of behavioral effects spatial repellents elicit in mosquito species will be critical to understanding the overall impact these products have on vector populations and will guide expectations of efficacy against DENV transmission. Methodology/Principal Findings: The current study quantified changes in attraction of gravid Ae. aegypti to experimental oviposition sites following exposure to the spatial repellent transfluthrin. Responses were measured with two-choice olfaction bioassays using ‘sticky-screens’ covering cups to prevent contact with the oviposition substrate. Two cups contained a bacterial attractant composed of four species of bacteria in calcium alginate beads in water and two cups contained only deionized water. Results from 40 replicates (n = 780 females total per treatment) indicated an estimated difference in attraction of 9.35% ± 0.18 (p ≤ 0.003), implying that the transfluthrin-exposed mosquitoes were more attracted to the experimental oviposition sites than the non-exposed mosquitoes. Conclusions/Significance: Findings from this study will further characterize the role of spatial repellents to modify Ae. aegypti behavior related to dengue prevention specifically, and encourage innovation in vector control product development more broadly. [ABSTRACT FROM AUTHOR]

The High Throughput Screening System (HITSS) has been applied in insecticide behavioral response studies with various mosquito species. In general, chemical or natural compounds can produce a range of insect responses: contact irritancy, spatial repellency, knock-down, and toxicity. This study characterized these actions in essential oils derived from citronella, hairy basil, catnip, and vetiver in comparison to DEET and picaridin against Aedes aegypti and Anopheles minimus mosquito populations. Results indicated the two mosquito species exhibited significantly different (P<0.05) contact irritant escape responses between treatment and control for all tested compound concentrations, except with the minimum dose of picaridin (P>0.05) against Ae. aegypti. Spatial repellency responses were elicited in both mosquito species when exposed to all compounds, but the strength of the repellent response was dependent on compound and concentration. Data show that higher test concentrations had greatest toxic effects on both mosquito populations, but vetiver had no toxic effect on Ae. aegypti and picaridin did not elicit toxicity in either Ae. aegypti or An. minimus at any test concentration. Ultimately, this study demonstrates the ability of the HITSS assay to guide selection of effective plant essential oils for repelling, irritating, and killing mosquitoes. [ABSTRACT FROM AUTHOR]

Background: New vector control paradigms expanding the use of spatial repellents are promising, but there are many gaps in our knowledge about how repellents work and how their long-term use might affect vector populations over time. Reported here are findings from a series of in vitro studies that investigated the plasticity and heritability of spatial repellent (SR) behaviors in Aedes aegypti exposed to airborne transfluthrin, including results that indicate a possible link between repellent insensitivity and insecticide resistance. Methodology/principal findings: A dual-choice chamber system was used to observe directional flight behaviors in Aedes aegypti mosquitoes exposed to passively emanating transfluthrin vapors (1.35 mg/m3). Individual SR responder and SR non-responder mosquitoes were identified, collected and maintained separately according to their observed phenotype. Subsequent testing included re-evaluation of behavioral responses in some mosquito cohorts as well as testing the progeny of selectively bred responder and non-responder mosquito strains through nine generations. At baseline (F0 generation), transfluthrin actively repelled mosquitoes in the assay system. F0 mosquitoes repelled upon initial exposure to transfluthrin vapors were no more likely to be repelled again by subsequent exposure 24h later, but repelled mosquitoes allowed to rest for 48h were subsequently repelled at a higher proportion than was observed at baseline. Selective breeding of SR responders for nine generations did not change the proportion of mosquitoes repelled in any generation. However, selective breeding of SR non-responders did produce, after four generations, a strain of mosquitoes that was insensitive to the SR activity of transfluthrin. Compared to the SR responder strain, the SR insensitive strain also demonstrated decreased susceptibility to transfluthrin toxicity in CDC bottle bioassays and a higher frequency of the V1016Ikdr mutation. Conclusions/significance: SR responses to volatile transfluthrin are complex behaviors with multiple determinants in Ae. aegypti. Results indicate a role for neurotoxic irritation of mosquitoes by sub-lethal doses of airborne chemical as a mechanism by which transfluthrin can produce SR behaviors in mosquitoes. Accordingly, how prolonged exposure to sub-lethal doses of volatile pyrethroids might impact insecticide resistance in natural vector populations, and how already resistant populations might respond to a given repellent in the field, are important considerations that warrant further monitoring and study. Results also highlight the critical need to develop new repellent active ingredients with novel mechanisms of action. Author Summary: There is growing evidence to support the expanded use of spatial repellents for vector control, but there are still many uncertainties about how repellents work and how their long term use may impact vector populations over time. Here, we conducted a series of in vitro experiments that investigated spatial repellent (SR) behaviors in Aedes aegypti mosquitoes exposed to airborne transfluthrin, a volatile pyrethroid commonly used in repellent products. We were able to show that repellent insensitivity is linked to reduced insecticide susceptibility and increased knock down resistance allele frequency, indicating that sub-lethal doses of airborne transfluthrin can elicit SR behaviors in mosquitoes by inducing an agitated state via neurotoxic pathways independent of olfactory stimulation. This raises questions about how the use of volatile pyrethroid repellents may impact insecticide resistance in target vectors over time, highlighting the need to further understand all of the physiological drivers of SR behaviors and emphasizing the requirement to develop new repellent active ingredients with novel, non-toxic mechanisms of action. [ABSTRACT FROM AUTHOR]

The behavioral responses of colony populations of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus to four essential oils (citronella, hairybasil, catnip, and vetiver), two standard repellents (DEET and picaridin), and two synthetic pyrethroids (deltamethrin and permethrin) were conducted in the laboratory using an excito-repellency test system. Results revealed that Cx. quinquefasciatus and An. minimus exhibited much stronger behavioral responses to all test compounds (65-98% escape for contact, 21.4-94.4% escape for non-contact) compared to Ae. aegypti (3.7-72.2% escape (contact), 0-31.7% (non-contact)) and Ae. albopictus (3.5-94.4% escape (contact), 11.2-63.7% (non-contact)). In brief, essential oil from vetiver elicited the greatest irritant responses in Cx. quinquefasciatus (96.6%) and An. minimus (96.5%) compared to the other compounds tested. The synthetic pyrethroids caused a stronger contact irritant response (65-97.8% escape) than non-contact repellents (0-50.8% escape for non-contact) across all four mosquito species. Picaridin had the least effect on all mosquito species. Findings from the current study continue to support the screening of essential oils from various plant sources for protective properties against field mosquitoes. [ABSTRACT FROM AUTHOR]

Background: An integrated approach to reduce densities of adult Aedes aegypti inside homes is currently being evaluated under experimentally controlled field conditions. The strategy combines a spatial repellent (SR) treatment (applied indoors) with the Biogents Sentinel™ (BGS) mosquito trap positioned in the outdoor environment. In essence, when combined, the goal is to create a push-pull mechanism that will reduce the probability of humanvector contact. The current study measured BGS recapture rates of Ae. aegypti test cohorts that were exposed to either SR or control (chemical-free) treatments within experimental huts. The objective was to define what, if any, negative impact SR may have on BGS trap efficacy (i.e., reduced BGS collection). Methods: Aedes aegypti females were exposed to SR compounds within experimental huts in the form of either treated fabric (DDT and transfluthrin) or mosquito coil (metofluthrin). Test cohorts were released within individual screen house cubicles, each containing 4 BGS traps, following SR exposure according to treatment. Two separate test cohorts were evaluated: (i) immediate release (IR) exposed from 06:00-12:00 hours and released at 12:00 hours and (ii) delayed release (DR) exposed from12:00-18:00 hours and released at 05:30 hours the following day. BGS recapture was monitored at 09:30, 13:30 and 15:30 hours and the cumulative recapture by time point quantified. Results: Exposure of Ae. aegypti females to either DDT or metofluthrin did not significantly impact BGS capture as compared to cohorts of non-exposed females. This was true for both IR and DR exposure populations. IR cohorts exposed to transfluthrin resulted in significantly lower BGS recapture compared to matched controls but this effect was primarily due to high mosquito mortality during transfluthrin trials. Conclusion: Our data indicate no more than minor and short-lived impacts (i.e., reduced attraction) on BGS trap catches following exposure to the pyrethroid compounds transfluthrin and metofluthrin and no change in recapture densities using DDT as compared to matched controls. These findings suggest a combined SR and BGS approach to vector control could function as a push-pull strategy to reduce Ae. aegypti adults in and around homes. [ABSTRACT FROM AUTHOR]

Background: Previous studies have demonstrated contact irritant and spatial repellent behaviors in Aedes aegypti following exposure to sublethal concentrations of chemicals. These sublethal actions are currently being evaluated in the development of a push-pull strategy for Ae. aegypti control. This study reports on mosquito escape responses after exposure to candidate chemicals for a contact irritant focused push-pull strategy using varying concentrations and focal application. Methods: Contact irritancy (escape) behavior, knockdown and 24 hour mortality rates were quantified in populations of female Ae. aegypti under laboratory conditions and validated in the field (Thailand and Peru) using experimental huts. Evaluations were conducted using varying concentrations and treatment surface area coverage (SAC) of three pyrethroid insecticides: alphacypermethrin, lambacyhalothrin and deltamethrin. Results: Under laboratory conditions, exposure of Ae. aegypti to alphacypermethrin using the standard field application rate (FAR) resulted in escape responses at 25% and 50% SAC that were comparable with escape responses at 100% SAC. Significant escape responses were also observed at <100% SAC using ½FAR of all test compounds. In most trials, KD and 24 hour mortality rates were higher in mosquitoes that did not escape than in those that escaped. In Thailand, field validation studies indicated an early time of exit (by four hours) and 40% increase in escape using ½FAR of alphacypermethrin at 75% SAC compared to a matched chemical-free control. In Peru, however, the maximum increase in Ae. aegypti escape from alphacypermethrin-treated huts was 11%. Conclusions/Significance: Results presented here suggest a potential role for sublethal and focal application of contact irritant chemicals in an Ae. aegypti push-pull strategy to reduce human–vector contact inside treated homes. However, the impact of an increase in escape response on dengue virus transmission is currently unknown and will depend on rate of biting on human hosts prior to house exiting. Author Summary: Dengue virus is spread by Aedes aegypti, a mosquito that prefers to feed on humans. Chemicals used at toxic levels is currently the only confirmed effective strategy for dengue vector control, but insecticide resistance issues are threatening this approach. Other chemical actions that break vector-human contact exist: contact irritant effects, causing escape from homes and spatial repellent effects, preventing house entry. These actions are being evaluated in the development of a push-pull strategy to target indoor resting sites (using contact irritants) and/or portals of house entry (using spatial repellents) at sublethal concentrations to push the vector away from the treated structure and associated human hosts. When combined with an attractant trap placed outdoors to pull the irritated/repelled vectors from the peridomestic environment, the integrated stimuli form a push-pull system that can be used to further enhance disruption of human-vector contact. Here we report on the quantification of contact irritancy (escape) behavior, knockdown and 24 hour mortality rates in populations of Ae. aegypti under both laboratory and field conditions. Evaluations were carried out against three pyrethroid insecticides. Findings indicate an increase in escape response using sublethal concentrations and focal application suggesting a potential role for contact irritants in a push-pull strategy. [ABSTRACT FROM AUTHOR]

We determined the feasibility of using the BG-Sentinel™ mosquito trap (BGS) as the pull component in a push-pull strategy to reduce indoor biting by Aedes aegypti. This included evaluating varying numbers of traps (1-4) and mosquito release numbers (10, 25, 50, 100, 150, 200, and 250) on recapture rates under screen house conditions. Based on these variations in trap and mosquito numbers, release intervals were rotated through a completely randomized design with environmental factors (temperature, relative humidity, and light intensity) and monitored throughout each experiment. Data from four sampling time points (05:30, 09:30, 13:30, and 17:30) indicate a recapture range among treatments of 66-98%. Furthermore, 2-3 traps were as effective in recapturing mosquitoes as 4 traps for all mosquito release numbers. Time trends indicate Day 1 (the day the mosquitoes were released) as the "impact period" for recapture with peak numbers of marked mosquitoes collected at 09:30 or 4 h post-release. Information from this study will be used to guide the configuration of the BGS trap component of a push-pull vector control strategy currently in the proof-of-concept stage of development in Thailand and Peru. [ABSTRACT FROM AUTHOR]

Background: Previous studies have identified the behavioral responses of Aedes aegypti to irritant and repellent chemicals that can be exploited to reduce man-vector contact. Maximum efficacy of interventions based on irritant chemical actions will, however, require full knowledge of variables that influence vector resting behavior and how untreated "safe sites" contribute to overall impact. Methods: Using a laboratory box assay, resting patterns of two population strains of female Ae. aegypti (THAI and PERU) were evaluated against two material types (cotton and polyester) at various dark:light surface area coverage (SAC) ratio and contrast configuration (horizontal and vertical) under chemical-free and treated conditions. Chemicals evaluated were alphacypermethrin and DDT at varying concentrations. Results: Under chemical-free conditions, dark material had significantly higher resting counts compared to light material at all SAC, and significantly increased when material was in horizontal configuration. Cotton elicited stronger response than polyester. Within the treatment assays, significantly higher resting counts were observed on chemical-treated dark material compared to untreated light fabric. However, compared to matched controls, significantly less resting observations were made on chemical-treated dark material overall. Most importantly, resting observations on untreated light material (or "safe sites") in the treatment assay did not significantly increase for many of the tests, even at 25% SAC. Knockdown rates were ≤5% for all assays. Significantly more observations of flying mosquitoes were made in test assays under chemical-treatment conditions as compared to controls. Conclusions/Significance: When preferred Ae. aegypti resting sites are treated with chemicals, even at reduced treatment coverage area, mosquitoes do not simply move to safe sites (untreated areas) following contact with the treated material. Instead, they become agitated, using increased flight as a proxy indicator. It is this contact irritant response that may elicit escape behavior from a treated space and is a focus of exploitation for reducing man-vector contact inside homes. Author Summary: Aedes aegypti, the primary vector mosquito of dengue virus, typically lives near or inside human dwellings, and feeds preferentially on humans. The control of this mosquito vector remains the most important dengue prevention method. The use of chemicals at levels toxic to mosquitoes is currently the only confirmed effective adult vector control strategy with interventions usually applied following epidemic onset. However, research indicates that sub-lethal chemical approaches to prevent human-vector contact at the house level exist: contact irritancy and spatial repellency. The optimum efficacy of an intervention based on contact irritant actions of chemicals will, however, require full knowledge of variables that will influence vector resting behavior and thereby chemical uptake from treated sources. Here we characterize the resting patterns of female Ae. aegypti on two material types at various dark:light surface area coverage ratios and contrast configurations under chemical-free and treated conditions using a laboratory behavioral assay. Change in resting behavior between baseline and treatment conditions was quantified to determine potential negative effects of untreated surfaces ("safe sites") when irritant responses are elicited. We show that treatment of preferred resting sites with known irritant compounds do not stimulate mosquitoes to move to safe sites after making contact with treated surfaces. [ABSTRACT FROM AUTHOR]

Insecticide toxicity is commonly evaluated for disease vectors by either the WHO test or Bottle assay. More recently, a high-throughput screening (HTS) system was developed for testing insecticide effects on mosquito behavior and mortality. We compared HTS with the Bottle assay to evaluate the toxicity of insecticides in a population of Aedes aegypti from Thailand. Both the HTS and Bottle assay system were determined to be equivalent. The two systems mainly differed (1) in reaction time, with mosquitoes reacting faster in the Bottle assay than HTS, (2) in knockdown and mortality at low doses. This information will guide the testing protocol for evaluating chemical effects on behavioral responses in various vector populations. [ABSTRACT FROM AUTHOR]

Mark-release-recapture experiments with Aedes aegypti were performed using experimental huts equipped with entrance and exit traps to evaluate their movement patterns during a two-year period in Thailand. Results indicate no significant differences in the patterns of movement between the two years of observation. Movement into the huts occurred during the early morning period (06:00-11:00) with a peak at 07:00 in the summer and rainy season and 09:00 in the winter. In contrast, the exit pattern was observed during the late morning (09:00-12:00) and early afternoon (12:00-16:00), with a peak at 16:00 in the winter, 11:00 in the summer, and 14:00 in the rainy season. Multiple regression analysis indicated that movements of Ae. aegypti females into and out of the huts were impacted by humidity and temperature during the day. [ABSTRACT FROM AUTHOR]

The main objective of this study was to find the optimal dosage of deltamethrin, cyphenothrin, d-tetramethrin, and tetramethrin that would elicit repellency and irritability responses of Aedes aegypti. The F1-F3 generations of field mosquitoes collected from Pu Teuy Village, Sai-Yok District, Kanchanaburi Province, Thailand, were tested with four pyrethroids to determine the LC25, LC50, and LC99. These concentrations were 0.010%, 0.020%, and 0.055%, respectively, for deltamethrin; 0.113%, 0.167%, and 0.353%, respectively, for cyphenothrin; 2.091%, 2.770%, and 5.114%, respectively, for d-tetramethrin; and 2.377%, 4.251%, and 10.715%, respectively, for tetramethrin. All dosages were tested in the excito-repellency system. Survival analysis was used to compare each chamber of the test. It was found that cyphenothrin had a stronger repellent effect than the other pyrethroids, while the contact irritant effect was similar among compounds tested. The LC50 of each pyrethroid was found to be the optimal dose for repelling Ae. aegypti. There was no significant difference in LC99 values for either non-contact or contact trials for each pyrethroid. [ABSTRACT FROM AUTHOR]

A modular and novel assay system for rapid mass screening of chemical compounds for contact irritant and spatial repellent actions against adult mosquitoes is described. The responses of Aedes aegypti to various concentrations of 3 topical repellents, deet, Bayrepel®, and SS220, were evaluated. At treatment concentrations ≥25 nmol/cm² of SS220, mosquitoes exhibited significant contact irritant (escape) and spatial repellent (movement away from the chemical source) responses, whereas, a 10-fold increase in the treatment concentration of deet and Bayrepel was required to produce similar responses. The novel bioassay system detected contact irritancy and spatial repellency activity with reproducible results and provided baseline data for determining minimum effective concentrations for other chemicals. The system is compact in size, easy to decontaminate, and requires only a minute quantity of chemical compound. [ABSTRACT FROM AUTHOR]

Background: Insecticides used against Aedes aegypti and other disease vectors can elicit a multitude of dose-dependent effects on behavioral and bionomic traits. Estimating the potential epidemiological impact of a product requires thorough understanding of these effects and their interplay at different dosages. Volatile spatial repellent (SR) products come with an additional layer of complexity due to the potential for altered movement of affected mosquitoes and diffusion of volatile particles of the product beyond the treated house. Here, we propose a paired experimental design and statistical inference framework for estimating these nuanced effects of volatile SRs. Method: We fitted a continuous-time Markov chain model in a Bayesian framework to data on marked mosquitoes released in interconnected experimental huts conducted in Iquitos, Peru. We estimated the effects of two dosages of transfluthrin on Ae. aegypti behaviors associated with human-vector contact: repellency, exiting and knockdown in the treated space and in connected, adjacent huts. We validated the framework using simulated data. Results: The odds of a female Ae. aegypti being repelled, and thus prevented from entering a treated hut (HT), increased at both dosages (low dosage: odds = 1.64, 95% highest density interval (HDI) = 1.30–2.09; high dosage: odds = 1.35, HDI = 1.04–1.67). The relative risk of exiting from the treated hut was reduced (low: RR = 0.70, HDI = 0.62–1.09; high: RR = 0.70, HDI = 0.40–1.06), with this effect carrying over to untreated spaces two huts away from the treated hut (H2) (low: RR = 0.79, HDI = 0.59–1.01; high: RR = 0.66, HDI = 0.50–0.87). Knockdown rates were increased in both treated and downstream huts, particularly under high dosage (HT: RR = 8.37, HDI = 2.11–17.35; H1: RR = 1.39, HDI = 0.52–2.69; H2: RR = 2.22, HDI = 0.96–3.86). Conclusions: Our framework is effective at elucidating multiple effects of volatile chemicals used in SR products, as well as their downstream effects. For the examined formulations of transfluthrin, we found notable dose-dependent effects on repellency, movement and knockdown that carry over to adjacent, untreated spaces. [ABSTRACT FROM AUTHOR]

Dengue fever is an acute disease caused by the dengue virus and transmitted primarily by the mosquito Aedes aegypti. The current strategy for dengue prevention is vector control including the use of topical repellents to reduce mosquito biting. Although N,N-diethyl-m-methylbenzamide (DEET) is the most common active ingredient in topical repellent products, para-menthane-3,8-diol (PMD) is also used commercially. Studies have indicated PMD reduced biting by 90–95% for up to 6–8 h, similar to the efficacy of DEET, depending on the testing environment. The purpose of this study was to evaluate the behavioral effects of PMD on Ae. aegypti blood feeding and fecundity to explore the potential impact of PMD on downstream mosquito life-history traits. Two experiments were performed. In both experiments, cohorts of female Ae. aegypti (Belize strain) were exposed to 20% PMD or ethanol for 10 min in a closed system and introduced to an artificial membrane feeding system. Following a 30min feed time, mosquitoes of Experiment 1 were killed and weighed as a proxy measure of blood meal, whereas mosquitoes of Experiment 2 were monitored for oviposition, a measure of fecundity. Results showed a statistically significant reduction (p < 0.001) in the percentage of Ae. aegypti that blood-fed when exposed to PMD (38%) compared to those non-exposed (49%). No significant difference in fecundity between test populations was indicated. These findings suggest that exposure of Ae. aegypti to 20% PMD may influence the probability of subsequent blood feeding but of those mosquitoes that do blood feed, egg-lay density is not affected. Further studies are warranted to investigate the full range of effects of PMD exposure on other Ae. aegypti life-history traits such as mating, to continue characterizing the potential effects of PMD to impact overall vector population dynamics. [ABSTRACT FROM AUTHOR]