Experimental setting
All experiments took place within a large enclosed living area (111 m3) below a typical Queenslander-style house in Cairns, Queensland, Australia (Fig. 1a–d). Throughout this study we will use the term “harbourage area” (HA) to describe an object, usually a suitcase, that is black or dark surfaced. Since Ae. aegypti have a tendency to gather and rest near, and on dark surfaces, we use these HAs to visually bait or direct the mosquito to a chosen part or parts of the room throughout this study.
The metofluthrin product used in this study was a small (9.5 × 15 × 1 cm), plastic frame containing a polyethylene mesh which the 10% AI w/w formulation is incorporated into (Sumitomo Chemical Australia Pty Ltd., Sydney, Australia) [31] (Fig. 2a). In previous longevity studies of this emanator, its efficacy was sustained up to 20 days [31].
Rearing, sexing, caging and blood-feeding mosquitoes
A Cairns colony (F1) of Ae. aegypti infected with wMel Wolbachia, derived from field populations, was used throughout the experiments. The colony was reared in a controlled temperature room at 25 °C and maintained at 70% relative humidity. Wolbachia-infected mosquitoes were used to avoid accidental introduction of uninfected mosquitoes in an area where the Eliminate Dengue program had established wMel in Ae. aegypti. Female mosquitoes were separated as pupae by size and then allowed to emerge into 500 ml containers or into a BugDorm (30 × 30 × 30 cm), depending on the requirements of the experiment. A 10% honey pad was provided and removed 24 h prior to use in experiments. During rearing, when blood meals were required, mosquitoes were offered a blood meal by resting the back of a human leg on top of the cage or container.
Human landing counts
During experiments where a human landing count (HLC) was performed, the lower half of both legs were exposed, and only landing/biting attempts on the exposed lower legs were recorded.
Twenty-two-hour exposure: recovery and survival (fan off)
To observe the impact of metofluthrin on Ae. aegypti survival, forty female Ae. aegypti were released into a large room (111 m3) (Fig. 1) for a 22-h period in both a metofluthrin-treated and an untreated room. An initial HLC was performed immediately prior to each treatment and replication to confirm the fitness of the released mosquitoes. During the treatment replications, the metofluthrin emanator was activated by removing the emanator from the packaging and placing it in centrally within the room to volatilize immediately following the pre-treatment HLC. After 22 h, all live mosquitoes were collected from the room using an insect net, and all knocked down mosquitoes were gently collected into paper towel-lined 500 ml cups using forceps. Observations for mortality and recovery occurred up to 3 h after the completion of each replication. A total of 5 replications were completed. Data from the experiment were analyzed using a Mann-Whitney test in Prism 6.
Effective spatial range of metofluthrin (fan off)
In a large room (111 m3) with four HAs placed in each corner of the room, and a centrally located metofluthrin emanator (Fig. 2d), 10 female Ae. aegypti were released for each replicate, and biting activity was observed over a 5-min period each at 1, 3 and 5 m intervals from the emanator. Once the mosquitoes were released, they were allowed to settle over a 10-min period. For each replication, a control was performed prior to the treatment. For the treatment, the metofluthrin emanator was activated for 10 min prior to the first HLC. A total of 4 replications were completed. Between each replicate, the room was aired out using strong fans with all windows and doors open over a 2-h period. Mosquitoes were not removed or replaced when rotating through the three locations (1, 3 and 5 m) in the room, however, the starting point for each replication was rotated. Data were analyzed using a Mann-Whitney test in Prism 6.
The effect of host and metofluthrin location relative to harbourage areas on human landing counts (fan on vs fan off)
Fan off
Here we visually baited female Ae. aegypti with one large HA, at one end of the room (Fig. 3a, b). We looked at the influence of the proximity of the host and the metofluthrin emanator to HAs on HLCs. In order to do this, we performed two treatments: (i) when the host was near, this meant that the host was within 1 m of the HA, and the emanator was placed on the opposite end of the room, 8 m away from the HA and the host; (ii) when the host was far from the HA, the host would be 8 m from the HA on the opposite end of the room, and the emanator located immediately next to the HA. During the control replicates for each of these treatments, near and far refer to the position of the host relative to the HA without the presence of an active emanator in the room.
For each replication, 10 Ae. aegypti females were released into the room (111 m3) and allowed to settle for 10 min. A 5-min HLC was performed prior to each treatment (activation of the metofluthrin emanator in the room), as a control, corresponding to the host location (near or far) of the subsequent treatment. Once the control was completed, the mosquitoes were again allowed 5 min to settle prior to activating the metofluthrin emanator. The metofluthrin, once activated, was allowed to volatilize for 10 min prior to the HLC. A total of 14 replications were performed. Halfway through the replications, the HAs were moved to the opposite end of the room to eliminate a location effect. Data were analyzed in SPSS using a negative binomial regression analysis.
Fan on
In a related experiment, we repeated the above protocols with the addition of a ceiling fan. We wanted to see if a ceiling fan on its lowest setting (0.8 m/s airflow when standing directly below the fan, measured with a Kestrel anemometer, 1000 model) would aid in the circulation of the metofluthrin throughout the room, thus increasing its efficacy against host-seeking Ae. aegypti. A total of 4 replications were completed. Data were analyzed in Prism 6 using a Mann-Whitney test.
Introduction of a new mosquito to a treated room (fan off)
In a 70 m3 bedroom, attached to the large main room (Fig. 4a, b), one female Ae. aegypti was released for each replication of the control (n = 16) and treatment (n = 16). For the treatment, a metofluthrin emanator was allowed to volatilize centrally within the room (without a fan) for a 30-min period (Fig. 4a, b). Each mosquito was released from a small covered (200 ml) cylindrical container at the corner of the room, approximately 3 m from the host (Fig. 4a, b). The host sat centrally in the room, at the end of the bed, next to the activated emanator (Fig. 4a, b). The time required for the mosquito to reach the host was recorded, for up to 10 min, for both the control and treatment. Whether or not the mosquito reached the host was also recorded. A total of 16 replications were completed. Data were analyzed in Prism 6 using a Fisher’s exact test.
Impact of protected mosquito resting areas on efficacy (fan on)
Groups of five female Ae. aegypti were placed into separate tambourine cages (Fig. 5b) and placed in selected exposed and protected locations within a large room (Fig. 5a-d). Exposed locations were in plain sight and unsheltered, where protected locations were sheltered within the room (e.g. under a bed or behind an object leaning against a wall).
Three protected locations were created within the room: (i) behind a large painting leaning against a wall (Fig. 6a); (ii) underneath a bed (Fig. 6b), and (iii) within a cupboard with its door 5 cm ajar (Fig. 6c). Two exposed locations were also included at 3 and 5 m distance from the emanator (Fig. 5a, c). Five female Ae. aegypti were kept in a tambourine cage in a separate untreated room as a control. Once in position, the metofluthrin emanator was activated in the room, and the time to 100% knockdown was recorded. One hundred percent knockdown was used as the indicator that the replication for the treatment (protected or exposed) was completed.
A ceiling fan on low setting (0.8 m/s) was used to aid in air circulation of the volatilizing compound. Using a flashlight, cages were visually assessed for knockdown every 15 min in order not to disrupt the tambourine cage or contaminate the airspace surrounding it. Once 100% of the mosquitoes were knocked down within a cage, it was immediately replaced with another cage of 5 mosquitoes, taking care not to disturb the protected space or expose the second cage prior to its placement. The second cage was observed every 15 min until 100% KD was achieved, and then removed. Between each replication, the bed and painting were moved and turned over, and the cupboard opened widely, to ensure the metofluthrin was evacuated from the protected spaces properly. Fans were turned on high and all doors and windows open for 2 h. A total of 5 replications were completed. The time to achieve 100% knockdown was compared between the protected and exposed locations for both the first and second exposure of tambourine cages. The second exposure allowed us to observe whether or not knockdown in the protected locations, over time, would be a result of a maintained low-dose exposure or if the concentration of the compound in the protected space would reach some level of equilibrium with the rest of the room. Five mosquitoes in a tambourine cage were kept in a separate room, outside of the treatment area as a control for each replicate. Data were analyzed using a negative binomial regression analysis in SPSS.
Statistical analysis
Data were analyzed, depending on the model required for each analysis, in IBM® SPSS® version 24.0 or Prism 6 for Mac OSX (v. 6.0 h, GraphPad Software Inc.).