A full understanding of adult vector ecology and behavior is vital in developing novel control strategies as well as optimizing existing tools. It is general knowledge that Ae. aegypti
adults prefer to rest in dark, damp locations in households, and are also attracted to black colors 
and, in fact, the development of oviposition, host-seeking and/or other adult traps are based on these observations 
. However, few standardized studies have been performed to quantify such behavioral patterns in an attempt to reduce adult mosquito densities inside homes, a site of disease transmission 
With current suggestions that sub-lethal chemical approaches to vector control (i.e. contact irritancy) may pose viable options to reduce disease 
, it is important to characterize minimal effective doses of irritant chemicals and the relationship between surface area coverage of these doses and the behavioral responses that they elicit (i.e., rapid escape from inside homes). Current adult vector control approaches such as insecticide treated bed nets, and/or clothing rely on human hosts as the attractant or bait to lure mosquitoes into contact with the treated material long enough to deliver the lethal dose of the insecticide 
. However, when relying on the treatment of resting sites, such as the interior house walls, to reduce man-vector contact through an irritant response, interaction of the vector with these treated surfaces is facultative. Untreated areas in the house or safe-sites may be available and/or preferred for resting 
thus minimizing the impact of the intervention. It is vital therefore, to understand the drivers of these resting preferences in order to exploit and maximize the effects of irritant chemicals on vector escape responses. Such strategies will guide development of cost-effective tools for the future.
The present study quantified the resting patterns of two Ae. aegypti female populations (THAI and PERU) under both chemical-free and treatment conditions using a simple laboratory assay. During the chemical-free baseline trials, several variables were evaluated to include material type (cotton and polyester), dark : light color surface area coverage (SAC), and fabric configuration (horizontal, vertical). Not surprisingly, results indicate that both mosquito strains were observed resting preferentially on dark versus light colored material against both material types. These patterns were consistent using both the vertical and horizontal configuration study designs. The magnitude of this response was measured as greater than expected proportions of resting observations on the dark material even at the 25% SAC ratio despite the availability of alternate resting sites, or other behavioral responses such as flight. Similar findings have been described during our experimental hut validation studies in Thailand [Thainchum et al. unpublished data] and Peru [Castro et al. unpublished data] where most Ae. aegypti preferred to rest on dark material rather than light, regardless of fabric type even at 25% and 50% SAC.
Although horizontal configuration enhanced resting on both dark cotton and polyester material strips in the current study, as well as under field conditions in experimental huts [Thainchum et al. unpublished data], no consistent preference was observed between upper and lower locations of the dark material within the laboratory assay chamber. This may be due to the relatively small size of the assay that created a spatial bias for the test system – i.e., the laboratory assay dimensions may have precluded substantial differences in height between upper and lower wall portions. However, similar observations have been made in our experimental hut studies where based on observations from upper and lower wall heights, greater proportions of female Aedes aegypti populations were observed resting on lower portions of the wall when exposed to cotton material whereas against polyester, upper wall portions were preferred (unpublished data).
An explanation for the variation in resting patterns between the two material types in the current study may include the variation in the microclimate within the test box. Previous studies under laboratory conditions have reported similar findings using Anopheles
mosquitoes in which they preferred to rest on lower portion of a test box that was cooler than that of the upper portion 
. Unfortunately, it is not possible to validate this theory using the datasets of the current study because environmental parameters were only measured from a central location inside the bioassay room rather than along the wall surfaces within the test box. Future experiments should integrate microclimate data to better understand behavioral responses.
The fact that cotton enhanced resting on the dark strips as compared to polyester indicates that: 1) the green color of the polyester did not provide as much contrast to the white background as the black color of the cotton; 2) the weave or texture of the cotton provides enhanced tactile cues; or 3) material-specific moisture absorption properties exist under the conditions in which the assay was conducted (i.e. cotton retains more moisture than polyester). When evaluating green cotton versus green polyester simultaneously, cotton still enhanced resting on the dark strips. This finding suggests that the differential resting preference observed between cotton and polyester may not be due to variations in color contrast between material types, but rather is the result of their texture and/or moisture absorption properties. Cotton exhibits greater moisture absorption than that of polyester 
. It is interesting to note that studies under field conditions in Thailand and Peru are also indicating an overall general decrease in resting when polyester is used versus cotton under chemical-free conditions [Thainchum et al. ; Castro et al. unpublished data]. Such information could be vital in optimizing various vector control tools and could be most beneficial for products designed to target attraction/resting behaviors.
Observations made within the treatment metal boxes during chemical trials indicate that, knockdown responses in all test assays were low (≤5%) even at high chemical dose and treatment area coverage (i.e., 75% and 100%). Low KD even at test doses higher than WHO recommended field application rate for alphacypermethrin (≈7 nm/cm2
) is probably due to a reduced resting on the treated material and consequentially an increase in proportion of mosquitoes flying (irritated/agitated). It must be noted that test populations were only exposed to the treated surfaces for a total of 10 min, well below the standard 1 hour used in toxicity assays 
. Also, as the THAI Ae. aegypti
strain has been characterized as pyrethroid tolerant and DDT resistant 
, it was expected that KD/mortality would be low in these test populations. More importantly, the THAI strain still exhibited a contact irritant response (indicated by increased flying) when exposed to both alphacypermethrin and DDT. These results indicate that sub-lethal approaches to vector control may be effective in resistance management.
Perhaps most important for operational significance is the observation that was made in the test chamber during chemical trials indicating that both mosquito strains continued to rest in greater proportions on dark chemical-treated material versus safe-sites (i.e. chemical-free light material, assay lids and floor) when any dose of either alphacypermethrin or DDT were used. Even under test conditions in which shifting to safe-sites were expected (i.e., 25% SAC), results show no consistent increase in resting counts on chemical-free material. As expected, however, when observations were compared between treatment and matched control assays, significantly fewer mosquitoes were observed resting overall on the dark material treated with chemical.
For all chemical evaluations, the proportion of mosquitoes observed flying was significantly increased in the treatment assay as compared to matched control regardless of the material type used, surface area coverage and configuration of the treated areas within the box. Again, these findings indicate that under current test conditions, Ae. aegypti
did not simply move to safe-sites (untreated areas) following contact with chemical-treated material but were clearly agitated as measured by an increased flight response. It is this contact irritant response that may elicit escape behavior from a treated space and can be exploited for reducing man-vector contact inside homes. Any residual chemical that is applied to indoor surfaces and has sufficiently strong irritant properties would potentially disrupt the normal resting and may affect the feeding pattern of a vector. These actions could consequently reduce vector – human contact because of rapid escape from inside human dwellings 
. Such a contact irritant response is well documented in previous field experimentation 
. While the designs were different in these studies, results from each indicate a rapid escape of mosquitoes from inside experimental huts in response to irritant chemical applications and is the basis for the current laboratory conclusion. The challenge is to ensure that agitation, observed in the current study, does not increase biting on humans prior to escape as this would be counterproductive to intervention impact. Ongoing laboratory studies using the box assay are evaluating escape responses under similar current test conditions to measure the effect of focal treatment on mosquito movement away from a treatment source.
It should be noted that it was not the aim of the current study to compare resting behavior patterns between THAI versus PERU Ae. aegypti strains. Each strain was evaluated independently as results from each are currently being validated under field conditions at strain-specific locales (i.e, Kanchanaburi and Iquitos, respectively). However, future studies could investigate the relationship between behavioral phenotype and genetic characteristics of each geographical strain to explore differences that may exist in the resting behavior in response to chemical actions. This information would be useful in understanding the varying challenges in successful implementation of sub-lethal vector control strategies designed to have impact on mosquito populations from different geographic locations.
In summary, results from the current study indicate that both strains of Ae. aegypti preferred to rest on dark versus light-colored surfaces during both chemical-free and treated assays, and that agitation (i.e., flight response) was elicited under chemical conditions rather than an increase in resting on untreated safe-sites, even at the lowest 25% D:L coverage. To our knowledge, this is the first attempt to quantify resting responses to sub-lethal doses of irritant chemicals at different treatment surface area coverage. A similar concept of using minimum chemical dose and coverage is also being applied to measure the spatial repellency actions of chemicals to prevent mosquito entry into homes. Pertinent to the larger Push-Pull project under evaluation, laboratory observations have identified those variables that may have the greatest effect in eliciting an escape response following tarsal contact with a chemical-treated surface under experimental conditions. These factors include which material (cotton versus polyester), and configuration (horizontal versus vertical) result in the highest resting response and thereby initiate flight when treated with chemical. Although encouraging, it is the increase in flying that needs to be optimized and to elicit this response in such a way as to minimize opportunities for biting humans. Quantifying vector avoidance of an irritant chemical, through observations of the resting response on untreated and treated surfaces, has been a vital initial component in estimating the likelihood of success of a contact irritant Push-Pull strategy, especially one focused on the use of minimal treatment coverage area. Findings in the current study, together with ongoing field validation, indicate such an approach could be successful.