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.
In a series of experiments, we evaluated a “push–pull” strategy against the primary dengue and chikungunya virus vector, Aedes aegypti (L.), to reduce its human contact in and around homes. The “push” component focused on using a spatial repellent (SR) and/or contact irritant (CI) chemical in sublethal doses to the mosquito (also rendering them safer for expended human exposure) applied to fabric to reduce indoor biting (Salazar et al. 2012). The Biogents Sentinel™ (BGS) trap (Biogents AG, Regensburg, Germany), was used as the preferred trapping method of adult female Ae. aegypti (Krockel et al. 2006, Maciel de Freitas et al. 2006, Williams et al. 2006, Barrera et al. 2013), representing the “pull” component to remove chemically repelled or excited/irritated mosquitoes from the test environment and thus further reduce human–vector contact.
Our previous studies demonstrated the effectiveness of BGS traps to recapture released Ae. aegypti in a screened house setting (Salazar et al. 2012) and confirmed that Ae. aegypti previously exposed to repellents/irritants (i.e., dichlorodiphenyltrichloroethane (DDT), metofluthrin, or transfluthrin) can be effectively captured by BGS traps (Salazar et al. 2013). This study aimed to determine the extent to which capture of released females is influenced by the positioning of BGS traps in relation to host-occupied experimental huts. Specifically, we quantified recapture rates for BGS traps positioned near hut windows or doors (entry points), for hut corners, and for traps placed at different distances outside the huts. Additionally, we used interception traps fixed to hut windows and doors to quantify attempts of mosquito entry to the hut in relation to BGS trap positioning.
MATERIALS AND METHODS
Studies were conducted in 2011 near Pu Teuy (14°17′N, 99°11′E), a small rural village (<1,500 inhabitants) located 150 km northwest of Bangkok in Sai Yok District, Kanchanaburi Province, Thailand. The village, where Ae. aegypti is naturally prevalent, is surrounded by dense primary forest, fruit orchards, and vegetable plots. The abundance of immature Ae. aegypti in water-holding containers was surveyed weekly by the local Thongpaphum District health clinic at the time this study was conducted. The clinic was also distributing organophosphate larvicide (temephos) in larval infested habitats as part of their mosquito control interventions. The experimental location was >800 m from the closest indigenous occupied home, creating a sufficient distance buffer for mark–release–recapture mosquito behavioral studies that exceeds the typical maximum flight range (<100 m) of Ae. aegypti (Muir and Kay 1998, Harrington et al. 2005).
Immature Ae. aegypti mosquitoes were collected from Pu Teuy village and reared to adults at an on-site field insectary. Nulliparous, 3–5-day-old, sugar-starved (24 h) females were used for all experimental trials. Mosquito test cohorts (control or treatment) were marked with a unique fluorescent color powder before release (Achee et al. 2005).
Experimental huts and mosquito trapping
Three experimental huts were used in the mark–release–recapture study. The experimental hut design and construction materials were the same as previously described by Chareonviriyaphap et al. (2010). The huts are intended to mimic indigenous Thai homes, both in materials and dimensions, and are the same huts used in other studies to evaluate daytime active Ae. aegypti entering and exiting behaviors as part of the development of the push–pull strategy. Huts were approximately 30–42 m apart from one another.
The windows and doors of the experimental huts were fitted with removable interception traps that captured mosquitoes as they entered the structure (Fig. 1). Interception trap collections were used to compare reduction in Ae. aegypti entry into the huts for each treatment used in this study (with BGS traps placed in different locations and at different distances) and to complement the results of BGS traps recapture rate.
Recaptures of released mosquitoes attempting to enter the experimental huts by means of interception traps followed established research protocols (Grieco et al. 2007). Two volunteers were assigned to stay in each hut to provide constant host cues and to serve as collectors. Thirty minutes before expected sunrise, 1 collector from each hut would release 100 females (marked with a unique identifying color) assigned to that hut at a fixed location, approximately 10 m diagonal from the front of the hut. Immediately afterward, the collector would join the other volunteer and would enter the hut through the door to begin observations. Interception trap baffles were opened 15 min before the first sampling interval.
Collections from interception traps affixed on the 3 windows and single door of the hut were conducted for approximately 1 min at each 20-min interval from 0600 to 1800 h. To avoid collector bias due to potential varying host attractiveness and collection skill, the collectors were moving from 1 hut to another in a counterclockwise manner (Latin square rotation scheme) every 6 h (∼0600 to 1200 h and 1200 to 1800 h time per hut) for the duration of the study.
At the time of collection, all window and door trap baffles were closed, and collectors would carefully search each trap for a period of approximately 1 min using a manual aspirator and flashlight. Mosquitoes were aspirated into individual plastic holding cups labeled by interception trap ID, sampling period, hut, and date. Trap baffles were then reopened at the end of each sampling period, and holding cups were given to another person for mosquito species identification. Captured mosquitoes (marked or not) were recorded by species, gender, feeding status (fed or unfed), and marking color per capture hut and sampling period. All collected mosquitoes were maintained inside humidified chambers at ambient temperatures, provided with 10% sucrose solution, and monitored for 24 h mortality.
BGS trap distance and location trials
Outdoor mosquito trapping was done using BGS traps equipped with the BG-Lure™ (Krockel et al. 2006, Maciel de Freitas et al. 2006). The BGS traps were placed at different set locations and distances outside of each experimental hut. The BGS lures were replaced before the indicated duration (75 days) of its usability upon opening. The trials were done from June to December 2011.
To examine the effect of trap location on the number of recaptured mosquitoes, 2 positions were tested: near hut vertices (corners) and near the window/door (portals of entry). Both locations were set at ∼0 m (i.e., less than 1 m) away from the hut, with traps positioned on the elevated hut platform (30 cm above ground; Fig. 2) either near each hut structure corner (1 BGS trap per corner) or opposite each portal of entry (1 trap at the door and 1 each opposite the windows) (Figs. 2–3).
For the evaluation of BGS position effect, 3 experimental huts were used. Likewise, 3 treatments were tested simultaneously: 1) hut with no BGS traps; 2) hut with BGS traps placed at the corners or vertices; and 3) hut with BGS traps placed opposite portals of entry (Figs. 2 and 3). These 3 treatments were rotated every day across the 3 experimental huts following a Latin square design, totaling 3 replicate trials each over a consecutive 3-day period; with a total of 300 Aedes aegypti released for recapture either by interception traps fitted to entry portals of experimental huts or by BGS placed in 2 different locations.
After establishing the best locations for BGS traps, the search for optimum distance of BGS traps from human-occupied huts commenced. With the earlier mentioned host set-up/procedure kept constant, a second set of experiments, designed to determine the optimum trap placement distance from the hut for recapture of released uniquely marked mosquitoes, followed.
The BGS traps were placed at 3 different distances from host-occupied experimental huts (∼0, 3, or 10 m; Figs. 3 and 4) with another hut left without BGS traps to serve as control; making it a total of 4 treatments. During this period, only 3 experimental huts were available at Pu Teuy, Kanchanaburi site. However, as earlier discussed, there were 4 separate treatments implemented for the BGS distance trial.
To accommodate all the treatments, experiments were conducted in batches: first batch–BGS traps at 0, 3, and 10 m from experimental huts (July 2011); second batch comprised of hut with no BGS trap, together with BGS trap positioned at 3-m and 10-m distances (July–August 2011). The third and last batch was comprised of BGS traps placed at 0, 3, and 10 m distances from host-occupied experimental huts (December 2011).
Trial 1 was designed to observe the recapture rate when traps were present at different distances from all 3 huts. Trial 2 was designed to observe number of recaptured mosquitoes attempting to enter huts via door and window interception traps in the absence of BGS trap near portals of entry. Trial 3 was a repeat of the first trial design to increase overall treatment replicates. However, owing to logistic problems (not enough huts in particular), the replicate (Trial 3) did not yield results comparable with Trial 1, since they were conducted in different seasons of the year, which may have affected the Aedes aegypti density in the area.
With these types of design, 2 sets of data were obtained. The first set of data represents recaptures from BGS traps at varying distances (0, 3, 10 m) from the hut, while the second set of data represents recaptures from experimental hut interception traps while the 4 treatments were implemented.
All experimental huts were thoroughly cleaned after each batch of treatments were left for at least a week before new sets of experiments were performed.
Temporary shelters were used to protect BGS traps from rain and direct sunlight when evaluating the 3 m and 10 m distances (Fig. 5). The shelter was elevated 10 cm above ground with a galvanized iron roofing 1.5 m above the platform. This provided a 15 cm space around the trap following the BGS trap manufacturer's specification.
Mosquito test populations (100 Ae. aegypti females each) were released at designated points outside each experimental hut on day 1 at approximately 0530 h. The BGS trap recapture collections were monitored at 0930, 1330, and 1730 h on the day of release, and 0500, 0930, 1330, and 1730 h on each subsequent testing day. The monitoring intervals were based on earlier quantification of recapture densities and rates using Ae. aegypti in semifield (screened house) conditions (Salazar et al. 2012). At each sampling interval, the BGS trap collection bags were removed and replaced. Recaptured mosquitoes were recorded according to marking color, time of collection, BGS trap number, and experimental hut.
Temperature (°C), % relative humidity (RH), and light intensity (lux/ft2) were measured during experiments, using constant data loggers set at 20 min collection time intervals (HOBO® U12-012 Model, Onset Computer Corporation, Pocasset, MA). Climatic data were collected to determine peaks of recapture periods between experiments to provide data on timing of trap placement for maximum Aedes aegypti trap recaptures. Furthermore, environmental parameters were recorded to have data to refer to, just in case a highly unexpected recapture (extremely high or extremely low) was observed.
The percentages of recaptured mosquitoes were calculated according to recapture time points following time of release (Day 1, 0930 h, 1330 h, 1730 h; Day 2, 0530 h) with mean cumulative percentage recapture ± standard deviation (SD) reported. The Kruskal–Wallis test compared the BGS recapture percentages between location points (hut corners and portals of entry) and distances (0, 3, 10 m), respectively. The percentage reduction in entry (%RE) for mosquitoes collected with window and door interception traps for each treatment was computed using the following formula:
Control huts (with no BGS trap) served as the basis for computation, with 0% RE assumed for the controls [1 − (% recaptures from control/% recaptures from control)]. For all statistical tests, a P ≤ 0.05 was considered significant. All statistical analyses were performed in STATA® 11.2 (StataCorp, College Station, TX) using the ranksum and kwallis syntax for Mann–Whitney and Kruskal–Wallis tests, respectively.
The use of human subjects as bait attraction and for collection of mosquitoes from hut interception traps inside the huts and outside BGS traps was approved by the Institutional Review Boards of Kasetsart University, Bangkok, Thailand, and the Uniformed Services University of the Health Sciences, Bethesda, Maryland.
The overall recapture of Ae. aegypti females with the BGS traps near the windows and door (38.7%; 116/300) was greater than those traps placed at the hut corners (23.7%; 71/300), although the difference was not statistically significant (Table 1). For both location and distance experiments (Tables 1 and 3), the highest Ae. aegypti trap recaptures were obtained immediately after release (0530–0930 h).
The interception traps showed greater reduction in Ae. aegypti entry into the experimental huts, as compared with a control, when BGS traps were located at primary portals of entry (68.9%) than when placed at the corners (31.1%) (Table 2).
In terms of distance from the hut, the overall BGS trap recapture of Ae. aegypti females was higher for placement at 0 and 10 m distances (18.5%, 111/600; 14.2%,128/900, respectively) as compared with 3 m distance (7.9%; 71/900), albeit not statistically significant (Table 3).
The interception trap collections showed greater reduction in Ae. aegypti entry to huts with BGS traps present at varying distances compared with control huts without BGS traps (Table 4). Moreover, the interception trap collections showed greatest reduction in Ae. aegypti entry (65.6%) at 0 m (less than 1 m) distance from the experimental huts compared with 3 m (17.2% reduction) and 10 m (14.6% reduction) distances (Table 4).
We demonstrate the importance of trap positioning, both location and distance, to optimize the efficiency of a push–pull strategy in which a mosquito trap is the pull component. Not surprisingly, we found that BGS traps were more effective in capturing the largely endophilic and anthropophilic Ae. aegypti (Edman et al. 1992, Van Handel et al. 1994) when placed near windows and doors, which serve as more likely entry points into human-occupied buildings compared with the corners or vertices of the experimental huts. Likewise, it was not surprising that BGS traps placed directly adjacent to the experimental huts captured more Ae. aegypti than traps placed 3 m away from the huts. This could be due to high concentration of attractants (host cues and BG Lure combined) that increased recapture. On the other hand, similarly high mosquito recapture numbers for BGS traps placed 10 m from the huts may be explained by their proximity to the mosquito release points (∼10 m diagonal from front of hut). The data from the interception traps fitted to hut windows and doors provide further evidence of the value of placing BGS traps close to windows and doors, since this resulted in greater reduction of mosquito entry into the huts as compared with traps placed at hut corners or farther from the huts. One limitation of the study design was the use of 1 fixed position release point per hut, which would potentially have an effect on both emanation and detection of host cues and BGS chemo-attraction lures.
Importantly, our trials showed a 3-fold reduction in hut entry by Ae. aegypti when BGS traps are positioned just outside and very near hut windows and door (Tables 1 and 2). This study therefore suggests that BGS traps continuously show a promising role in the proof of concept of push–pull strategy for Aedes aegypti as an excellent candidate trap when placed closest to portals of entry of human-occupied huts (Fig. 6).
Highest Ae. aegypti trap recaptures were obtained immediately after release (0530–0930 h) for both location and distance experiments (Tables 1 and 3). This indicates that host seeking possibly starts at the earliest suitable time when at least minimal required light, ambient temperatures, and RH permits host seeking (Kawada et al. 2005). It did match, however, the most productive period of operation for Ae. aegypti BGS trapping in the natural setting experiments (between 1330 and 1730 h). The BGS trap in the said trial appeared to be an effective pull device in the push–pull control strategy in natural (local community) settings (Salazar et al. 2017).
It was interesting to observe that mosquitoes were captured by the BGS traps rather than directly entering the human-occupied huts. The BGS trap with lure appears to be sufficiently attractive to Ae. aegypti for this trapping device to be an effective pull component even when human hosts are present nearby. Since individual homes are unlikely to use more than 1 or 2 of the relatively expensive BGS traps in a push–pull control strategy, the deployment of the minimal number of traps and optimal placement will be crucially important to maximize effectiveness. Downstream studies will move from experimental huts to local homes in Thailand to address the issue of trap placement in real life scenarios. Additionally, it would be advantageous to design smaller, less expensive trapping devices that could function with the same efficiency as the BGS traps.
We thank the Armed Forces Development Command, Sai Yok District, Kanchanaburi Province, Thailand, for support of the research program by providing land for the study site. Funding for this research was provided by the Bill and Melinda Gates Foundation (Grant 48513) and the Thailand Research Fund (RTA5280007).