Large-scale deployment of pyriproxyfen-treated Lehmann’s Funnel Entry Traps to control malaria mosquito populations

Large-scale deployment of pyriproxyfen-treated Lehmann’s Funnel Entry Traps to control malaria mosquito populations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Large-scale deployment of pyriproxyfen-treated Lehmann’s Funnel Entry Traps to control malaria mosquito populations Roger Sanou, Hamidou Maïga, Bazoumana D. Sow, Adama Ouema, Abdoul Azize Millogo, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2432364/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background There is a great need to find new effective tools to prevent mosquitoes from biting humans. As part of this search, the Lehmann Funnel Entry Trap, a window screen, was tested and has proven to be effective in mosquito density reduction in low and high vector density settings. Here we aimed to assess whether a large-scale deployment of pyriproxyfen-treated traps can control malaria mosquito populations and how well the traps were accepted at community level. Method Overall, 1,313 traps impregnated with Pyriproxyfen (PPF) were deployed. Of these, 12 traps in 12 houses were randomly selected across the intervention village, Vallée du Kou 3 (VK3), for indoor mosquito density assessment compared to houses without traps in the control village, Vallée du Kou 5 (VK5). Traps were placed in the windows, while doors were blocked with curtains. From July until October 2015, for nine days per month, mosquitoes were collected from the traps and matching houses in VK3, and in houses only in VK5. Anopheles coluzzii collected from the field as adults and as larvae reared through to adults and An. gambiae were used or released into traps to assess the bio-efficacy effect of PPF in VK3 against control mosquitoes from VK5. Then, mosquitoes that were in contact with the PPF trap netting were allowed to oviposit in the laboratory for fecundity and egg fertility reduction evaluation as compared to a control. The mosquitoes collected as part of the monthly programme were morphologically identified, counted, and preserved in 80% ethanol vials for subsequent analyses, including resistance genes and female mosquito age structure. The impact of the trap on mosquito density at community level was assessed by performing a pyrethrum spray catch (PSC). The integrity of net fabric as well as some sociological points of view on the trap’s efficacy and design were then reported. Results Overall mosquito density was reduced by ~ 90% in all houses equipped with traps in VK3. At the community level, while mosquito density before intervention was 33% higher in VK3 than in VK5, it was 47% higher in VK5 after the intervention. Old female mosquito numbers increased in VK5 by 12% in October but not in VK3, indicating that the traps were cumulatively killing older females. The additional effect of PPF was to limit egg-laying, with a smaller number of eggs counted, and with a lower hatching rate than in VK5. Mosquitoes were highly resistant to pyrethroids with a ~ 0.9 frequency of the kdr mutation. The trap was well accepted by the communities as most of the interviewees in VK3 found the traps reduced mosquito bites with peaceful sleep. They hoped to keep the traps in place beyond the study period. Conclusion The Lehmann Funnel Entry Trap has real potential to control malaria mosquito populations and can be widely used to sustain the global effort of malaria elimination. Malaria vectors Lehmann’s Funnel Entry trap control density Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Vector control has contributed to the reduction of malaria burden over the last few years. According to a recent WHO report, the number of deaths due to malaria in 2019 is estimated to be 409,000 [ 1 ]. Several vector control related factors may have contributed to this reduction, such as Indoor Residual Spraying (IRS) and the mass distribution campaigns of Insecticide Treated Nets (ITNs) [ 2 ]. However, the development of the emergence and spread of insecticide resistance in malaria vectors is threatening the future of vector control [ 3 – 5 ], making it clear that alternative tools are needed to sustain vector control toward malaria elimination. Thus, discovering new tools or improving the effectiveness of the existing ones should be prioritized [ 3 ]. Since the link was established between malaria and mosquitoes, window screening and nets have been used as part of malaria vector control management [ 6 , 7 ]. More recently, studies using screens within windows, on ceilings, and on doors have proven to be effective in reducing the number of mosquitoes entering homes. Consequently, there is added personal protection against mosquito bites as well as community protection through added mosquito mortality [ 8 – 11 ]. Whilst some studies have established a relation between malaria illness and the type and quality of dwelling construction [ 12 , 13 ], other studies have demonstrated that in addition to quality of construction of the home, modifications to prevent mosquitoes from entering houses can help reduce malaria transmission by lowering human exposure to infectious bites [ 7 , 10 , 11 , 14 – 18 ]. Interestingly, the use of mosquito-repellent plants in the home has also been shown to reduce mosquito densities [ 19 ], and there is a significant reduction of anaemia in children living in homes with window screens [ 20 ]. Although these methods deny mosquitoes’ entry to a dwelling, mosquitoes are not killed and so they can still bite outdoors and transmit malaria parasites [ 21 – 24 ]. To date, most malaria transmission occurs indoors, and several malaria vectors bite indoors for their blood meal in human dwellings [ 14 , 15 , 25 ]. However, some malaria transmission may occur outdoors [ 26 , 27 ]. As such, denying mosquito access to indoor living spaces is a worthy cause, but killing blood-seeking (and resting-site-seeking) mosquitoes could be a more effective goal. Other alternative tools for insecticide resistance management include the use of molecules such as pyriproxyfen 10EC (PPF), which is a juvenile hormone analogue that mimics a natural insect hormone, essential for the normal physiological growth, development, and maturation of juvenile insects. As mosquitoes are holometabolous insects, the growth regulator hormone that PPF replaces is essential for larval development and its removal leads to undeveloped miniated pupal or adult stages [ 27 , 28 ]. Formerly used as a larvicide, PPF has been tested on adult mosquito species and has shown an interesting negative effect on female fecundity and egg fertilization [ 28 – 30 ]. Adding a PPF-treated net to a trap may therefore increase the effects on mosquito density reduction by additionally sterilizing any mosquito encountering the net. This proposal led to the development of a new approach, which considers mosquito behavior and typical house entry routes for mosquitoes. Within this approach, Lehmann Funnel Entry traps (LFETs) were tested in high and low vector density settings as a malaria vector control tool in development [ 9 ]. Recently, three new prototypes were created and successfully tested in two ecological settings to the southwest and northwest of Bobo-Dioulasso, Burkina Faso [ 31 ], in view of further use against malaria vectors. Once installed into a window, a LFET catches and kills all mosquitoes that attempt to access the house through the relevant entry point. Consequently, members of the household may be prevented from being bitten if all windows are secured with traps. Once a mosquito enters the small opening of the trap on the outside of the home, it becomes trapped and will remain so until dehydration kills it. In this trial, the traps were all installed at the same time in the intervention village, which allowed the blocking of all window entries in all of the houses, and as a consequence, mosquito densities were reduced at community level. Blocking all windows within a house with screens, helps to remove indoor resting and anthropophagic mosquitoes. Most vector control interventions against malaria require active participation from the local population to ensure proper adherence [ 32 , 33 ]. Recent studies have demonstrated the important role of stakeholder’ engagement in the successful control and elimination of many infectious diseases. Communities should have detailed information on the intervention or program in development at the beginning of any given study, perhaps even early on the design of the program, as well as during the study and at the end. An accurate timeline is also important for transparency reasons [ 34 – 36 ]. Furthermore, as the space within the household has multiple functions, the homeowner’s full buy-in is required, as LFETs (or any other materials important to the study) may require reorganization at home. Given that the LFET is an indoor intervention tool, community engagement is especially important to ensure the traps are sustainable and fully operational throughout the use period. The objectives of this trial were to assess the impact of a large-scale deployment of PPF-treated traps in terms of mosquito density reduction, to evaluate the effects of PPF on female An. gambiae mosquitoes from the laboratory and the field in an intervention village compared to a control village. In addition, the age of mosquitoes, as well as the physical integrity of the traps’ nets, were assessed over the study period. Sociological endpoints were also reported in this study. Materials And Methods Study area The study was carried out in Vallée du Kou, Burkina Faso (11˚23' N, 4˚24' W, Fig. 1), in an irrigated rice field area. The site is characterized by wooded savannah across 1,200 ha and contains seven discrete villages. The mosquito population in the village is resistant to insecticides and so a solution is required. Relatively high mosquito densities are observed annually during August and September, corresponding to the peak of the rainy season. Anopheles coluzzii is predominant throughout the year and An. gambiae is observed toward the end of the rainy season (frequencies fluctuating between five and 20%). Both species are highly resistant to pyrethroids and DDT ( kdr frequency (0.8–0.95) [ 9 , 37 – 39 ]. Study Design And Period The study was carried out in three sequential phases that encapsulated product optimisation through to large-scale field deployment as follows: Selection of the most effective PPF dose (May 2015, in the laboratory) Trap manufacturing and impregnation (May – June 2015) Large-scale field evaluation of the LFET (July to October 2015, in the field site.) (Table 1: Gantt chart of large-scale field evaluation of LFET) Two villages, Vallée du Kou 3 (VK3) and Vallée du Kou 5 (VK5), were selected as intervention village (IV) and control village (CV), respectively. The control site was selected as it presents the same ecological characteristics in terms of mosquito densities and species as VK3, and was located 1 km away, which helped minimize any potential PPF contamination. Prior to trap fabrication, a general survey of the houses and windows was conducted in VK3 using a Global Positioning System (GPS) [ 40 ] to count all the inhabited houses. Traps were then produced and deployed across the entire VK3 village. Phase I: Selection Of The Most Effective Pyriproxyfen-dose Laboratory mosquitoes Mosquitoes were maintained at the Institut de Recherche en Sciences de la Santé (IRSS) insectary under controlled conditions (Temperature 27 ± 2 o C and relative humidity 80 ± 10%). For testing purposes, female and male mosquito cages were set up with pupae on the same day, maintained together until the day of testing (three days later at least), when females were removed from the cage. This time allowed them to mate before testing. Previous work done on the same laboratory colonies from the IRSS insectary showed an insemination rate of > 90%. Prior to cone testing, some females were checked to calculate the insemination rate [ 29 ]. Spermathecae were dissected under a magnifying glass 24 hours before bioassay to check this. Females were then blood fed when it was shown that the insemination rate was up to 90%. Three- to five-day-old female mosquitoes were starved for six hours by removing the 5% glucose (weight/volume, w/v) solution prior to blood feeding. The blood feeding took place at 18:00 for 45 minutes using direct rabbit feeding in the laboratory. The cage was blood-fed with males still inside. The non-blood-fed females were sorted and discarded, and only blood-fed mosquitoes were kept overnight and provided with glucose solution in cotton balls for the experiments. The laboratory mosquitoes were made up of susceptible An. gambiae -Kisumu, resistant laboratory strain An. coluzzii , and field An. gambiae s.l. collected at larval stage from breeding sites in both villages. Effects of selected pyriproxyfen doses on mosquito fecundity and fertility Prior to impregnating the trap net, preliminary laboratory testing with two doses of PPF liquid (10EC) was conducted to assess PPF efficacy on susceptible An. gambiae -Kisumu. After this, the most effective sterilizing dose was selected. A piece of net was impregnated at 20 mg/m 2 or 30 mg/m 2 of active ingredient (ai) selected according to previous studies [ 41 , 42 ]. Three- to five-day-old blood-fed female mosquitoes were put in contact with impregnated pieces of net in WHO cones for three minutes. Then, these mosquitoes were transferred individually into cups (200 mL) containing a filter paper for oviposition 24 hours post exposure. A daily manual count of the number of eggs laid was performed to evaluate the fecundity. In cups where eggs were observed, water was added in order to hatch the eggs. The number of larvae were also counted. After the subsequent three days following oviposition, the number of females that laid eggs, and those that died before laying were counted. The fecundity and fertility of treated mosquitoes were assessed as compared to a control under laboratory conditions. It was shown that the PPF dose with the most desired effect on the tested mosquitoes was 30 mg/m 2 ai. This was also the case in previous studies [ 29 , 41 , 42 ]. Phase Ii: Trap Manufacturing And Impregnation With Pyriproxyfen Trap manufacturing The traps were made from a metal frame (69 × 51 × 82.5 cm) and were fitted from the bottom to the top with a regular mosquito net to prevent any mosquitoes or other insects from escaping the trap once inside (see Sanou et al., 2021, [ 31 ] for more details) (Fig. 2). All of the windows of the inhabited houses (non-inhabited house windows were secured simply with a net, to reduce the number of mosquito-resting sites) were counted and measured to manufacture the traps accordingly. Each manufactured net covers the trap entirely, which itself fits into the window perfectly. Each trap’s net also has a sleeve for easy access, to open and/or close the window. The metal manufacturer first produced a sample of each trap size. Then, the remainder of the traps were produced, painted with neutral oil (Fig. 3a), labelled according to dimensions, before transportation to VK3. The trap samples were also sent to a tailor in order for the nets to be made to size (Fig. 3b). Trap impregnation with pyriproxyfen After the traps were manufactured, they were impregnated with PPF. Through weighing the equipment, the amount of water and PPF solution needed for each net size was determined (Supplementary Table 1) according to the WHO insecticide impregnation process with pyriproxyfen 30 mg ai /m 2 (PPF). For each set of dimensions, calculations were made to ensure the right amount of insecticide and water necessary to entirely coat the net (Supplementary Table 2). After the nets were coated, all trap nets were dried overnight indoors and wrapped into labelled sachets for easy identification prior to their transfer into the field for installation by the team and local workers (Fig. 3c). Phase Iii: Large-scale Field Evaluation Of The Lehmann Funnel Entry Trap (Dup: Abstract ?) Experimental design The large-scale field trial was designed with one intervention area and one control area, and the entomological endpoints were assessed simultaneously in both villages after the traps were deployed. The villages selected were: Vallée du Kou 3 (VK3) for the intervention village (IV) and Vallée du Kou 5 (VK5) for the control village (CV). These two villages were selected because they had similar ecological properties in terms of mosquito densities and species [ 43 , 44 ] and were situated 1 km apart, which minimizes any potential PPF contamination between the two sites. A general survey of the houses and windows was conducted in VK3 using a Global Positioning System (GPS) [ 40 ] to count all the inhabited houses. Each household was georeferenced (Fig. 4a, b, c). Deployment and installation of the traps in VK3 For trap installation, the village was divided into six line-bands from the north to the south separated by green areas used for circulation in the village (Fig. 4d). All eaves and holes in the houses were blocked using cloth or sponge and a curtain was placed at each door by a large team of local and technical workers. In total, 1,313 traps were placed within the windows of houses to intercept incoming mosquitoes, and a new curtain made from regular cloth was provided to each house in VK3 to block mosquitoes from entering through the door. No constraints were required on the use of the doors or windows, and occupants were free to go to bed at any time. Mosquito collection To assess the trap performance, a monthly mosquito collection was performed from 12 selected traps (trapped mosquitoes) and matching houses (indoor resting mosquitoes) in VK3, while only indoor resting mosquitoes from eight houses were collected in VK5. Single-room houses (a single house with one window and one door) were randomly selected according to their geographic location (central, north, east, west, and south) in both villages. Houses were located far from each other, spaced at least 10 m apart to avoid human attractivity bias, and were monitored over nine days per month in both villages from July to October 2015. Mosquitoes were manually collected (on 36 collection days over the four months of the trial) with mouth aspirators in the traps and matching houses (for two hours) by three experienced collectors. The mosquito collection was simultaneously carried out in VK3 and VK5. To provide evidence of the impact of the traps being deployed on mosquito density reduction at village level, a pyrethrum spray catch (PSC) [ 45 ] was carried out simultaneously one day per month over the four month-trial, in 10 randomly selected houses in each village. These houses were different ones to the regular study houses in the villages. Assessment of mosquito species identification and allelic frequencies of kdr mutation in trap and house collection from Vallée du Kou over the study period Collected mosquitoes (traps and houses) were morphologically identified to genus, species, and physiological status [ 46 ], and then counted. A sub-sample was then preserved in 80% ethanol vials for subsequent genotyping to species level and to check on the frequency of the knock down resistance ( kdr) mutation [ 47 , 48 ]. Assessing wild female mosquito parity during trap deployment In addition, to assess mosquito population age structure [ 41 ] from July to October 2015, around 55 unfed female An. gambiae collected from the traps and houses were dissected and classified into parous and nulliparous mosquitoes, according to Detinova’s protocol [ 49 ]. Efficacy bioassays The efficacy of PPF-treated traps was assessed on two mosquito strains including susceptible An. gambiae- Kisumu and wild adult An. coluzzii collected at larval stage. The susceptible An. gambiae- Kisumu strain mosquitoes were released into traps to assess how long a PPF-treated net effect can last, and to follow the degradation dynamic over time. The morning following the blood meal, between 06:00 and 07:00 am, mosquitoes were transported by car while covered with a wet cloth (to maintain humidity) to the field. Once in the field, one hour rest time was given to the mosquitoes prior to the release. This rest time coincided with the monthly mosquito collection, where traps were emptied. Around 150 blood-fed female An. gambiae- Kisumu were then released 12 h after a blood meal once per month in VK3 (~ 50 females in each of two out of 12 monthly selected monitored traps) and in VK5 (50 females into one house). Mosquitoes were allowed to be in contact with the trap net for 30 mins, after which all of these mosquitoes were recaptured and brought to the laboratory for oviposition (fecundity) and egg hatchability (fertility) assessment. Only valid mosquitoes were oviposited. Two releases (July and August 2015) of the An. gambiae -Kisumu susceptible strain were performed over the study period. After each release-recapture process in VK5, an indoor spray with insecticide (Kaltox Paalga, SAPHYTO, Burkina) was performed to kill the non-recaptured mosquitoes. Anopheles gambiae s.l. mosquito larvae were randomly collected from breeding sites in VK3 and VK5 to evaluate the effect of PPF over time on the wild mosquito population. These larvae were reared at the IRSS insectary until adulthood. Three to-five-day-old female mosquitoes were processed in the same way as described in the above section prior to their release. A total of 100 blood-fed female An. gambiae were simultaneously released in VK3 (in two selected traps in the third and fourth regular monthly collection traps) and in VK5 (50 blood-fed females into one house), once a month over the two-month trial. Effect of pyriproxyfen-treated traps on wild blood-fed mosquitoes Blood-fed mosquitoes were collected from traps and houses in VK3 and VK5 to assess whether females that encountered PPF-treated traps overnight had reduced fecundity and fertility. These blood-fed mosquitoes were collected from each of the 12 randomly selected traps once per month over three months in VK3. Similar mosquito sampling was performed inside eight houses in VK5 and considered as controls. About 25 blood-fed female mosquitoes per trap per day in VK3 and the same per house in VK5 were morphologically identified to species. They were then brought back to the IRSS laboratory on the same day and were allowed to lay eggs into a single cup containing Whatman filter paper and 5 mL of water for a week. Cups were checked daily, and females that laid eggs were subsequently removed, killed, dried, and preserved into silica 1.5 mL cryotubes labelled for subsequent analysis. Eggs were counted under stereomicroscope and then hatched into rearing trays (43 × 26 × 15 cm) filled with 1 liter of tap water with TetraMin baby fish food (TetraMin®, Germany). Furthermore, to evaluate the impact of PPF on egg development as compared to a control as previously described [ 29 , 30 ], about 100 field blood-fed An. gambiae s.l. were randomly collected from the monthly collection traps in VK3 and compared to about 100 blood-fed females collected from VK5 testing houses. These mosquitoes were kept individually in 20 mL cups with Whatman paper and 5 mL of water, and around 30 of them were dissected per day at 24 h, 48 h and 72 h post-collection over three months. Physical conditions and cleanliness of the traps during the trial Immediately after trap installation, 50 nets were sampled and were checked for their physical integrity in 50 selected households over two months (one and two months after installation). The assessment of the integrity of the fabric was performed by visual examination without removing the nets. Any holes observed were assigned to one of four size categories according to WHO guidelines [ 50 ]: a hole size of 0.5-2.0 cm or ‘ a thumb but a fist but 2.5 cm or ‘> a head’. General trap integrity was assessed based on two measurements: The proportion of nets with any observed hole(s). The integrity of the nets was determined by counting the number of tears and holes as described: total number of coded nets with at least one hole of size (1-4) ×100 / total number of nets assessed in surveyed households. The proportionate holes index (pHI) for each net, calculated as the sum of the holes weighted by size for each net. For this group, the weights used to calculate the pHI were 1, 23, 196 and 576 as described below: pHI = 1 × number of size − 1 holes + 23 × number of size − 2 holes + 196 × number of size − 3 holes + 576 × number of size − 4 holes. To better correlate the holes index to an integrity status (net condition) for each sampled net, the pHI was categorized into ‘good’ (pHI ≤ 64), ‘serviceable’ (pHI ≤ 768) and ‘replace’ (pHI > 768). The trap net dirtiness (/cleanness) was also evaluated, and nets were classified and categorized into ‘clean’, ‘a bit dirty’, ‘dirty’, and ‘very dirty’. When the net was deemed irreparably damaged by dirtiness, it was replaced by another net of the same size. Socio-anthropological investigation on the use of the traps Qualitative and quantitative surveys were conducted from March to August 2015 from the beneficiaries of the LFET traps in VK3. The qualitative survey consisted of individual interviews with members of the community about their perception of the traps. There was direct observation of trap being used in the village. The usage of traps was witnessed elsewhere. The quantitative survey covered 276 inhabitants and was based on the level of acceptance of the trap by its users, the trap’s perceived effectiveness, and the limits of the trap. In addition, all inconveniences reported by users of the traps were recorded by a social worker and reported for subsequent remedial measures. A follow-up survey according to the WHO indices [ 50 ] was conducted once the traps were installed, any mishandling was also reported, and action was taken to resolve any problems raised either by a social worker or the users themselves. Parameters Measured And Statistical Analysis The outcomes of the large-scale field trial were: Mosquito density – calculated as the number of mosquitoes caught in the trap out of the total number of mosquitoes collected in the trap in the matching house (house where a LFET was placed and where the daily mosquito collection was performed) Direct mortality – calculated as the number of dead mosquitoes caught in the traps out of collected mosquitoes from the trap. Allele frequency – calculated as the number of resistant mosquito homozygote resistant (2nRR) and hybrid resistant (nRS) out of the 2x total of homozygote resistant (RR), and susceptible (SS), and RS the hybrid Resistant-susceptible. Age structure (parity) – defined as the number of female mosquitoes having laid eggs (parous) and nulliparous out of a total of dissected mosquitoes. Physical integrity of the traps – defined as a check of how a trap’s net was maintained in the intervention village. This physical integrity data was analysed using descriptive statistics (mean, median, interquartile range) to compare pHI values. Acceptability – defined as a socio-anthropological investigation on the use of traps by end-users and calculated by counting the “Yes” or “No” answers to the relevant questions, relating to the total number of interviewees. Descriptive data was summarized, inputted, and cross-checked in Microsoft Excel 2007 (Microsoft®, New York, USA), and R-4.0.4 was used to produce tables, graphs, counts, means and standard errors. A Generalized Linear Mixed Models (GLMM) with a Poisson or negative binomial distribution was used to choose the suitable distribution of the mosquitoes collected in the traps and houses from VK3 and VK5 respectively. A zero inflated Poisson mixed regression modelling tool was used to estimate the intervention (trap) effect on daily numbers of mosquitoes collected while accounting for a possible spatial variation in terms of total mosquitoes collected between VK5 and VK3. This was to check for a possible difference in terms of physiological status (gravid, blood fed, unfed), or for a possible temporal variation induced by monthly weather conditions (rainfall, humidity, or temperature). Therefore, two models were built including two random-intercepts, one random coefficient and zero inflation terms. The model structure is defined with trap, gonotrophic _status, status, rainfall, and humidity considered as the fixed effects (fixed effects = trap + gonotrophic _status + status + rainfall + humidity) and the total number of collected female mosquitoes as the response variable. Model 1: The probability of inflation terms was constant Y ∼ fixed-effects + random¬ (month) + random (village) + zero-inflation (∼1) + residual error Model 2: The probability of inflation terms was dynamic depending on the trap variable Y ∼ fixed-effects + random¬ (village /month) + zero-inflation (∼trap) + residual error, where Y is the total number of collected female mosquitoes. The sub-model of model 2 was built on basic count, zero inflated, and altered models to account for zero values in the data. In these models, the rainfall and humidity fixed effects were removed to compare their Root Mean-Square Error (RMSE) and the Median Absolute Error (MAE) used to choose the best model. The lower the RMSE, the better the model. The model with the lowest AIC (Akaike information criterion) was considered the best model to fit the data [ 51 ]. Akaike information criterion and recent developments in information complexity were used as these methods consider both suitability and complexity of the model to check the performance of zero-inflated models. To prevent the model from overfitting, trap data was split into a training set (0.8) and a test set (0.2). The different models were then tested on the training set and confirmed with the test set. For all analyses, the level of significance chosen was 5% (Supplementary Fig. 1). To assess the dynamic of mosquitoes between VK3 and VK5, pyrethrum spray catch data were analysed using a non-parametric pairwise test, Anova-glmmTMB. A one-way Anova was used to compare the age distribution of mosquitoes between VK3 and VK5. The PPF effect on female mosquito fecundity was calculated as the mean number of eggs per female that contributed to the oviposition. The fertility was measured as the mean number of larvae per female that contributed to oviposition. Following this, a non-parametric pairwise test, Anova-glmmTMB, was used to compare the reduction of fecundity and fertility between the group exposed to PPF-treated net and the control group. As the number of eggs and larvae from the different treatments did not follow a normal distribution, a one-way non-parametric analysis of variance (Kruskal Wallis test) was used to determine whether there was any difference between 30mg/m 2 and 20 mg/m 2 ai doses in terms of fecundity and fertility reduction. Weather related temporal measures such as the mean temperature, the mean relative humidity and rainfall, were collected from Burkina Faso’s meteorological station ( http://www.meteoburkina.bf ). Results Phase I: Selection of the most effective pyriproxyfen-dose Effects of selected pyriproxyfen doses on mosquito fecundity and fertility With susceptible An. gambiae- Kisumu strain and two pieces of net impregnated with 20 and 30 mg/m 2 , the dose 30mg/m 2 ai showed a better reduction in fecundity with a lower fecundity and egg fertility rate compared to 20 mg/m 2 (Kruskal-Wallis chi-squared = 15.2, df = 3, p-value = 0.0017). This 30 mg/m 2 ai dose was then selected to impregnate the tailored nets for the traps. Phase Iii: Large-scale Field Evaluation Of The Lehmann Funnel Entry Trap Mosquito collection A total of 18,884 and 15,650 An. gambiae mosquitoes were collected over the study period in VK3 (from selected traps and matched houses) and in VK5 (inside houses), respectively. Out of this, 87% (16,437 of the 18,884) were collected from the traps, including the following live mosquitoes: unfed (49%), blood-fed (40%), and gravid females (8%); and the following dead mosquitoes: unfed (1%), blood-fed (1%) and gravid females (1%). This compared to 13% (2,447 of the 18,884) from the matching houses in VK3. In VK5, of the 15,650 mosquitoes collected, 67% were found unfed, 28% blood-fed and 5% were gravid females. The sampling on average per night per trap in VK3 for dead mosquitoes ranged from 0.95 ± 0.19 to 31.76 ± 4.40 (means ± se), and for live mosquitoes from 8.96 ± 0.92 to 54.99 ± 5.34, caught in trap in VK3. This sampling average varied from 2.77 ± 0.43 to 10.18 ± 2.12 for live mosquitoes collected indoor in VK3, compared to between 1.65 ± 0.18 and 21.44 ± 2.02 for live mosquitoes collected indoors in VK5 (Table 2, supplementary Fig. 2). No dead mosquitoes were collected from the houses in either site. Table 2: Descriptive table of gonotrophic status of An. gambiae female mosquitoes in VK3 and VK5 over the study period. In terms of mosquito density reduction, there were significantly fewer mosquitoes collected from the houses in VK3 as compared to VK5, regardless of the gonotrophic status (unfed, df = 0.4964, 𝜒 2 = 2175, p < 0.0001, blood fed, df = 0.3525, 𝜒 2 = 1193.2, p < 0.0001, and gravid, df = 0.1439, 𝜒 2 = 665.27, p < 0.0001) (Table 3). Table 3: Comparison and proportions of Anopheles gambiae female mosquitoes collected per house in VK3 and VK5 over the study period. Other mosquito species collection in VK3 and VK5 over the study period During the study, 1,169 other mosquitoes were caught in traps, including species as An. coustani (2.6%), An. pharoensis (6.0%), Culex sp (87.3%), Mansonia sp (4.1%) and Aedes (0%) in VK3. In addition, 89 mosquitoes were collected in matched houses and identified as An. coustani (2.6%), An. pharoensis (6.7%), Culex sp (87.6%), Mansonia sp (1.1%). In VK5, 707 mosquitoes were collected from the houses comprising An. coustani (0.4%), An. pharoensis (4.2%), An. rufipes (0.1%), Culex sp (87.7%), and Mansonia sp (0.4%) (Table 4). Table 4: Numbers and proportions of other mosquito species caught in trap and house in VK3 and VK5 over the study period. Assessment of the mosquito density reduction over the three months of study in VK3 and VK5 The PSC with Kaltox Paalga showed that resting mosquito density (Table 5) in VK5 was significantly higher than in VK3 (rate = 0.662 se = 0.0113, df = 176, t-ratio = -24.131, p < 0.0001) over three consecutive months (from August to October 2015) (Table 6). These PSC results highlighted the effectiveness of traps in removing mosquitoes from the environment. In VK3, mosquitoes entered houses and were not able to get out, whereas in VK5, there were no traps and so the mosquito population density remained more and less constant. Table 5: Assessment of mosquitoes collected through pyrethrum spray catch over the three months of study period in VK3 and VK5. Table 6: Comparison of the mosquito density reduction over three months of study period in VK3 and VK5 Assessment of mosquito species identification and allelic frequency of kdr mutation in Vallée du Kou over the study period Anopheles coluzzii (formerly An. gambiae form M) was identified as the only Anopheles species in VK3 and VK5. The kdr mutation was genotyped in mosquitoes that were both exposed or not exposed to PPF-treated nets in order to estimate their frequencies in the natural populations from Vallée du Kou. Out of 512 sub-samples analyzed, PCR assay revealed that most of the mosquitoes were highly resistant to pyrethroids and DDT, 0.9 ( kdr based mechanism), as shown in Table 7. Table 7: Assessment of mosquito species identification and allelic frequencies of kdr mutation in trap and house collection from Vallée du Kou over the study period. Assessing wild female mosquito parity during trap deployment Ovary dissection indicated that the mosquito population kept a stable age structure as compared to the beginning of the intervention with ~ 2% (1/55) of the population being parous females in both VK3 and VK5 (𝜒 2 = 0.00016532, df = 1, p-value = 0.9897). Toward the end of the intervention, 12% (7/58) of the females were parous in VK5 as compared to ~ 3% (2/69) in VK3 (𝜒 2 = 4.0247, df = 1, p-value = 0.04484) (Table 8). Table 8: Assessing wild female mosquito parity during trap deployment in July, August and September 2015. Evaluation of environmental effects on mosquito collection between VK3 and VK5 In Table 9, the intercept estimates the (Log-scale) expected mean number of mosquitoes collected in the trapping area, in a village at given months, and the fixed slope coefficient estimates the change in the expectation associated with rainfall, humidity, status and mosquito gonotrophic status. The model ZIPMM results indicate that following fixed trapping effects (trap), status (dead), rainfall, humidity, gono_status (blood-fed), gono_status (gravid) is significantly different from 0 at 5% level. In addition, these results reveal a statistically significant positive relationship between total mosquitoes and trapping area, and a statistically significant negative relationship between total mosquitoes and status, rainfall, humidity, gono_status. The power of a model to fit the data was considered based on the lower AIC and BIC basic models results. Table 9: Model fitting of Poisson models for number of collected mosquito count dataset. From the above metrics, the zero inflated negative binomial model was chosen as the best predicting model, fitting the data with the lowest RMSE value 33.05, and lowest MAE value 13.32. This result is consistent with the fact that this kind of model deals with zero inflated data (Table 10). Table 10: Model fitting of the ZIP and ZINB model for the dataset on the trap collection. The ten deciles test set Zero Inflated Negative Binomial (ZINB) (Fig. 5) shows that the actual (original) and predicted values of mosquitoes collected are very close at the beginning and diverge in the middle. This is, on average, over or under predicting in the middle deciles, while in the last decile, actual and predicted, there is a better match. Efficacy bioassay Anopheles gambiae -Kisumu strain released in July and August 2015 into the traps for 30 minutes showed a significant higher reduction of fecundity (mean number of eggs per female) in VK3 as compared to VK5 (egg Control / (PPF_30mg/m 2 ) rate: 1.34, se: 0.091, df: 174, t-ratio: 4.318, p < 0.0001). Similarly, a greater female fertility reduction (mean number of larvae per female) was observed in VK3 as compared to VK5 (larvae Control / (PPF_30mg/m 2 ) rate: 0.681, se: 0.078, df: 174, t-ratio: -3.358, p < 0.0001). With An. gambiae mosquitoes collected at larval stage in both sites in August and September 2015, there was a greater fecundity and fertility reduction in VK3 than in VK5 (egg Control / (PPF_30mg/m 2 ) rate: 1.45, se: 0.041, df: 214, t-ratio: 13.005, p < 0.0001 and larvae Control / (PPF_30mg/m 2 ) rate: 3.27, se: 0.026, df: 214, t-ratio: 14.750, p < 0.0001). Effect of pyriproxyfen-treated traps on field blood-fed mosquitoes With field blood-fed female An. gambiae collected from the traps in July, August and September 2015, the fecundity rate was similar in both sites (egg Control / (PPF_30mg/m 2 ) rate: 1.01, se: 0.009, df: 1147, t-ratio: 1.292, p = 0.1967), while a greater reduction in fertility was observed between VK3 and VK5 (larvae Control / (PPF_30mg/m 2 ) rate: 1.29, se: 0.019, df: 1147, t-ratio: 17.034, p < 0.0001) (Table 11). Furthermore, of 100 field blood-fed An. gambiae mosquitoes (collected from the traps monthly in VK3 and dissected after 24h, 48h, and 72h), 80 females had non-developed ovaries compared to normal female ovaries from VK5 (Supplementary Fig. 3). The PPF effects on fecundity and fertility reduction are summarized in Supplementary Table 3. Table 11: Comparison of pyriproxyfen effects on susceptible Anopheles gambiae -Kisumu and field mosquitoes tested over two months and three months respectively in VK5 and VK3. Physical condition and cleanliness of the of the traps during the trial The proportion of nets with at least one hole was ~ 76% over the two months of the study. Moreover, the number of holes (141) was higher in the second month of the survey than the first month (91). The mean score of the Hole index was significantly higher in the second month at 2.86 (95% CI = 2.079–3.641) as compared to the first month with 1.82 (95% CI = 1.244–2.396; p = 0.0298). The number of holes did not seem to change significantly based on the location of the net, with 69 holes found on entry window nets and 74 holes on rear window nets (p > 0.05). The data showed a greater number of holes in category hole-1 than that observed in the two other groups (categories hole-2 and hole-3) (121:20:1 respectively) (p < 0.0001). The location of holes in the net was explored during the second month. Of the 141 counted holes, ~ 53 were found on the front of the nets, 38 on the seams, 30 on the length, and 20 on the width. Twenty five percent (25%) of the holes were found along the length and width of the net followed by 10% and less than 1% respectively on the front side and the seams of the net. The median proportionate hole index (pHI) was 3.5 with IQR (2-27.5). The pHI for each net showed good net utilization as 66% (33/50) of nets were found in a good condition (pHI ≤ 64) and only 1% was found just serviceable (pHI ≤ 768 – serviceable). About 75% of the nets were deemed to be clean. The dirty nets were typically installed at the windows of houses built with bricks made of mud. Moreover, the dirtiest nets were located at the front of the house. At the end of each survey, nets with several holes were replaced by new ones with the same dimensions. Socio-anthropological investigation on the use of the traps: Acceptance and benefits attached to the trap Qualitative and quantitative data showed a good level of acceptance of the traps by the interviewees. About 80.8% (223/276) of the beneficiaries declared that the trap reduced the number of mosquitoes in the house. This was appreciated by the users, as shown by the following quote, which is taken from an interview with a resident: "when you look into the object, there is a lot of mosquitoes like that. If all these mosquitoes should enter the home! Good gracious!" Table 12 below shows the details of the various benefits that the traps brought to the population of VK3. Importantly 98.55% (272/276) of the respondents claim to have had peaceful sleep since trap installation, and 98.91% of the respondents believe that the trap will help reduce disease in the area. Table 12: Socio-anthropological investigation on the use of the Lehmann Funnel Entry Traps over the study period in VK3, Acceptance and benefits attached to the Lehmann Trap. Discussion The main objective of this study was to show the potential of the Lehmann Funnel Entry Trap to control malaria vectors in insecticide-resistant mosquito population settings. In addition, one challenge of this study was to produce as many LFETs as was necessary to cover the entire intervention village of VK3. A total of 1,313 traps were deployed in VK3, which contributed to a reduction of indoor mosquito density of 90% throughout the four months of the trial. Traps have proven to be effective in suppressing An. gambiae species and have successfully caught a variety of other mosquito species, including species that might have bitten the villagers if there were no traps deployed. Traps protected the population of the village from indoor mosquito biting in addition to the other malaria control methods already in use such as bed nets, IRS, repellent plants, etc. This could have consequently reduced malaria transmission in the village. This study result is consistent with a study showing how screening reduced malaria mosquito house entry and contributed to prevention of anaemia in children [ 16 ]. Ensuring complete trap coverage in VK3 may have contributed to mosquito density reduction as mosquitoes were repeatedly trapped after emerging from rice breeding sites. However, as the intervention village is surrounded by water, it would be a challenge to reduce mosquito density to a very low level. This challenge could have been minimized somewhat if the study site design was randomized, as the suppressing effect of the trap could have been more visible with a study designed to take place in different ecological settings. The successful implementation of LFET trap installation as a malaria tool may focus on the direct monitoring of the adult mosquito population, as this provides a real-time picture of the effects of the intervention [ 52 , 53 ]. As part of this direct monitoring, the PSC results indicated that traps have significantly reduced the mosquito population density at the community level in VK3 as compared to the control village, VK5. In this study, the mosquito population was exclusively composed of An. coluzzii (the former M molecular form) in Vallée du Kou, which is in alignment with the previous studies [ 29 , 54 ]. The kdr L1014F mutation conferring resistance to pyrethroids and to DDT was found in the samples analysed to be very high (~ 0.9) when the study was carried out. This result is consistent with previous studies reporting a high level of resistance in mosquito populations due to the extensive use of pesticides in rice and cotton fields in the village [ 37 , 55 , 56 ]. In this study, environmental factors such as rainfall, humidity, and temperature had a great impact on mosquito densities. Interestingly, the dissection of the ovaries of mosquitoes collected indicated that the mosquito population had the same age structure at the beginning of the intervention (~ 2% female parous) in both villages. However, toward the end of the intervention, 12% of the dissected females were scored parous in the control village against ~ 3% in the intervention village, indicating that the trap was cumulatively extracting and killing old females capable of transmitting malaria in the intervention village. Moreover, traps removed and killed a proportion of older outdoor mosquitoes from the village, which may have impacted an outdoor malaria transmission, as recently shown in other studies [ 24 , 57 – 59 ]. Furthermore, adult dissection results showed that 80% of the females dissected had failed to develop their ovaries as previously reported [ 29 , 30 , 53 ]. As the LFETs were impregnated with PPF, this could have increased the performance of the trap by sterilizing female mosquitoes that encountered the trap net, preventing them from having offspring and consequently decreasing the malaria vector population in the village. For this trial, susceptible mosquitoes exposed to PPF-treated nets over two months of the study showed a fewer number of eggs per female and a lower egg hatch rate in the intervention village as compared to the mosquitoes from the control village. Similarly, field An. coluzzii collected as larvae reared through to adulthood in the laboratory indicated a significant reduction of eggs per female and a lower egg hatch rate over two months. This result is consistent with the study performed by Harris et al. (2013), where they found that female mosquitoes blood-fed one day prior to PPF exposure produced no viable offspring during this gonotrophic cycle [ 41 ].The few female spermathecae dissected during this assessment showed an insemination rate of up to 90% which is consistent with the results from previous work, which showed that the insemination rate of susceptible An. gambiae -Kisumu females, maintained in cages with males over 3–5 days in the insectary, was above 90% [ 29 ]. This insemination rate was used as a baseline to analyse female fecundity and egg fertility. However, the fecundity of the mosquitoes collected from the traps in the intervention village was similar to the control mosquitoes. This may be due to the short contact time of some mosquitoes, as the entry rate and time spent on the trap’s netting are not evenly spread and similar for all mosquitoes. Additionally, the number of blood feedings and the age of females caught in traps may differ and impact their fecundity and ultimately the egg fertility. These aspects were not recorded and were considered as some of the limitations of this study. The impact of PPF on other trapped mosquito species was not assessed in this study. Nevertheless, recent studies have demonstrated a PPF sterilizing effect on Aedes spp, and Culex spp etc. when it is transported and spread by mosquito tarsi to breeding sites [ 53 ]. Therefore, this could have impacted other mosquitoes within and around the LFET trial site. VK3 is one of seven discrete villages (VK1 to VK7) within the Vallée du Kou area. The village is close to its sister villages VK2 and VK4, on the western and eastern side respectively, and is surrounded by rice fields. The poor isolation of VK3 has contributed to its sustained high mosquito density. It has been shown that mosquitoes can disperse up to two kilometers looking for a blood meal and so this could have impacted mosquito density in VK3 [ 60 ]. The LFET has proven efficacy in controlling highly resistant malaria vectors. A recent study completed in the same ecological setting in Burkina Faso has shown that newer branded nets struggled to kill mosquitoes in this area, which makes it clear that additional intervention tools are needed to control resistant mosquito populations [ 61 ]. The fact that the trap can further help to control the mosquito population makes it very attractive. Once installed, the trap requires minimal adaptations in human behavior, and unlike mosquito bite prevention tools such as bednets, it protects everyone who is sleeping within the dwelling. Importantly, most of the interviewees acknowledged that the trap reduced mosquito biting and allowed them to sleep peacefully, which is one of the most important criteria that impacts the acceptability of conventional intervention tools such as bed nets [ 61 ]. The high acceptance of the LFET may explain the overall good condition of the traps seen at one to two months post-deployment. Although the retail price of a single trap was estimated to be too high by the villagers, it is worth noting that they all wanted to keep the trap after the evaluation, meaning that the trap is very useful to them. It is common that most households receive malaria preventive methods, including bed nets, for free in Burkina Faso [ 62 ]. A cost-effectiveness study has not yet been done but it is important to note that a single trap installed in a house protects everyone sleeping within that house. Conclusion The Lehmann Funnel Entry Trap has proven efficacy in controlling highly resistant malaria vectors. The additional effect of PPF also enhanced its efficacy. The acceptance of the trap was high, as the beneficiaries could see the number of mosquitoes trapped in it every day, and it was acknowledged that the trap reduced mosquito biting and allowed peaceful sleep. Although the deployment of the LFET across an entire village showed a positive impact, a trial in an isolated site with a high mosquito population could better show the suppressing effect of the trap. Additionally, a randomized controlled design of the study could better show the effects of the traps on mosquito density reduction. No survey data on parasitemia and malaria cases were collected during this trial and so a trial with malaria epidemiological end points could link the performance of the trap against malaria. Nevertheless, there is growing evidence that the trap installation in VK3 may have reduced malaria transmission [ 63 ]. Assuming that the size of the trap can be further reduced and the price to the user can be lowered, LFETs may represent a viable business opportunity to entrepreneurs. The next step is to build a business plan and work alongside local window manufacturers to help them to start manufacturing the traps at a large scale. Efforts will be devoted toward the national malaria control program in Burkina Faso to integrate the Lehmann Funnel Entry Trap into the malaria control toolbox. Abbreviations Matching house: a house where a LFET was placed and where a daily mosquito collection was performed. PPF treated trap net: a net impregnated with a PPF dose with an active ingredient (a.i.) mg/m 2 and used to cover a LFET metal frame. ZIP : Zero inflated Poisson. ZIPMM : Zero inflated Poisson Mixed Model ZINB : Zero Inflated Negative Binomial RMSE : Root Mean-Square Error MAE : Median Absolute Error AIC: Akaike Information Criterion VK3: Vallée du Kou 3 VK5: Vallée du Kou 3 GLMM: Generalised Linear Mixed Models LFET: Lehmann Funnel Entry Trap Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Availability of data and materials All data generated and analysed during this study are available on Github: https://github.com/RogerSANOU/Lehmann-trap-dataset.git. Authors’ contributions RS and HM carried out the field work, participated in the analysis of the data, and wrote the original manuscript. AO and AAM carried out the sociological field and geographic information system work respectively and revised the manuscript. ASH carried out the physical integrity work and revised the manuscript. SPS, KB, SAM, AMGB, LPT and RKD revised the manuscript. BDS carried out the statistical analyses. AD supervised the entire work. All authors have read and approved the final manuscript. Acknowledgements We are grateful to Tovi Lehmann, Adama Dao and Alpha Yaro, whose previous work inspired us to develop these new traps and with whom we had fruitful discussion. Authors thank Guel Z. Hyacinthe, Diabaté Noufou for their contribution to the fieldwork. We are also grateful to Kieran Callanan for proofreading and editing. The authors thank the villagers who accepted the traps to be tested in their homes. Funding This work was funded by Grand Challenges Canada (GCC, Grant ID: S6 0510-01-10) to DA. Author details 1 Institut de Recherche en Sciences de la Santé/Centre Muraz, Bobo-Dioulasso, Burkina Faso. 2 Université Nazi BONI de Bobo-Dioulasso, PO 1091. References WHO. World malaria report 2020- WHO. 2020. Jill N, Ulrich DP 1, Naranjo2 TO. Alimi1, Günter C. Müller3 and JCB. 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BioMed Central; 2020;1–15. Available from: https://doi.org/10.1186/s13071-020-04221-z . Traoré A, Badolo A, Guelbeogo MW, Sanou A, Viana M, Nelli L, et al. Anopheline species composition and the 1014F-genotype in different ecological settings of Burkina Faso in relation to malaria transmission. Malar J [Internet]. BioMed Central; 2019;18:1–10. Available from: https://doi.org/10.1186/s12936-019-2789-8 . Dabire RK, Diabat A, Namountougou M, Djogbenou L, Wondji C, Chandre F, et al. Trends in Insecticide Resistance in Natural Populations of Malaria Vectors in Burkina Faso, West Africa: 10 Years’ Surveys. Insectic - Pest Eng. 2012. Toé KH, Falé SN, Dabiré RK, Ranson H, Jones CM. The recent escalation in strength of pyrethroid resistance in Anopheles coluzzi in West Africa is linked to increased expression of multiple gene families. 2015;1–11. Durnez L, Coosemans M. Residual Transmission of Malaria: An Old Issue for New Approaches. Anopheles mosquitoes - New insights into Malar vectors. 2013. Hiscox A, Homan T, Mweresa CK, Maire N, Di Pasquale A, Masiga D, et al. Mass mosquito trapping for malaria control in western Kenya: Study protocol for a stepped wedge cluster-randomised trial. Trials [Internet]. Trials; 2016;17:1–12. Available from: http://dx.doi.org/10.1186/s13063-016-1469-z . Killeen GF. Characterizing, controlling, and eliminating residual malaria transmission. Malar J. 2014;13:1–22. Epopa PS, Millogo AA, Collins CM, North AR, Benedict MQ, Tripet F, et al. Anopheles gambiae (s.l.) is found where few are looking: assessing mosquito diversity and density outside inhabited areas using diverse sampling methods. Parasites and Vectors. 2020;13:1–11. Toé LP, Skovmand O, Dabiré KR, Diabaté A, Diallo Y, Guiguemdé TR, et al. Decreased motivation in the use of insecticide-treated nets in a malaria endemic area in Burkina Faso. Malar J. 2009;8. Bocoum FY, Belemsaga D, Adjagba A, Walker D, Kouanda S, Tinto H. Malaria prevention measures in Burkina Faso: Distribution and households’ expenditures. Int J Equity Health. 2014;13:1–6. Furnival-Adams J, Olanga EA, Napier M, Garner P. House modifications for preventing malaria. Cochrane Database Syst Rev. 2021;2021. Tables Table 1 to 12 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files LFET2SupplementaryFigures.pptx LFET2SupplementaryTables.pptx LFET2Tables2015.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2432364","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":164309009,"identity":"731bc473-e2de-44d7-99fd-89e53456aa34","order_by":0,"name":"Roger Sanou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDADfgYGNihNLJBsgGgB0sQCgwPEapFvP3vwMe8eG7vNN5KfPfhQwSBhTkiPwZm8ZGOeZ2nJ226kmRvOOMMgIXOAkBaGHDNpngOHk81uJJhJ87Yx1EkQdFj/G4gW4xnp30BaJAhqYbgBscXOQCLHjDgtBjfeGBvOOZCWIHHmTZnkjDMShLXI9+cYPnhzwMaevz19m8SHChsiHAYFiQ0CCSCaaA0MDPYM/AeIVz0KRsEoGAUjCwAAHOw6YZMyLIUAAAAASUVORK5CYII=","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Roger","middleName":"","lastName":"Sanou","suffix":""},{"id":164309010,"identity":"a00c6539-d9d0-41a0-affb-7e1115b2b0e2","order_by":1,"name":"Hamidou Maïga","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hamidou","middleName":"","lastName":"Maïga","suffix":""},{"id":164309013,"identity":"1587f3c0-e12c-4c96-8655-dad1317a0e29","order_by":2,"name":"Bazoumana D. Sow","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bazoumana","middleName":"D.","lastName":"Sow","suffix":""},{"id":164309014,"identity":"a82b9224-3382-4f2e-ab05-19e0e73912f2","order_by":3,"name":"Adama Ouema","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Adama","middleName":"","lastName":"Ouema","suffix":""},{"id":164309016,"identity":"daf5c248-ebf6-4fdb-af1e-25773a7bd055","order_by":4,"name":"Abdoul Azize Millogo","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abdoul","middleName":"Azize","lastName":"Millogo","suffix":""},{"id":164309017,"identity":"2d558614-24c3-4f24-a12e-2a4e39083410","order_by":5,"name":"Koama Bayili","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Koama","middleName":"","lastName":"Bayili","suffix":""},{"id":164309018,"identity":"6eade634-391d-43c2-8226-06ddae6ad44b","order_by":6,"name":"Aristide Sawdetuo Hien","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aristide","middleName":"Sawdetuo","lastName":"Hien","suffix":""},{"id":164309019,"identity":"74240a0a-2c48-4be4-a119-af1c8881adba","order_by":7,"name":"Simon P. 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Dabiré","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Roch","middleName":"K.","lastName":"Dabiré","suffix":""},{"id":164309024,"identity":"42e4ecf8-46eb-494c-94da-1106f8f0f28f","order_by":12,"name":"Abdoulaye Diabaté","email":"","orcid":"","institution":"Institut de Recherche en Sciences de la Santé (IRSS) / Centre Muraz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abdoulaye","middleName":"","lastName":"Diabaté","suffix":""}],"badges":[],"createdAt":"2022-12-31 23:59:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2432364/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2432364/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":31179696,"identity":"7b96159c-effa-4b8c-b570-fb3814ff52df","added_by":"auto","created_at":"2023-01-05 18:31:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":584703,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Vallée du Kou and its different villages\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/8f5be01dea985bb08e9fdbe7.png"},{"id":31179939,"identity":"cff61d97-ea55-44f3-896e-bb44389daa80","added_by":"auto","created_at":"2023-01-05 18:39:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":418066,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Lehmann Funnel Entry Trap within a house, (B) outward view through a window, (C) and a curtain at the door, (D) dimensions of the trap.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/2e647efa05233f6b2bf297f0.png"},{"id":31179942,"identity":"a0362b3c-96dd-44bf-a7bd-65ca5d7aea83","added_by":"auto","created_at":"2023-01-05 18:39:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1472296,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Local window manufacturers producing the traps, (b) tailors sewing nets, and (c) field entomology local workers recruited to install the traps.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/a69aef4e697c6e4a4e472f5e.png"},{"id":31179940,"identity":"9613f060-b4c9-4d41-a505-1746af8fdbc6","added_by":"auto","created_at":"2023-01-05 18:39:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":606853,"visible":true,"origin":"","legend":"\u003cp\u003eGeoreferencing (a) of house distribution, (b) the number of windows, (c) human density, and (d) the distribution of the households and colored bands showing the scheme of trap installation in VK3.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/a9b74aeac7a118d21843e7eb.png"},{"id":31179702,"identity":"31199a77-a68b-4da3-ab6d-a39e5f8f9eaa","added_by":"auto","created_at":"2023-01-05 18:31:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":58476,"visible":true,"origin":"","legend":"\u003cp\u003eTen deciles test set (actual and predicted values) for ZINB.pngprediction of mosquito catches in VK3\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/f1f0545319607041243bed4e.png"},{"id":44494125,"identity":"2d76ec44-9531-4ccc-bb81-c36086117072","added_by":"auto","created_at":"2023-10-12 08:52:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3885185,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/847c3b96-d6e1-40a2-b6e4-534062f30202.pdf"},{"id":31179700,"identity":"0ace2720-cd42-435c-a205-63371f5c905a","added_by":"auto","created_at":"2023-01-05 18:31:08","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1032359,"visible":true,"origin":"","legend":"","description":"","filename":"LFET2SupplementaryFigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/cc047a26d550f645fd344696.pptx"},{"id":31179697,"identity":"5a461b83-d9f0-48c0-b575-fdfb15a96ad8","added_by":"auto","created_at":"2023-01-05 18:31:08","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":50632,"visible":true,"origin":"","legend":"","description":"","filename":"LFET2SupplementaryTables.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/492bcde037ac454b2b1cebf3.pptx"},{"id":31180288,"identity":"b7834a8a-8cc1-4e2d-b6ed-ab202206cca2","added_by":"auto","created_at":"2023-01-05 18:47:08","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":91485,"visible":true,"origin":"","legend":"","description":"","filename":"LFET2Tables2015.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2432364/v1/32be2b28dd473c6cc014518e.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Large-scale deployment of pyriproxyfen-treated Lehmann’s Funnel Entry Traps to control malaria mosquito populations","fulltext":[{"header":"Background","content":"\u003cp\u003eVector control has contributed to the reduction of malaria burden over the last few years. According to a recent WHO report, the number of deaths due to malaria in 2019 is estimated to be 409,000 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Several vector control related factors may have contributed to this reduction, such as Indoor Residual Spraying (IRS) and the mass distribution campaigns of Insecticide Treated Nets (ITNs) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the development of the emergence and spread of insecticide resistance in malaria vectors is threatening the future of vector control [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], making it clear that alternative tools are needed to sustain vector control toward malaria elimination. Thus, discovering new tools or improving the effectiveness of the existing ones should be prioritized [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince the link was established between malaria and mosquitoes, window screening and nets have been used as part of malaria vector control management [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. More recently, studies using screens within windows, on ceilings, and on doors have proven to be effective in reducing the number of mosquitoes entering homes. Consequently, there is added personal protection against mosquito bites as well as community protection through added mosquito mortality [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Whilst some studies have established a relation between malaria illness and the type and quality of dwelling construction [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], other studies have demonstrated that in addition to quality of construction of the home, modifications to prevent mosquitoes from entering houses can help reduce malaria transmission by lowering human exposure to infectious bites [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Interestingly, the use of mosquito-repellent plants in the home has also been shown to reduce mosquito densities [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and there is a significant reduction of anaemia in children living in homes with window screens [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough these methods deny mosquitoes\u0026rsquo; entry to a dwelling, mosquitoes are not killed and so they can still bite outdoors and transmit malaria parasites [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To date, most malaria transmission occurs indoors, and several malaria vectors bite indoors for their blood meal in human dwellings [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, some malaria transmission may occur outdoors [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. As such, denying mosquito access to indoor living spaces is a worthy cause, but killing blood-seeking (and resting-site-seeking) mosquitoes could be a more effective goal.\u003c/p\u003e \u003cp\u003eOther alternative tools for insecticide resistance management include the use of molecules such as pyriproxyfen 10EC (PPF), which is a juvenile hormone analogue that mimics a natural insect hormone, essential for the normal physiological growth, development, and maturation of juvenile insects. As mosquitoes are holometabolous insects, the growth regulator hormone that PPF replaces is essential for larval development and its removal leads to undeveloped miniated pupal or adult stages [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Formerly used as a larvicide, PPF has been tested on adult mosquito species and has shown an interesting negative effect on female fecundity and egg fertilization [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Adding a PPF-treated net to a trap may therefore increase the effects on mosquito density reduction by additionally sterilizing any mosquito encountering the net. This proposal led to the development of a new approach, which considers mosquito behavior and typical house entry routes for mosquitoes. Within this approach, Lehmann Funnel Entry traps (LFETs) were tested in high and low vector density settings as a malaria vector control tool in development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, three new prototypes were created and successfully tested in two ecological settings to the southwest and northwest of Bobo-Dioulasso, Burkina Faso [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], in view of further use against malaria vectors. Once installed into a window, a LFET catches and kills all mosquitoes that attempt to access the house through the relevant entry point. Consequently, members of the household may be prevented from being bitten if all windows are secured with traps. Once a mosquito enters the small opening of the trap on the outside of the home, it becomes trapped and will remain so until dehydration kills it. In this trial, the traps were all installed at the same time in the intervention village, which allowed the blocking of all window entries in all of the houses, and as a consequence, mosquito densities were reduced at community level. Blocking all windows within a house with screens, helps to remove indoor resting and anthropophagic mosquitoes.\u003c/p\u003e \u003cp\u003eMost vector control interventions against malaria require active participation from the local population to ensure proper adherence [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Recent studies have demonstrated the important role of stakeholder\u0026rsquo; engagement in the successful control and elimination of many infectious diseases. Communities should have detailed information on the intervention or program in development at the beginning of any given study, perhaps even early on the design of the program, as well as during the study and at the end. An accurate timeline is also important for transparency reasons [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, as the space within the household has multiple functions, the homeowner\u0026rsquo;s full buy-in is required, as LFETs (or any other materials important to the study) may require reorganization at home. Given that the LFET is an indoor intervention tool, community engagement is especially important to ensure the traps are sustainable and fully operational throughout the use period.\u003c/p\u003e \u003cp\u003eThe objectives of this trial were to assess the impact of a large-scale deployment of PPF-treated traps in terms of mosquito density reduction, to evaluate the effects of PPF on female \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes from the laboratory and the field in an intervention village compared to a control village. In addition, the age of mosquitoes, as well as the physical integrity of the traps\u0026rsquo; nets, were assessed over the study period. Sociological endpoints were also reported in this study.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eThe study was carried out in Vall\u0026eacute;e du Kou, Burkina Faso (11˚23' N, 4˚24' W, Fig.\u0026nbsp;1), in an irrigated rice field area. The site is characterized by wooded savannah across 1,200 ha and contains seven discrete villages. The mosquito population in the village is resistant to insecticides and so a solution is required. Relatively high mosquito densities are observed annually during August and September, corresponding to the peak of the rainy season. \u003cem\u003eAnopheles coluzzii\u003c/em\u003e is predominant throughout the year and \u003cem\u003eAn. gambiae\u003c/em\u003e is observed toward the end of the rainy season (frequencies fluctuating between five and 20%). Both species are highly resistant to pyrethroids and DDT (\u003cem\u003ekdr\u003c/em\u003e frequency (0.8\u0026ndash;0.95) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Design And Period\u003c/h3\u003e\n\u003cp\u003eThe study was carried out in three sequential phases that encapsulated product optimisation through to large-scale field deployment as follows:\u003c/p\u003e \u003cp\u003e \u003col style=\"list-style-type: upper-roman;\"\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSelection of the most effective PPF dose (May 2015, in the laboratory)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTrap manufacturing and impregnation (May \u0026ndash; June 2015)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLarge-scale field evaluation of the LFET (July to October 2015, in the field site.) (Table\u0026nbsp;1: Gantt chart of large-scale field evaluation of LFET)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eTwo villages, Vall\u0026eacute;e du Kou 3 (VK3) and Vall\u0026eacute;e du Kou 5 (VK5), were selected as intervention village (IV) and control village (CV), respectively. The control site was selected as it presents the same ecological characteristics in terms of mosquito densities and species as VK3, and was located 1 km away, which helped minimize any potential PPF contamination.\u003c/p\u003e \u003cp\u003ePrior to trap fabrication, a general survey of the houses and windows was conducted in VK3 using a Global Positioning System (GPS) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] to count all the inhabited houses. Traps were then produced and deployed across the entire VK3 village.\u003c/p\u003e\n\u003ch3\u003ePhase I: Selection Of The Most Effective Pyriproxyfen-dose\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory mosquitoes\u003c/h2\u003e \u003cp\u003eMosquitoes were maintained at the Institut de Recherche en Sciences de la Sant\u0026eacute; (IRSS) insectary under controlled conditions (Temperature 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u003csup\u003eo\u003c/sup\u003e C and relative humidity 80\u0026thinsp;\u0026plusmn;\u0026thinsp;10%). For testing purposes, female and male mosquito cages were set up with pupae on the same day, maintained together until the day of testing (three days later at least), when females were removed from the cage. This time allowed them to mate before testing. Previous work done on the same laboratory colonies from the IRSS insectary showed an insemination rate of \u0026gt;\u0026thinsp;90%. Prior to cone testing, some females were checked to calculate the insemination rate [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Spermathecae were dissected under a magnifying glass 24 hours before bioassay to check this. Females were then blood fed when it was shown that the insemination rate was up to 90%. Three- to five-day-old female mosquitoes were starved for six hours by removing the 5% glucose (weight/volume, w/v) solution prior to blood feeding. The blood feeding took place at 18:00 for 45 minutes using direct rabbit feeding in the laboratory. The cage was blood-fed with males still inside. The non-blood-fed females were sorted and discarded, and only blood-fed mosquitoes were kept overnight and provided with glucose solution in cotton balls for the experiments. The laboratory mosquitoes were made up of susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e-Kisumu, resistant laboratory strain \u003cem\u003eAn. coluzzii\u003c/em\u003e, and field \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. collected at larval stage from breeding sites in both villages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffects of selected pyriproxyfen doses on mosquito fecundity and fertility\u003c/h2\u003e \u003cp\u003ePrior to impregnating the trap net, preliminary laboratory testing with two doses of PPF liquid (10EC) was conducted to assess PPF efficacy on susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e-Kisumu. After this, the most effective sterilizing dose was selected. A piece of net was impregnated at 20 mg/m\u003csup\u003e2\u003c/sup\u003e or 30 mg/m\u003csup\u003e2\u003c/sup\u003e of active ingredient (ai) selected according to previous studies [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Three- to five-day-old blood-fed female mosquitoes were put in contact with impregnated pieces of net in WHO cones for three minutes. Then, these mosquitoes were transferred individually into cups (200 mL) containing a filter paper for oviposition 24 hours post exposure. A daily manual count of the number of eggs laid was performed to evaluate the fecundity. In cups where eggs were observed, water was added in order to hatch the eggs. The number of larvae were also counted. After the subsequent three days following oviposition, the number of females that laid eggs, and those that died before laying were counted. The fecundity and fertility of treated mosquitoes were assessed as compared to a control under laboratory conditions. It was shown that the PPF dose with the most desired effect on the tested mosquitoes was 30 mg/m\u003csup\u003e2\u003c/sup\u003e ai. This was also the case in previous studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhase Ii: Trap Manufacturing And Impregnation With Pyriproxyfen\u003c/h3\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTrap manufacturing\u003c/h2\u003e \u003cp\u003eThe traps were made from a metal frame (69 \u0026times; 51 \u0026times; 82.5 cm) and were fitted from the bottom to the top with a regular mosquito net to prevent any mosquitoes or other insects from escaping the trap once inside (see Sanou et al., 2021, [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] for more details) (Fig.\u0026nbsp;2). All of the windows of the inhabited houses (non-inhabited house windows were secured simply with a net, to reduce the number of mosquito-resting sites) were counted and measured to manufacture the traps accordingly. Each manufactured net covers the trap entirely, which itself fits into the window perfectly. Each trap\u0026rsquo;s net also has a sleeve for easy access, to open and/or close the window.\u003c/p\u003e \u003cp\u003eThe metal manufacturer first produced a sample of each trap size. Then, the remainder of the traps were produced, painted with neutral oil (Fig.\u0026nbsp;3a), labelled according to dimensions, before transportation to VK3. The trap samples were also sent to a tailor in order for the nets to be made to size (Fig.\u0026nbsp;3b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTrap impregnation with pyriproxyfen\u003c/h2\u003e \u003cp\u003eAfter the traps were manufactured, they were impregnated with PPF. Through weighing the equipment, the amount of water and PPF solution needed for each net size was determined (Supplementary Table\u0026nbsp;1) according to the WHO insecticide impregnation process with pyriproxyfen 30 mg ai /m\u003csup\u003e2\u003c/sup\u003e (PPF). For each set of dimensions, calculations were made to ensure the right amount of insecticide and water necessary to entirely coat the net (Supplementary Table\u0026nbsp;2). After the nets were coated, all trap nets were dried overnight indoors and wrapped into labelled sachets for easy identification prior to their transfer into the field for installation by the team and local workers (Fig.\u0026nbsp;3c).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhase Iii: Large-scale Field Evaluation Of The Lehmann Funnel Entry Trap (Dup: Abstract ?)\u003c/h3\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eThe large-scale field trial was designed with one intervention area and one control area, and the entomological endpoints were assessed simultaneously in both villages after the traps were deployed. The villages selected were: Vall\u0026eacute;e du Kou 3 (VK3) for the intervention village (IV) and Vall\u0026eacute;e du Kou 5 (VK5) for the control village (CV). These two villages were selected because they had similar ecological properties in terms of mosquito densities and species [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and were situated 1 km apart, which minimizes any potential PPF contamination between the two sites. A general survey of the houses and windows was conducted in VK3 using a Global Positioning System (GPS) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] to count all the inhabited houses. Each household was georeferenced (Fig.\u0026nbsp;4a, b, c).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDeployment and installation of the traps in VK3\u003c/h2\u003e \u003cp\u003eFor trap installation, the village was divided into six line-bands from the north to the south separated by green areas used for circulation in the village (Fig.\u0026nbsp;4d). All eaves and holes in the houses were blocked using cloth or sponge and a curtain was placed at each door by a large team of local and technical workers. In total, 1,313 traps were placed within the windows of houses to intercept incoming mosquitoes, and a new curtain made from regular cloth was provided to each house in VK3 to block mosquitoes from entering through the door. No constraints were required on the use of the doors or windows, and occupants were free to go to bed at any time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMosquito collection\u003c/h2\u003e \u003cp\u003eTo assess the trap performance, a monthly mosquito collection was performed from 12 selected traps (trapped mosquitoes) and matching houses (indoor resting mosquitoes) in VK3, while only indoor resting mosquitoes from eight houses were collected in VK5. Single-room houses (a single house with one window and one door) were randomly selected according to their geographic location (central, north, east, west, and south) in both villages. Houses were located far from each other, spaced at least 10 m apart to avoid human attractivity bias, and were monitored over nine days per month in both villages from July to October 2015. Mosquitoes were manually collected (on 36 collection days over the four months of the trial) with mouth aspirators in the traps and matching houses (for two hours) by three experienced collectors. The mosquito collection was simultaneously carried out in VK3 and VK5.\u003c/p\u003e \u003cp\u003eTo provide evidence of the impact of the traps being deployed on mosquito density reduction at village level, a pyrethrum spray catch (PSC) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] was carried out simultaneously one day per month over the four month-trial, in 10 randomly selected houses in each village. These houses were different ones to the regular study houses in the villages.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAssessment of mosquito species identification and allelic frequencies of kdr mutation in trap and house collection from Vall\u0026eacute;e du Kou over the study period\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCollected mosquitoes (traps and houses) were morphologically identified to genus, species, and physiological status [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and then counted. A sub-sample was then preserved in 80% ethanol vials for subsequent genotyping to species level and to check on the frequency of the \u003cem\u003eknock down\u003c/em\u003e resistance (\u003cem\u003ekdr)\u003c/em\u003e mutation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAssessing wild female mosquito parity during trap deployment\u003c/h2\u003e \u003cp\u003eIn addition, to assess mosquito population age structure [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] from July to October 2015, around 55 unfed female \u003cem\u003eAn. gambiae\u003c/em\u003e collected from the traps and houses were dissected and classified into parous and nulliparous mosquitoes, according to Detinova\u0026rsquo;s protocol [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy bioassays\u003c/h2\u003e \u003cp\u003eThe efficacy of PPF-treated traps was assessed on two mosquito strains including susceptible \u003cem\u003eAn. gambiae-\u003c/em\u003eKisumu and wild adult \u003cem\u003eAn. coluzzii\u003c/em\u003e collected at larval stage.\u003c/p\u003e \u003cp\u003eThe susceptible \u003cem\u003eAn. gambiae-\u003c/em\u003eKisumu strain mosquitoes were released into traps to assess how long a PPF-treated net effect can last, and to follow the degradation dynamic over time. The morning following the blood meal, between 06:00 and 07:00 am, mosquitoes were transported by car while covered with a wet cloth (to maintain humidity) to the field. Once in the field, one hour rest time was given to the mosquitoes prior to the release. This rest time coincided with the monthly mosquito collection, where traps were emptied. Around 150 blood-fed female \u003cem\u003eAn. gambiae-\u003c/em\u003eKisumu were then released 12 h after a blood meal once per month in VK3 (~\u0026thinsp;50 females in each of two out of 12 monthly selected monitored traps) and in VK5 (50 females into one house). Mosquitoes were allowed to be in contact with the trap net for 30 mins, after which all of these mosquitoes were recaptured and brought to the laboratory for oviposition (fecundity) and egg hatchability (fertility) assessment. Only valid mosquitoes were oviposited. Two releases (July and August 2015) of the \u003cem\u003eAn. gambiae\u003c/em\u003e-Kisumu susceptible strain were performed over the study period. After each release-recapture process in VK5, an indoor spray with insecticide (Kaltox Paalga, SAPHYTO, Burkina) was performed to kill the non-recaptured mosquitoes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. mosquito larvae were randomly collected from breeding sites in VK3 and VK5 to evaluate the effect of PPF over time on the wild mosquito population. These larvae were reared at the IRSS insectary until adulthood. Three to-five-day-old female mosquitoes were processed in the same way as described in the above section prior to their release. A total of 100 blood-fed female \u003cem\u003eAn. gambiae\u003c/em\u003e were simultaneously released in VK3 (in two selected traps in the third and fourth regular monthly collection traps) and in VK5 (50 blood-fed females into one house), once a month over the two-month trial.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pyriproxyfen-treated traps on wild blood-fed mosquitoes\u003c/h2\u003e \u003cp\u003eBlood-fed mosquitoes were collected from traps and houses in VK3 and VK5 to assess whether females that encountered PPF-treated traps overnight had reduced fecundity and fertility. These blood-fed mosquitoes were collected from each of the 12 randomly selected traps once per month over three months in VK3. Similar mosquito sampling was performed inside eight houses in VK5 and considered as controls. About 25 blood-fed female mosquitoes per trap per day in VK3 and the same per house in VK5 were morphologically identified to species. They were then brought back to the IRSS laboratory on the same day and were allowed to lay eggs into a single cup containing Whatman filter paper and 5 mL of water for a week. Cups were checked daily, and females that laid eggs were subsequently removed, killed, dried, and preserved into silica 1.5 mL cryotubes labelled for subsequent analysis. Eggs were counted under stereomicroscope and then hatched into rearing trays (43 \u0026times; 26 \u0026times; 15 cm) filled with 1 liter of tap water with TetraMin baby fish food (TetraMin\u0026reg;, Germany).\u003c/p\u003e \u003cp\u003eFurthermore, to evaluate the impact of PPF on egg development as compared to a control as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], about 100 field blood-fed \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. were randomly collected from the monthly collection traps in VK3 and compared to about 100 blood-fed females collected from VK5 testing houses. These mosquitoes were kept individually in 20 mL cups with Whatman paper and 5 mL of water, and around 30 of them were dissected per day at 24 h, 48 h and 72 h post-collection over three months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePhysical conditions and cleanliness of the traps during the trial\u003c/h2\u003e \u003cp\u003eImmediately after trap installation, 50 nets were sampled and were checked for their physical integrity in 50 selected households over two months (one and two months after installation). The assessment of the integrity of the fabric was performed by visual examination without removing the nets. Any holes observed were assigned to one of four size categories according to WHO guidelines [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]: a hole size of 0.5-2.0 cm or \u0026lsquo;\u0026lt; a thumb-sized opening\u0026rsquo;; a hole size of 2.0\u0026ndash;10.0 cm or \u0026lsquo;\u0026gt; a thumb but \u0026lt;\u0026thinsp;a fist\u0026rsquo;; a hole size of 10\u0026ndash;25 cm or \u0026lsquo;\u0026gt; a fist but \u0026lt;\u0026thinsp;a head\u0026rsquo;; and a hole size of \u0026gt;\u0026thinsp;2.5 cm or \u0026lsquo;\u0026gt; a head\u0026rsquo;.\u003c/p\u003e \u003cp\u003eGeneral trap integrity was assessed based on two measurements:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe proportion of nets with any observed hole(s). The integrity of the nets was determined by counting the number of tears and holes as described: total number of coded nets with at least one hole of size (1-4) \u0026times;100 \u003cb\u003e/\u003c/b\u003e total number of nets assessed in surveyed households.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe proportionate holes index (pHI) for each net, calculated as the sum of the holes weighted by size for each net. For this group, the weights used to calculate the pHI were 1, 23, 196 and 576 as described below: pHI\u0026thinsp;=\u0026thinsp;1 \u0026times; number of size\u0026thinsp;\u0026minus;\u0026thinsp;1 holes\u0026thinsp;+\u0026thinsp;23 \u0026times; number of size\u0026thinsp;\u0026minus;\u0026thinsp;2 holes\u0026thinsp;+\u0026thinsp;196 \u0026times; number of size\u0026thinsp;\u0026minus;\u0026thinsp;3 holes\u0026thinsp;+\u0026thinsp;576 \u0026times; number of size\u0026thinsp;\u0026minus;\u0026thinsp;4 holes. To better correlate the holes index to an integrity status (net condition) for each sampled net, the pHI was categorized into \u0026lsquo;good\u0026rsquo; (pHI\u0026thinsp;\u0026le;\u0026thinsp;64), \u0026lsquo;serviceable\u0026rsquo; (pHI\u0026thinsp;\u0026le;\u0026thinsp;768) and \u0026lsquo;replace\u0026rsquo; (pHI\u0026thinsp;\u0026gt;\u0026thinsp;768).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe trap net dirtiness (/cleanness) was also evaluated, and nets were classified and categorized into \u0026lsquo;clean\u0026rsquo;, \u0026lsquo;a bit dirty\u0026rsquo;, \u0026lsquo;dirty\u0026rsquo;, and \u0026lsquo;very dirty\u0026rsquo;. When the net was deemed irreparably damaged by dirtiness, it was replaced by another net of the same size.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSocio-anthropological investigation on the use of the traps\u003c/h2\u003e \u003cp\u003eQualitative and quantitative surveys were conducted from March to August 2015 from the beneficiaries of the LFET traps in VK3. The qualitative survey consisted of individual interviews with members of the community about their perception of the traps. There was direct observation of trap being used in the village. The usage of traps was witnessed elsewhere. The quantitative survey covered 276 inhabitants and was based on the level of acceptance of the trap by its users, the trap\u0026rsquo;s perceived effectiveness, and the limits of the trap.\u003c/p\u003e \u003cp\u003eIn addition, all inconveniences reported by users of the traps were recorded by a social worker and reported for subsequent remedial measures. A follow-up survey according to the WHO indices [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] was conducted once the traps were installed, any mishandling was also reported, and action was taken to resolve any problems raised either by a social worker or the users themselves.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParameters Measured And Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eThe outcomes of the large-scale field trial were:\u003c/p\u003e \u003cp\u003e\u003col style=\"list-style-type: lower-alpha;\"\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMosquito density \u0026ndash; calculated as the number of mosquitoes caught in the trap out of the total number of mosquitoes collected in the trap in the matching house (house where a LFET was placed and where the daily mosquito collection was performed)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eDirect mortality \u0026ndash; calculated as the number of dead mosquitoes caught in the traps out of collected mosquitoes from the trap.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAllele frequency \u0026ndash; calculated as the number of resistant mosquito homozygote resistant (2nRR) and hybrid resistant (nRS) out of the 2x total of homozygote resistant (RR), and susceptible (SS), and RS the hybrid Resistant-susceptible.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAge structure (parity) \u0026ndash; defined as the number of female mosquitoes having laid eggs (parous) and nulliparous out of a total of dissected mosquitoes.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ePhysical integrity of the traps \u0026ndash; defined as a check of how a trap\u0026rsquo;s net was maintained in the intervention village. This physical integrity data was analysed using descriptive statistics (mean, median, interquartile range) to compare pHI values.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAcceptability \u0026ndash; defined as a socio-anthropological investigation on the use of traps by end-users and calculated by counting the \u0026ldquo;Yes\u0026rdquo; or \u0026ldquo;No\u0026rdquo; answers to the relevant questions, relating to the total number of interviewees.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eDescriptive data was summarized, inputted, and cross-checked in Microsoft Excel 2007 (Microsoft\u0026reg;, New York, USA), and R-4.0.4 was used to produce tables, graphs, counts, means and standard errors.\u003c/p\u003e \u003cp\u003eA Generalized Linear Mixed Models (GLMM) with a Poisson or negative binomial distribution was used to choose the suitable distribution of the mosquitoes collected in the traps and houses from VK3 and VK5 respectively. A zero inflated Poisson mixed regression modelling tool was used to estimate the intervention (trap) effect on daily numbers of mosquitoes collected while accounting for a possible spatial variation in terms of total mosquitoes collected between VK5 and VK3. This was to check for a possible difference in terms of physiological status (gravid, blood fed, unfed), or for a possible temporal variation induced by monthly weather conditions (rainfall, humidity, or temperature). Therefore, two models were built including two random-intercepts, one random coefficient and zero inflation terms. The model structure is defined with trap, gonotrophic _status, status, rainfall, and humidity considered as the fixed effects (fixed effects\u0026thinsp;=\u0026thinsp;trap\u0026thinsp;+\u0026thinsp;gonotrophic _status\u0026thinsp;+\u0026thinsp;status\u0026thinsp;+\u0026thinsp;rainfall\u0026thinsp;+\u0026thinsp;humidity) and the total number of collected female mosquitoes as the response variable.\u003c/p\u003e \u003cp\u003eModel 1: The probability of inflation terms was constant\u003c/p\u003e \u003cp\u003eY \u0026sim; fixed-effects\u0026thinsp;+\u0026thinsp;random\u0026not; (month)\u0026thinsp;+\u0026thinsp;random (village)\u0026thinsp;+\u0026thinsp;zero-inflation (\u0026sim;1)\u0026thinsp;+\u0026thinsp;residual error\u003c/p\u003e \u003cp\u003eModel 2: The probability of inflation terms was dynamic depending on the trap variable\u003c/p\u003e \u003cp\u003eY \u0026sim; fixed-effects\u0026thinsp;+\u0026thinsp;random\u0026not; (village /month)\u0026thinsp;+\u0026thinsp;zero-inflation (\u0026sim;trap)\u0026thinsp;+\u0026thinsp;residual error,\u003c/p\u003e \u003cp\u003ewhere Y is the total number of collected female mosquitoes.\u003c/p\u003e \u003cp\u003eThe sub-model of model 2 was built on basic count, zero inflated, and altered models to account for zero values in the data. In these models, the rainfall and humidity fixed effects were removed to compare their Root Mean-Square Error (RMSE) and the Median Absolute Error (MAE) used to choose the best model. The lower the RMSE, the better the model. The model with the lowest AIC (Akaike information criterion) was considered the best model to fit the data [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Akaike information criterion and recent developments in information complexity were used as these methods consider both suitability and complexity of the model to check the performance of zero-inflated models.\u003c/p\u003e \u003cp\u003eTo prevent the model from overfitting, trap data was split into a training set (0.8) and a test set (0.2). The different models were then tested on the training set and confirmed with the test set. For all analyses, the level of significance chosen was 5% (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eTo assess the dynamic of mosquitoes between VK3 and VK5, pyrethrum spray catch data were analysed using a non-parametric pairwise test, Anova-glmmTMB.\u003c/p\u003e \u003cp\u003eA one-way Anova was used to compare the age distribution of mosquitoes between VK3 and VK5.\u003c/p\u003e \u003cp\u003eThe PPF effect on female mosquito fecundity was calculated as the mean number of eggs per female that contributed to the oviposition. The fertility was measured as the mean number of larvae per female that contributed to oviposition. Following this, a non-parametric pairwise test, Anova-glmmTMB, was used to compare the reduction of fecundity and fertility between the group exposed to PPF-treated net and the control group. As the number of eggs and larvae from the different treatments did not follow a normal distribution, a one-way non-parametric analysis of variance (Kruskal Wallis test) was used to determine whether there was any difference between 30mg/m\u003csup\u003e2\u003c/sup\u003e and 20 mg/m\u003csup\u003e2\u003c/sup\u003e ai doses in terms of fecundity and fertility reduction.\u003c/p\u003e \u003cp\u003eWeather related temporal measures such as the mean temperature, the mean relative humidity and rainfall, were collected from Burkina Faso\u0026rsquo;s meteorological station (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.meteoburkina.bf\u003c/span\u003e\u003cspan address=\"http://www.meteoburkina.bf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePhase I: Selection of the most effective pyriproxyfen-dose\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eEffects of selected pyriproxyfen doses on mosquito fecundity and fertility\u003c/h2\u003e \u003cp\u003eWith susceptible \u003cem\u003eAn. gambiae-\u003c/em\u003e Kisumu strain and two pieces of net impregnated with 20 and 30 mg/m\u003csup\u003e2\u003c/sup\u003e, the dose 30mg/m\u003csup\u003e2\u003c/sup\u003e ai showed a better reduction in fecundity with a lower fecundity and egg fertility rate compared to 20 mg/m\u003csup\u003e2\u003c/sup\u003e (Kruskal-Wallis chi-squared\u0026thinsp;=\u0026thinsp;15.2, df\u0026thinsp;=\u0026thinsp;3, p-value\u0026thinsp;=\u0026thinsp;0.0017). This 30 mg/m\u003csup\u003e2\u003c/sup\u003e ai dose was then selected to impregnate the tailored nets for the traps.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhase Iii: Large-scale Field Evaluation Of The Lehmann Funnel Entry Trap\u003c/h3\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eMosquito collection\u003c/h2\u003e \u003cp\u003eA total of 18,884 and 15,650 \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes were collected over the study period in VK3 (from selected traps and matched houses) and in VK5 (inside houses), respectively. Out of this, 87% (16,437 of the 18,884) were collected from the traps, including the following live mosquitoes: unfed (49%), blood-fed (40%), and gravid females (8%); and the following dead mosquitoes: unfed (1%), blood-fed (1%) and gravid females (1%). This compared to 13% (2,447 of the 18,884) from the matching houses in VK3. In VK5, of the 15,650 mosquitoes collected, 67% were found unfed, 28% blood-fed and 5% were gravid females.\u003c/p\u003e \u003cp\u003eThe sampling on average per night per trap in VK3 for dead mosquitoes ranged from 0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 to 31.76\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40 (means\u0026thinsp;\u0026plusmn;\u0026thinsp;se), and for live mosquitoes from 8.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 to 54.99\u0026thinsp;\u0026plusmn;\u0026thinsp;5.34, caught in trap in VK3. This sampling average varied from 2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 to 10.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12 for live mosquitoes collected indoor in VK3, compared to between 1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 and 21.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02 for live mosquitoes collected indoors in VK5 (Table\u0026nbsp;2, supplementary Fig.\u0026nbsp;2). No dead mosquitoes were collected from the houses in either site.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;2: Descriptive table of gonotrophic status of \u003cem\u003eAn. gambiae\u003c/em\u003e female mosquitoes in VK3 and VK5 over the study period.\u003c/p\u003e \u003cp\u003eIn terms of mosquito density reduction, there were significantly fewer mosquitoes collected from the houses in VK3 as compared to VK5, regardless of the gonotrophic status (unfed, df\u0026thinsp;=\u0026thinsp;0.4964, \u0026#120594;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;2175, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, blood fed, df\u0026thinsp;=\u0026thinsp;0.3525, \u0026#120594;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1193.2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, and gravid, df\u0026thinsp;=\u0026thinsp;0.1439, \u0026#120594;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;665.27, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;3: Comparison and proportions of \u003cem\u003eAnopheles gambiae\u003c/em\u003e female mosquitoes collected per house in VK3 and VK5 over the study period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eOther mosquito species collection in VK3 and VK5 over the study period\u003c/h2\u003e \u003cp\u003eDuring the study, 1,169 other mosquitoes were caught in traps, including species as \u003cem\u003eAn. coustani\u003c/em\u003e (2.6%), \u003cem\u003eAn. pharoensis\u003c/em\u003e (6.0%), \u003cem\u003eCulex\u003c/em\u003e sp (87.3%), \u003cem\u003eMansonia\u003c/em\u003e sp (4.1%) and \u003cem\u003eAedes\u003c/em\u003e (0%) in VK3. In addition, 89 mosquitoes were collected in matched houses and identified as \u003cem\u003eAn. coustani\u003c/em\u003e (2.6%), \u003cem\u003eAn. pharoensis\u003c/em\u003e (6.7%), \u003cem\u003eCulex\u003c/em\u003e sp (87.6%), \u003cem\u003eMansonia\u003c/em\u003e sp (1.1%).\u003c/p\u003e \u003cp\u003eIn VK5, 707 mosquitoes were collected from the houses comprising \u003cem\u003eAn. coustani\u003c/em\u003e (0.4%), \u003cem\u003eAn. pharoensis\u003c/em\u003e (4.2%), \u003cem\u003eAn. rufipes\u003c/em\u003e (0.1%), \u003cem\u003eCulex\u003c/em\u003e sp (87.7%), and \u003cem\u003eMansonia\u003c/em\u003e sp (0.4%) (Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;4: Numbers and proportions of other mosquito species caught in trap and house in VK3 and VK5 over the study period.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAssessment of the mosquito density reduction over the three months of study in VK3 and VK5\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe PSC with Kaltox Paalga showed that resting mosquito density (Table\u0026nbsp;5) in VK5 was significantly higher than in VK3 (rate\u0026thinsp;=\u0026thinsp;0.662 se\u0026thinsp;=\u0026thinsp;0.0113, df\u0026thinsp;=\u0026thinsp;176, t-ratio = -24.131, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) over three consecutive months (from August to October 2015) (Table\u0026nbsp;6). These PSC results highlighted the effectiveness of traps in removing mosquitoes from the environment. In VK3, mosquitoes entered houses and were not able to get out, whereas in VK5, there were no traps and so the mosquito population density remained more and less constant.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;5: Assessment of mosquitoes collected through pyrethrum spray catch over the three months of study period in VK3 and VK5.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;6: Comparison of the mosquito density reduction over three months of study period in VK3 and VK5\u003c/p\u003e \u003cp\u003e \u003cem\u003eAssessment of mosquito species identification and allelic frequency of kdr mutation in Vall\u0026eacute;e du Kou over the study period\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAnopheles coluzzii\u003c/em\u003e (formerly \u003cem\u003eAn. gambiae\u003c/em\u003e form M) was identified as the only \u003cem\u003eAnopheles\u003c/em\u003e species in VK3 and VK5. The kdr mutation was genotyped in mosquitoes that were both exposed or not exposed to PPF-treated nets in order to estimate their frequencies in the natural populations from Vall\u0026eacute;e du Kou. Out of 512 sub-samples analyzed, PCR assay revealed that most of the mosquitoes were highly resistant to pyrethroids and DDT, 0.9 (\u003cem\u003ekdr\u003c/em\u003e based mechanism), as shown in Table\u0026nbsp;7.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;7: Assessment of mosquito species identification and allelic frequencies of kdr mutation in trap and house collection from Vall\u0026eacute;e du Kou over the study period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eAssessing wild female mosquito parity during trap deployment\u003c/h2\u003e \u003cp\u003eOvary dissection indicated that the mosquito population kept a stable age structure as compared to the beginning of the intervention with ~\u0026thinsp;2% (1/55) of the population being parous females in both VK3 and VK5 (\u0026#120594;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.00016532, df\u0026thinsp;=\u0026thinsp;1, p-value\u0026thinsp;=\u0026thinsp;0.9897). Toward the end of the intervention, 12% (7/58) of the females were parous in VK5 as compared to ~\u0026thinsp;3% (2/69) in VK3 (\u0026#120594;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;4.0247, df\u0026thinsp;=\u0026thinsp;1, p-value\u0026thinsp;=\u0026thinsp;0.04484) (Table\u0026nbsp;8).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;8: Assessing wild female mosquito parity during trap deployment in July, August and September 2015.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of environmental effects on mosquito collection between VK3 and VK5\u003c/h2\u003e \u003cp\u003eIn Table\u0026nbsp;9, the intercept estimates the (Log-scale) expected mean number of mosquitoes collected in the trapping area, in a village at given months, and the fixed slope coefficient estimates the change in the expectation associated with rainfall, humidity, status and mosquito gonotrophic status. The model ZIPMM results indicate that following fixed trapping effects (trap), status (dead), rainfall, humidity, gono_status (blood-fed), gono_status (gravid) is significantly different from 0 at 5% level. In addition, these results reveal a statistically significant positive relationship between total mosquitoes and trapping area, and a statistically significant negative relationship between total mosquitoes and status, rainfall, humidity, gono_status. The power of a model to fit the data was considered based on the lower AIC and BIC basic models results.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;9: Model fitting of Poisson models for number of collected mosquito count dataset.\u003c/p\u003e \u003cp\u003eFrom the above metrics, the zero inflated negative binomial model was chosen as the best predicting model, fitting the data with the lowest RMSE value 33.05, and lowest MAE value 13.32. This result is consistent with the fact that this kind of model deals with zero inflated data (Table\u0026nbsp;10).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;10: Model fitting of the ZIP and ZINB model for the dataset on the trap collection.\u003c/p\u003e \u003cp\u003eThe ten deciles test set Zero Inflated Negative Binomial (ZINB) (Fig.\u0026nbsp;5) shows that the actual (original) and predicted values of mosquitoes collected are very close at the beginning and diverge in the middle. This is, on average, over or under predicting in the middle deciles, while in the last decile, actual and predicted, there is a better match.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy bioassay\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAnopheles gambiae\u003c/em\u003e-Kisumu strain released in July and August 2015 into the traps for 30 minutes showed a significant higher reduction of fecundity (mean number of eggs per female) in VK3 as compared to VK5 (egg Control / (PPF_30mg/m\u003csup\u003e2\u003c/sup\u003e) rate: 1.34, se: 0.091, df: 174, t-ratio: 4.318, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similarly, a greater female fertility reduction (mean number of larvae per female) was observed in VK3 as compared to VK5 (larvae Control / (PPF_30mg/m\u003csup\u003e2\u003c/sup\u003e) rate: 0.681, se: 0.078, df: 174, t-ratio: -3.358, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eWith \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes collected at larval stage in both sites in August and September 2015, there was a greater fecundity and fertility reduction in VK3 than in VK5 (egg Control / (PPF_30mg/m\u003csup\u003e2\u003c/sup\u003e) rate: 1.45, se: 0.041, df: 214, t-ratio: 13.005, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and larvae Control / (PPF_30mg/m\u003csup\u003e2\u003c/sup\u003e) rate: 3.27, se: 0.026, df: 214, t-ratio: 14.750, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pyriproxyfen-treated traps on field blood-fed mosquitoes\u003c/h2\u003e \u003cp\u003eWith field blood-fed female \u003cem\u003eAn. gambiae\u003c/em\u003e collected from the traps in July, August and September 2015, the fecundity rate was similar in both sites (egg Control / (PPF_30mg/m\u003csup\u003e2\u003c/sup\u003e) rate: 1.01, se: 0.009, df: 1147, t-ratio: 1.292, p\u0026thinsp;=\u0026thinsp;0.1967), while a greater reduction in fertility was observed between VK3 and VK5 (larvae Control / (PPF_30mg/m\u003csup\u003e2\u003c/sup\u003e) rate: 1.29, se: 0.019, df: 1147, t-ratio: 17.034, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table\u0026nbsp;11). Furthermore, of 100 field blood-fed \u003cem\u003eAn. gambiae\u003c/em\u003e mosquitoes (collected from the traps monthly in VK3 and dissected after 24h, 48h, and 72h), 80 females had non-developed ovaries compared to normal female ovaries from VK5 (Supplementary Fig.\u0026nbsp;3). The PPF effects on fecundity and fertility reduction are summarized in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;11: Comparison of pyriproxyfen effects on susceptible \u003cem\u003eAnopheles gambiae\u003c/em\u003e-Kisumu and field mosquitoes tested over two months and three months respectively in VK5 and VK3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003ePhysical condition and cleanliness of the of the traps during the trial\u003c/h2\u003e \u003cp\u003eThe proportion of nets with at least one hole was ~\u0026thinsp;76% over the two months of the study. Moreover, the number of holes (141) was higher in the second month of the survey than the first month (91). The mean score of the Hole index was significantly higher in the second month at 2.86 (95% CI\u0026thinsp;=\u0026thinsp;2.079\u0026ndash;3.641) as compared to the first month with 1.82 (95% CI\u0026thinsp;=\u0026thinsp;1.244\u0026ndash;2.396; p\u0026thinsp;=\u0026thinsp;0.0298). The number of holes did not seem to change significantly based on the location of the net, with 69 holes found on entry window nets and 74 holes on rear window nets (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The data showed a greater number of holes in category hole-1 than that observed in the two other groups (categories hole-2 and hole-3) (121:20:1 respectively) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The location of holes in the net was explored during the second month. Of the 141 counted holes, ~\u0026thinsp;53 were found on the front of the nets, 38 on the seams, 30 on the length, and 20 on the width. Twenty five percent (25%) of the holes were found along the length and width of the net followed by 10% and less than 1% respectively on the front side and the seams of the net. The median proportionate hole index (pHI) was 3.5 with IQR (2-27.5). The pHI for each net showed good net utilization as 66% (33/50) of nets were found in a good condition (pHI\u0026thinsp;\u0026le;\u0026thinsp;64) and only 1% was found just serviceable (pHI\u0026thinsp;\u0026le;\u0026thinsp;768 \u0026ndash; serviceable). About 75% of the nets were deemed to be clean. The dirty nets were typically installed at the windows of houses built with bricks made of mud. Moreover, the dirtiest nets were located at the front of the house. At the end of each survey, nets with several holes were replaced by new ones with the same dimensions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eSocio-anthropological investigation on the use of the traps: Acceptance and benefits attached to the trap\u003c/h2\u003e \u003cp\u003eQualitative and quantitative data showed a good level of acceptance of the traps by the interviewees. About 80.8% (223/276) of the beneficiaries declared that the trap reduced the number of mosquitoes in the house. This was appreciated by the users, as shown by the following quote, which is taken from an interview with a resident: \"when you look into the object, there is a lot of mosquitoes like that. If all these mosquitoes should enter the home! Good gracious!\" Table\u0026nbsp;12 below shows the details of the various benefits that the traps brought to the population of VK3. Importantly 98.55% (272/276) of the respondents claim to have had peaceful sleep since trap installation, and 98.91% of the respondents believe that the trap will help reduce disease in the area.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;12: Socio-anthropological investigation on the use of the Lehmann Funnel Entry Traps over the study period in VK3, Acceptance and benefits attached to the Lehmann Trap.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main objective of this study was to show the potential of the Lehmann Funnel Entry Trap to control malaria vectors in insecticide-resistant mosquito population settings. In addition, one challenge of this study was to produce as many LFETs as was necessary to cover the entire intervention village of VK3. A total of 1,313 traps were deployed in VK3, which contributed to a reduction of indoor mosquito density of 90% throughout the four months of the trial.\u003c/p\u003e \u003cp\u003eTraps have proven to be effective in suppressing \u003cem\u003eAn. gambiae\u003c/em\u003e species and have successfully caught a variety of other mosquito species, including species that might have bitten the villagers if there were no traps deployed. Traps protected the population of the village from indoor mosquito biting in addition to the other malaria control methods already in use such as bed nets, IRS, repellent plants, etc. This could have consequently reduced malaria transmission in the village. This study result is consistent with a study showing how screening reduced malaria mosquito house entry and contributed to prevention of anaemia in children [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnsuring complete trap coverage in VK3 may have contributed to mosquito density reduction as mosquitoes were repeatedly trapped after emerging from rice breeding sites. However, as the intervention village is surrounded by water, it would be a challenge to reduce mosquito density to a very low level. This challenge could have been minimized somewhat if the study site design was randomized, as the suppressing effect of the trap could have been more visible with a study designed to take place in different ecological settings.\u003c/p\u003e \u003cp\u003eThe successful implementation of LFET trap installation as a malaria tool may focus on the direct monitoring of the adult mosquito population, as this provides a real-time picture of the effects of the intervention [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. As part of this direct monitoring, the PSC results indicated that traps have significantly reduced the mosquito population density at the community level in VK3 as compared to the control village, VK5.\u003c/p\u003e \u003cp\u003eIn this study, the mosquito population was exclusively composed of \u003cem\u003eAn. coluzzii\u003c/em\u003e (the former M molecular form) in Vallée du Kou, which is in alignment with the previous studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The \u003cem\u003ekdr\u003c/em\u003e L1014F mutation conferring resistance to pyrethroids and to DDT was found in the samples analysed to be very high (~ 0.9) when the study was carried out. This result is consistent with previous studies reporting a high level of resistance in mosquito populations due to the extensive use of pesticides in rice and cotton fields in the village [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In this study, environmental factors such as rainfall, humidity, and temperature had a great impact on mosquito densities.\u003c/p\u003e \u003cp\u003eInterestingly, the dissection of the ovaries of mosquitoes collected indicated that the mosquito population had the same age structure at the beginning of the intervention (~ 2% female parous) in both villages. However, toward the end of the intervention, 12% of the dissected females were scored parous in the control village against ~ 3% in the intervention village, indicating that the trap was cumulatively extracting and killing old females capable of transmitting malaria in the intervention village. Moreover, traps removed and killed a proportion of older outdoor mosquitoes from the village, which may have impacted an outdoor malaria transmission, as recently shown in other studies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e–\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, adult dissection \u003cspan refid=\"Sec21\" class=\"InternalRef\"\u003eresults\u003c/span\u003e showed that 80% of the females dissected had failed to develop their ovaries as previously reported [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs the LFETs were impregnated with PPF, this could have increased the performance of the trap by sterilizing female mosquitoes that encountered the trap net, preventing them from having offspring and consequently decreasing the malaria vector population in the village. For this trial, susceptible mosquitoes exposed to PPF-treated nets over two months of the study showed a fewer number of eggs per female and a lower egg hatch rate in the intervention village as compared to the mosquitoes from the control village. Similarly, field \u003cem\u003eAn. coluzzii\u003c/em\u003e collected as larvae reared through to adulthood in the laboratory indicated a significant reduction of eggs per female and a lower egg hatch rate over two months. This result is consistent with the study performed by Harris et al. (2013), where they found that female mosquitoes blood-fed one day prior to PPF exposure produced no viable offspring during this gonotrophic cycle [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].The few female spermathecae dissected during this assessment showed an insemination rate of up to 90% which is consistent with the results from previous work, which showed that the insemination rate of susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e-Kisumu females, maintained in cages with males over 3–5 days in the insectary, was above 90% [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This insemination rate was used as a baseline to analyse female fecundity and egg fertility. However, the fecundity of the mosquitoes collected from the traps in the intervention village was similar to the control mosquitoes. This may be due to the short contact time of some mosquitoes, as the entry rate and time spent on the trap’s netting are not evenly spread and similar for all mosquitoes. Additionally, the number of blood feedings and the age of females caught in traps may differ and impact their fecundity and ultimately the egg fertility. These aspects were not recorded and were considered as some of the limitations of this study. The impact of PPF on other trapped mosquito species was not assessed in this study. Nevertheless, recent studies have demonstrated a PPF sterilizing effect on \u003cem\u003eAedes\u003c/em\u003e spp, and \u003cem\u003eCulex\u003c/em\u003e spp etc. when it is transported and spread by mosquito tarsi to breeding sites [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, this could have impacted other mosquitoes within and around the LFET trial site.\u003c/p\u003e \u003cp\u003eVK3 is one of seven discrete villages (VK1 to VK7) within the Vallée du Kou area. The village is close to its sister villages VK2 and VK4, on the western and eastern side respectively, and is surrounded by rice fields. The poor isolation of VK3 has contributed to its sustained high mosquito density. It has been shown that mosquitoes can disperse up to two kilometers looking for a blood meal and so this could have impacted mosquito density in VK3 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe LFET has proven efficacy in controlling highly resistant malaria vectors. A recent study completed in the same ecological setting in Burkina Faso has shown that newer branded nets struggled to kill mosquitoes in this area, which makes it clear that additional intervention tools are needed to control resistant mosquito populations [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The fact that the trap can further help to control the mosquito population makes it very attractive. Once installed, the trap requires minimal adaptations in human behavior, and unlike mosquito bite prevention tools such as bednets, it protects everyone who is sleeping within the dwelling. Importantly, most of the interviewees acknowledged that the trap reduced mosquito biting and allowed them to sleep peacefully, which is one of the most important criteria that impacts the acceptability of conventional intervention tools such as bed nets [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The high acceptance of the LFET may explain the overall good condition of the traps seen at one to two months post-deployment.\u003c/p\u003e \u003cp\u003eAlthough the retail price of a single trap was estimated to be too high by the villagers, it is worth noting that they all wanted to keep the trap after the evaluation, meaning that the trap is very useful to them. It is common that most households receive malaria preventive methods, including bed nets, for free in Burkina Faso [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. A cost-effectiveness study has not yet been done but it is important to note that a single trap installed in a house protects everyone sleeping within that house.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eThe Lehmann Funnel Entry Trap has proven efficacy in controlling highly resistant malaria vectors. The additional effect of PPF also enhanced its efficacy. The acceptance of the trap was high, as the beneficiaries could see the number of mosquitoes trapped in it every day, and it was acknowledged that the trap reduced mosquito biting and allowed peaceful sleep. Although the deployment of the LFET across an entire village showed a positive impact, a trial in an isolated site with a high mosquito population could better show the suppressing effect of the trap. Additionally, a randomized controlled design of the study could better show the effects of the traps on mosquito density reduction. No survey data on parasitemia and malaria cases were collected during this trial and so a trial with malaria epidemiological end points could link the performance of the trap against malaria. Nevertheless, there is growing evidence that the trap installation in VK3 may have reduced malaria transmission [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAssuming that the size of the trap can be further reduced and the price to the user can be lowered, LFETs may represent a viable business opportunity to entrepreneurs. The next step is to build a business plan and work alongside local window manufacturers to help them to start manufacturing the traps at a large scale. Efforts will be devoted toward the national malaria control program in Burkina Faso to integrate the Lehmann Funnel Entry Trap into the malaria control toolbox.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eMatching house:\u0026nbsp;\u003c/strong\u003ea\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehouse where a LFET was placed and where a daily mosquito collection was performed.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePPF treated trap net:\u003c/strong\u003e a net impregnated with a PPF dose with an active ingredient (a.i.) mg/m\u003csup\u003e2\u003c/sup\u003e and used to cover a LFET metal frame.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZIP\u003c/strong\u003e: Zero inflated Poisson.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZIPMM\u003c/strong\u003e: Zero inflated Poisson Mixed Model\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZINB\u003c/strong\u003e: Zero Inflated Negative Binomial\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRMSE\u003c/strong\u003e: Root Mean-Square Error\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAE\u003c/strong\u003e: Median Absolute Error\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAIC:\u0026nbsp;\u003c/strong\u003eAkaike Information Criterion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVK3:\u0026nbsp;\u003c/strong\u003eVall\u0026eacute;e du Kou 3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVK5:\u0026nbsp;\u003c/strong\u003eVall\u0026eacute;e du Kou 3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGLMM:\u0026nbsp;\u003c/strong\u003eGeneralised Linear Mixed Models\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLFET:\u0026nbsp;\u003c/strong\u003eLehmann Funnel Entry Trap\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analysed during this study are available on Github: https://github.com/RogerSANOU/Lehmann-trap-dataset.git.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRS and HM carried out the field work, participated in the analysis of the data, and wrote the original manuscript. AO and AAM carried out the sociological field and geographic information system work respectively and revised the manuscript. ASH carried out the physical integrity work and revised the manuscript. SPS, KB, SAM, AMGB, LPT and RKD revised the manuscript. BDS carried out the statistical analyses. AD supervised the entire work. All authors have read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong style=\"text-align: inherit;\"\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Tovi Lehmann, Adama Dao and Alpha Yaro, whose previous work inspired us to develop these new traps and with whom we had fruitful discussion. Authors thank Guel Z. Hyacinthe, Diabat\u0026eacute; Noufou for their contribution to the fieldwork. We are also grateful to Kieran Callanan for proofreading and editing. The authors thank the villagers who accepted the traps to be tested in their homes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong style=\"text-align: inherit;\"\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Grand Challenges Canada (GCC, Grant ID: S6 0510-01-10) to DA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong style=\"text-align: inherit;\"\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eInstitut de Recherche en Sciences de la Santé/Centre Muraz, Bobo-Dioulasso, Burkina Faso.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eUniversit\u0026eacute; Nazi BONI de Bobo-Dioulasso, PO 1091.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO. World malaria report 2020- WHO. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJill N, Ulrich DP 1, Naranjo2 TO. Alimi1, G\u0026uuml;nter C. 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Cochrane Database Syst Rev. 2021;2021.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 12 are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Malaria, vectors, Lehmann’s Funnel Entry trap, control, density ","lastPublishedDoi":"10.21203/rs.3.rs-2432364/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2432364/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThere is a great need to find new effective tools to prevent mosquitoes from biting humans. As part of this search, the Lehmann Funnel Entry Trap, a window screen, was tested and has proven to be effective in mosquito density reduction in low and high vector density settings. Here we aimed to assess whether a large-scale deployment of pyriproxyfen-treated traps can control malaria mosquito populations and how well the traps were accepted at community level.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e \u003cp\u003eOverall, 1,313 traps impregnated with Pyriproxyfen (PPF) were deployed. Of these, 12 traps in 12 houses were randomly selected across the intervention village, Vall\u0026eacute;e du Kou 3 (VK3), for indoor mosquito density assessment compared to houses without traps in the control village, Vall\u0026eacute;e du Kou 5 (VK5). Traps were placed in the windows, while doors were blocked with curtains. From July until October 2015, for nine days per month, mosquitoes were collected from the traps and matching houses in VK3, and in houses only in VK5. \u003cem\u003eAnopheles coluzzii\u003c/em\u003e collected from the field as adults and as larvae reared through to adults and \u003cem\u003eAn. gambiae\u003c/em\u003e were used or released into traps to assess the bio-efficacy effect of PPF in VK3 against control mosquitoes from VK5. Then, mosquitoes that were in contact with the PPF trap netting were allowed to oviposit in the laboratory for fecundity and egg fertility reduction evaluation as compared to a control. The mosquitoes collected as part of the monthly programme were morphologically identified, counted, and preserved in 80% ethanol vials for subsequent analyses, including resistance genes and female mosquito age structure. The impact of the trap on mosquito density at community level was assessed by performing a pyrethrum spray catch (PSC). The integrity of net fabric as well as some sociological points of view on the trap\u0026rsquo;s efficacy and design were then reported.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOverall mosquito density was reduced by ~\u0026thinsp;90% in all houses equipped with traps in VK3. At the community level, while mosquito density before intervention was 33% higher in VK3 than in VK5, it was 47% higher in VK5 after the intervention. Old female mosquito numbers increased in VK5 by 12% in October but not in VK3, indicating that the traps were cumulatively killing older females. The additional effect of PPF was to limit egg-laying, with a smaller number of eggs counted, and with a lower hatching rate than in VK5. Mosquitoes were highly resistant to pyrethroids with a\u0026thinsp;~\u0026thinsp;0.9 frequency of the \u003cem\u003ekdr\u003c/em\u003e mutation. The trap was well accepted by the communities as most of the interviewees in VK3 found the traps reduced mosquito bites with peaceful sleep. They hoped to keep the traps in place beyond the study period.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe Lehmann Funnel Entry Trap has real potential to control malaria mosquito populations and can be widely used to sustain the global effort of malaria elimination.\u003c/p\u003e","manuscriptTitle":"Large-scale deployment of pyriproxyfen-treated Lehmann’s Funnel Entry Traps to control malaria mosquito populations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-05 18:31:03","doi":"10.21203/rs.3.rs-2432364/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"63194640-a7ce-41f9-a865-316306a49c55","owner":[],"postedDate":"January 5th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-10-12T08:44:37+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-05 18:31:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2432364","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2432364","identity":"rs-2432364","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}