One of the key issues in the development of novel chemical approaches for vector control – to include spatial repellency – is defining the underlying mechanism that results in the reduction of mosquito densities within a treated space and therefore prevention of human-vector contact. Both toxic (mortality) and sublethal (repellency) actions will produce a vector free space, however, one is due to a direct killing action while the other is not. If the use of spatial repellents for disease prevention is to be supported, and thereby drive screening paradigms for novel chemical modes of action , clear evidence must be presented that: 1) the concentration of a repellent AI in a treated air space is below toxic thresholds and 2) that sublethal air concentrations reduce human-vector contact to include measures of deterring vector entry into the treated space. In essence, correlating AI concentration with key entomological measures of disease impact (i.e., human-vector contact) will guide standardization for the evaluation of spatial repellent products . This study outlines initial steps in determining methodologies and study designs for achieving this goal.
Our initial air sampling studies conducted in the laboratory against DDT indicated a thermal desorption (TD) method could detect volatilized DDT within the air space of a mosquito behavior monitoring assay . Results from these experiments also showed that the TD method was able to detect a gradient of DDT air concentration among the three chambers (unpublished data). Based on the ability of the TD technique to detect and quantify DDT, as well as the relatively quick turn-around time for processing air samples, this technique was selected as one possible method for field validation in the current study. The second air sampling method employed here, the USEPA TO-10A, uses a chemical rather than heat desorption process to collect AIs from a set volume of air and has been validated for the atmospheric determination of pyrethrins and the pyrethroids d-allethrin, d-trans-allethrin, permethrin, resmethrin, bifenthrin, prallethrin, transfluthrin and metofluthrin - all of which are currently registered repellent actives . Both air sampling and chemical analysis methods utilized in the current study may have utility in future spatial repellent evaluations. A direct comparison of the performance (LOD-limits of detection/LOQ-limits of quantification) of the two methods is not appropriate as different analytes were measured and there are a number of parameters that can be varied in the TO-10A method (detector type, final volume following solvent extraction, injection volume, and field vs lab analysis) that can impact the LOD and LOQ. Thermal desorption methods, similar to the one described here and elsewhere , demonstrate short term (≤8h), low-volume (<1m3) sampling can be used to measure the concentration of semi-volatile compounds allowing hour by hour measures of spatial repellent generation. The greatest limitation of these TD methods is they have not been assessed with the same rigors as the USEPA TO-10A method. Likewise, while the TO-10A method represents a standard air sampling and analysis method published by the USEPA, hourly variations in the concentration of semi-volatile SR cannot be reported as the method is intended for sampling periods of no less than 4h . Such characteristics, in addition to cost and logistics, are important to consider when evaluating chemical presence under field conditions.
A 58% reduction in Ae. aegypti entry occurred in the hut containing a 0.00625% metofluthrin coil but deterrency, albeit attenuated, was also observed using a blank coil (48%). This indicates that smoke alone has some effect on mosquito entry. While similar effects have been reported through the use of traditional repellent methods (i.e., burning of organic material, etc.), additional replicates beyond that performed in the current study are required to fully determine any significant differences in mosquito behavior in response to blank and metofluthrin coils. The total number of marked Ae. aegypti collected at any of the huts was low (range of 20–48) during metofluthrin evaluation, therefore rigorous analyses to correlate mosquito entry with chemical concentration in the air space could not be performed. In addition, only one sentinel cohort was positioned in the hut without a coil versus four used in the huts containing active and blank coils therefore direct comparisons of KD and 24h mortality between treatment and negative control may be biased. However, observed knock down and 24h mortality were similar between the two coil-containing huts (i.e., active and blank) indicating no greater toxic effect in the chemical treated space as compared to that containing inert ingredients alone. In addition, overall KD inside the hut containing the metofluthrin coil was minimal although AI was detected in the indoor air space at higher concentrations than outdoors. Combined, these results suggest that toxicity is having little, if any, impact on the number of Ae. aegypti entering the hut containing a 0.00625% metofluthrin coil and indicates deterrency measured was due primarily to a sublethal repellent effect. This conclusion is supported by the fact that air samples collected adjacent to both indoor and outside KD sentinel cohort sites measured metofluthrin at means well below the toxic ‘effective’ rate of 1.0 μg/m3 as specified by the manufacturer (Ken Welch, pers comm).
DDT evaluations also indicated a significant deterrent effect in Ae. aegypti release populations in response to chemical treatment as compared to a chemical-free control. This response occurred in the absence of KD observations in sentinel cohorts. Given the THAI Ae. aegypti strain used in the current study have been characterized as DDT resistant , the lack of KD and mortality was not unexpected but instead strengthens the conclusion that a sublethal repellent mechanism was responsible for the reduction in mosquito entry rather than toxicity. Results also showed a difference in the median concentration of airborne DDT measured between the two treatment huts despite the fact that both received the same treatment. The concentration of airborne DDT is temperature dependent and therefore can vary significantly over the range of temperatures that may occur during field evaluations . The significant difference in DDT air concentration between treatment huts therefore, was most likely due to the comparison of samples that were collected at various times (i.e., different hours) and locations (center vs. window etc.) and therefore at potentially different specific ambient temperatures and relative humidity conditions. The influence of microclimate conditions and airborne repellent chemical should be explored further in subsequent field studies. Regardless, the detection of DDT at the two treatment huts indicated AI was within the air space and the quantification was higher than that observed in the control, chemical-free hut.
The authors note that the placement of metofluthrin and blank coils (i.e. center of the floor) in relation to air sampling and KD sentinel cage locations (i.e. 1m above the floor) may have biased chemical particle dispersion and/or air space dosing and thereby mosquito KD outcomes. We chose this coil positioning to determine the atmospheric concentration of metofluthrin delivered from a mosquito-coil product based on one probable consumer-use scenario in which coils would be positioned on the floor of a house. The KD sentinel cohorts – and therefore air sampling – were fixed at the mid-way height of the windows and door of the experimental huts to reflect potential flight patterns during initial mosquito entry into the hut. In addition, air sampling and KD measurements directly outside the doors and windows during metofluthrin evaluations could have been confounded by interception traps and/or cages used to hold sentinel mosquito populations. The mesh netting on these structures could have prevented passage of some chemical particles and thereby diluted the chemical concentration and/or KD effects. However, we chose to use sentinel cages to evaluate KD behavioral effects as it represents a standard methodology in chemical exposure tests as outlined in WHO efficacy guidelines . Regardless, future studies could explore KD sentinel structures that better match chemical exposure under free-flying field conditions. Other methodologies to evaluate KD outside the huts could include white plastic sheeting at various distances away from the treated space although the challenge of such an approach will be to minimize the disturbance of incapacitated mosquitoes by predators.
Most important to the goal of the study, both DDT and metofluthrin were detectable and quantifiable inside treated experimental huts and resulted in reduced Ae. aegypti entry into corresponding treated spaces. In addition, both air sampling techniques employed in the current study were able to measure chemical actives in the air space, and indicated concentrations below thresholds required for toxic responses (mortality). In fact, spatial repellent (i.e., deterrent) responses using 0.00625% metofluthrin coils occurred at AI air concentrations representing ~2.8ppt (0.06μg/m3) and ~0.9ppt (0.02μg/m3) inside and outside, of huts, respectively, and at ~35ppt (0.74μg/m3) inside a hut treated with 2g/m2 DDT at 50% surface area coverage, highlighting the sensitivity of potential mosquito receptors that may drive behavioral modifications that underlie spatial repellent mechanisms of action . Definitive correlations between spatial repellent versus toxic properties of these two test chemicals that result in a vector reduced area will require more evaluations similar to those reported here as the limited number of replicates performed in the current study precluded rigorous analyses.