Study areas
The study was carried out in two ecological settings, Vallée du Kou and Soumousso (Fig. 1), two villages close to Bobo-Dioulasso, Burkina Faso.
Vallée du Kou (11˚23' N, 4˚24' W) is a village located to the north-west of Bobo-Dioulasso, characterized by over 1,200 hectares of wooded savannah. It is a rice growing area with high mosquito densities throughout the year. Because of the extensive use of insecticides in rice and cotton fields, mosquitoes exhibited high insecticide resistance levels. The trial was conducted in one of the village’s seven neighbourhoods separated from one another by rice fields, Vallée du Kou 3 (‘VK3’). This site was chosen due to its proximity to the main tarred road. Given the presence of surface water all year round, mosquitoes are found with ease, with a peak density observed in August-September, during the rainy season. Anopheles gambiae and An. coluzzii are present with a predominance of An. coluzzii throughout the year. Both species in this area are highly resistant to pyrethroids and DDT (kdr based mechanism, 0.8-0.95), and a rise in ace-1 resistance frequency has also been reported [20,21].
Soumousso (11˚04' N, 4˚03' W), located to the south of Bobo-Dioulasso, sits on arid fields, in complete contrast with VK3. It is a drier setting where the most dominant species are An. gambiae and a mixture of An. funestus, An. arabiensis, An. coluzzii, etc [22]. In this area, the mosquito density is lower compared to that of Vallée du Kou, and the dynamics of the mosquito population follow the two main seasons, with fewer mosquitoes in the dry season compared to the rainy season.
Description of the traps
The original LFET (prototype 1, P1) was created by Diabaté and collaborators in 2013 [19] and was made from a metal frame (length = 69 cm, width = 51 cm, height = 165 cm) fitted with a regular mosquito net to prevent mosquitoes and other insects from escaping the trap once they enter it (Fig. 2). A funnel made from a metal frame was inserted at the top of the trap in such a way that mosquitoes approaching the window go first through the larger opening of the funnel and enter the trap through the small and rectangular opening. The large opening of the funnel is 70 cm long and 54cm diagonally, while the small opening 13.3cm long and large of 11.2cm. The small opening of the funnel is 10cm away from the backside of the trap. The funnel is inserted in the frame in a way that allows the mosquitoes to enter the trap easily but prevents them from escaping. Once the mosquitoes enter the trap, they have a large space beneath the funnel where they can find refuge. For a mosquito to escape, it would have to fly all the way up towards the small opening of the funnel and be able to navigate through the 10 cm space separating the small opening of the funnel and the back side of the frame. Ultimately, mosquitoes will continue to fly to exhaustion before finding a way out. The principle of the LFET is to confine the mosquito inside the trap until dehydration and finally death. For the purpose of this experiment, traps were fitted with three sleeves on the side (one below, one in the middle and one on the top) through which mosquitoes were aspirated. The trap was secured to the windows using nails.
The Medium (prototype 2, P2) is a smaller version of the original LFET. The funnel dimensions are similar but its height (82.5cm) is half that of the original LFET (165cm) (Fig. 3). Similarly, the whole trap was also covered with a net, fitted with three sleeves for mosquito collection.
The Prototype 3 (P3) was made using a metal funnel frame with the following measurements: length = 81cm, width = 16cm, height = 80cm (Fig. 4). Here, the small funnel opening used as entry is a circular metal funnel, instead of rectangular as in the previous traps, with an opening size of 16.5cm in diameter. Two circular openings on the right and on the left (the distance between both circular openings is 12.5cm) were made to fit the window size. Depending on the space available around the window of the house, the trap position may allow the use of only one of the openings (either right or left). When one opening is used as entry, the second would be closed and covered with netting. The volume of the trap was made of a horizontal and rectangular metal frame (length = 110cm, width = 16cm, height = 56cm). A net covered the trap on the backside of the funnel and was fitted with five sleeves allowing mosquito collection.
The prototype 4 (P4) is similar to P3 in terms of size and funnel type but has an additional circular funnel frame. This small (9cm long) circular funnel gives access to the space beneath.
The distance between the end of the circular pipe and the back side of the trap is 10 cm. In addition, P4 was equipped with a mirror for the inhabitants’ personal use which would make it valuable for other functions beyond controlling mosquito populations. This may help increase the acceptability and sustained use of the trap (Fig. 5). As with P3, this trap was also outfitted with five sleeves enabling mosquito collection.
Study design and mosquito collection
Three of each of the new LFET prototypes (Medium/P2), P3 and P4) were tested in the two selected ecological settings along with three of the original LFET design (P1) for comparison purposes. The performance of the traps was assessed in terms of the number of malaria mosquitoes trapped as well as other mosquitoes entering the house through the window. A total of 12 houses, corresponding to 12 traps to install, were chosen in each testing site. Twelve traps were designed for each site giving a total number of 24 traps produced over the study for both sites. Only houses with a single room, single window (similar size and metal frame type) and single door were selected for the study. Each of the houses were 10m apart from each other. All of the traps were installed the same day between 15:00 and 17:00 with a two-day rotation of trap testing according to a Latin square plan, to reduce the bias of house inhabitants’ attraction. After installation, all the traps were simultaneously used per day, and checked every morning over eight consecutive days per month, for collection of all mosquitoes dead or alive in the traps for morphological identification. To ensure that mosquitoes had no other alternative except the windows to enter the house, any small holes in ceilings and walls were blocked by sponges or cloth, and a curtain was placed at the entrance of each house (Fig. 6). The inhabitants were informed of the aim of the study and therefore free to use their doors as they wished. The window where the LFET was fitted was left open throughout to allow mosquitoes to enter the house. Traps were installed the day before prior to mosquito sampling over eight consecutive days per month, from September to November in VK3 and Soumousso.
Traps were emptied from 07:00 to 09:00, using a mouth aspirator in addition to pyrethrum spray catches [23] performed in the corresponding house. All the mosquitoes caught were kept in a single cup, then killed with chloroform and morphologically identified on site. The daily work on site consisted of mosquito collection, sorting, identification, sexing according to Gillies and De Meillon [24], counting and scoring per genus, species and physiological status (unfed, blood fed, gravid), and the numbers were recorded on a spreadsheet. All traps set up each month were removed at the end of the trial after the day collection.
Statistical analysis
Microsoft Excel 2007 (Microsoft®, New York, USA) was used to record the data, and R-3.6.2 (package dplyr, questionr, and coin) was used to produce the graphs and statistical analyses.
In the study, three main variables were analyzed: (i) T = Tt + Th, T = Total number of An. gambiae s.l. collected in the trap and in the house, Tt = total number of mosquitoes collected in trap, Th = total number of mosquitoes collected in the house; (ii) P (%) = (Tt/T)*100, P = Proportion (%) of mosquito entry reduction in the house and (iii) Dr = T/24, Dr = daily removal of mosquitoes per site over 24 days.
The number of mosquitoes in the traps and the matching houses did not follow a normal distribution. Therefore, a non-parametric test, Kruskal-wallis sum test was used to assess the overall performance of the traps. The effects of monthly collection in mosquito density reduction were evaluated using a Levene’s test for homogeneity of variance (center = median) with a Wilcoxon rank sum test and a p-value bonferroni adjustment method. A Tukey multiple comparison of means with 95% family-wise confidence level test was used for pair-wise comparison between LFET prototypes.
To assess whether the traps caught more mosquitoes than those that entered in the matching houses, a comparison using Wilcoxon rank sum test with a holm p-value adjustment method was used.