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Indoor residual spraying with a mixture of clothianidin (a neonicotinoid insecticide) and deltamethrin provides improved control and long residual activity against pyrethroid resistant Anopheles gambiae sl in Southern Benin

PMCID: PMC5734732

PMID: 29252986

Abstract

Introduction There is an urgent need for new insecticides for indoor residual spraying (IRS) which can provide improved and prolonged control of malaria vectors that have developed resistance to existing insecticides. The neonicotinoid, clothianidin represents a class of chemistry new to public health. Clothianidin acts as an agonist on nicotinic acetyl choline receptors. IRS with a mixture of Clothianidin and another WHO approved insecticide such as deltamethrin could provide improved control of insecticide resistant malaria vector populations and serve as a tool for insecticide resistance management. Methods The efficacy and residual activity of a novel IRS mixture of deltamethrin and clothianidin was evaluated against wild pyrethroid resistant An . gambiae sl in experimental huts in Cove, Benin. Two application rates of the mixture were tested and comparison was made with clothianidin and deltamethrin applied alone. To assess the residual efficacy of the treatments on different local wall substrates, the inner walls of the experimental huts were covered with either cement, mud or plywood. Results Clothianidin demonstrated a clear delayed expression in mortality of wild pyrethroid resistant An . gambiae sl in the experimental huts which reached its full effect 120 hours after exposure. Overall mortality over the 12-month hut trial was 15% in the control hut and 24–29% in the deltamethrin-treated huts. The mixture of clothianidin 200mg/m 2 and deltamethrin 25mg/m 2 induced high overall hut mortality rates (87% on mud walls, 82% on cement walls and 61% on wooden walls) largely due to the clothianidin component and high hut exiting rates (67–76%) mostly due to the deltamethrin component. Mortality rates remained >80% for 8–9 months on mud and cement walls. The residual activity trend was confirmed by results from monthly in situ cone bioassays with laboratory susceptible An . gambiae Kisumu strain. Conclusion IRS campaigns with the mixture of clothianidin plus deltamethrin have the potential to provide prolonged control of malaria transmitted by pyrethroid resistant mosquito populations.

Full Text

With increased funding and impetus from the Global Fund and the US President’s Malaria Initiative (PMI), the use of indoor residual spraying (IRS) for malaria vector control in Africa has increased significantly in recent years [1]. This together with a massive scale up in the distribution of LLINs has contributed significantly to the substantial reductions in malaria morbidity and mortality seen today [1]. Twelve insecticides belonging to four classes of chemistry can be used for IRS [2]. Apart from DDT which lasts over 6 months most WHO-recommended IRS insecticides have a short residual life (2–6 months) on local wall substrates and thus would require multiple application rounds in holoendemic areas where transmission occurs year-round. This poses a major challenge to the effective use of IRS in most of sub-Saharan Africa given the operational resources required for the implementation of re-current IRS campaigns.
Of the four classes of insecticides approved for IRS, pyrethoids have been the most widely used owing to their low cost, low mammalian toxicity and efficacy [3]. However, resistance to pyrethroids and DDT is now widespread across sub-Saharan Africa [3, 4] and this has led to an increased interest in the use of carbamate and organophosphate insecticides within the existing portfolio of WHO approved insecticides for IRS [5, 6]. Nevertheless, resistance to organophosphates and carbamates especially due to the insensitive acetyl cholinesterase gene (Ace1R) has been reported in several areas in West Africa and is spreading [7–10]. The identification of novel long-lasting IRS insecticides which do not show any cross resistance to current insecticides is thus vital.
Clothiandin is a neonicotinoid insecticide which represents a new class of chemistry for public health. Neonicotinoids are widely used in crop protection against piercing/sucking insects and in veterinary medicine against cat and dog fleas [11]. They exhibit great biological efficacy against a broad spectrum of insect pests among the Hemiptera, Coleoptera, Thysanoptera, Lepidoptera and Diptera. Clothianidin acts as an agonist on nicotinic actetylcholine receptors thus demonstrating a different mode of action to pyrethroids, organophosphates and carbamates. Because neonicotinoids have very low affinity for vertebrate nicotinic receptors relative to insect nicotinic receptors, they generally show very low toxicity to mammals [12, 13] and are thus promising for public health use. Considering the novel mode of action, they show potential to provide improved control of vector populations that have developed resistance to existing public health insecticides.
The use of mixtures of unrelated insecticides in co-formulations for IRS has the potential to improve malaria vector control through the differential effects of the insecticides involved and manage insecticide resistance in areas where resistance to either active ingredient is not yet established [4]. The latter is based on the concept that insect vectors which are resistant to one component of the mixture can be killed by the other; thus, preventing selection of insecticide resistant genotypes [10, 14]. Because IRS targets the insect vector at a stage when it is more likely to make longer contact with the insecticide (fed and resting on the wall), the use of mixtures for IRS could be very effective.
The study was performed in a newly constructed experimental hut station in Cove, Central Benin belonging to the LSHTM/CREC Collaborative Research Programme which is being used for the evaluation of several indoor vector control tools to WHOPES Phase 2 standards [15–17]. The station is situated at the center of a large rice growing field. The rice paddies provide extensive breeding sites for An gambiae sl throughout the year. The vector population consists of both An coluzzi and An gambiae ss with the latter occurring at much lower frequencies (~23%) and mostly in the drier seasons. The rainy season extends from March to October and the dry season from November to February. The local vector population is highly resistant to pyrethroids. Molecular genotyping and microarray studies showed that pyrethroid resistance in the Cove population is mediated by high levels of L1014F gene (90%) and overexpression of metabolic genes including CYP6P3, a P450 which metabolizes pyrethroids [16].
The trial was carried out over 12 months between December 2013 and November 2014 in 11 experimental huts of the WHO-approved West African design [18]. The experimental huts are built on concrete plinths surrounded by water-filled moats to prevent entry of scavenging ants, and have veranda traps to capture the exiting mosquitoes. They are made of brick plastered with cement on the inside, with a corrugated iron roof. The huts have a ceiling of palm thatch and four window slits (1cm gap) on the walls through which mosquitoes enter.
The trial followed WHOPES guidelines [19]. Treatments were randomly allocated to the experimental huts. Eleven consenting adult human volunteers slept in the huts from 20:00 to 05:00 each night of the study to attract mosquitoes into the hut and were rotated between huts on successive nights to adjust for any variation in individual attractiveness to mosquitoes. In the morning of each day of the trial, mosquitoes were collected from the rooms and verandahs of the hut and brought to the laboratory where they were identified and scored for blood feeding status and mortality. Mortality was recorded for up to 120 hours across all the hut treatments including the control.
To assess residual activity of the insecticide treatments on the different substrates, 2–5 days old mosquitoes of the susceptible An. gambiae Kisumu laboratory strain were tested in WHO cone bioassays in situ on the interior treated walls of each hut 3 days after application of the treatment and subsequently every month of the trial. A total of 50 mosquitoes were tested per hut in batches of 10 mosquitoes per cone and one cone was tested per wall/ceiling surface. Mosquitoes were exposed for 30 min according to WHO IRS guidelines [19]. Knockdown was recorded after 60 minutes and mortality after a 120-hour holding period.
Knock down and mortality rates of the susceptible laboratory An gambiae Kisumu strain after exposure to deltamethrin 0.05% treated papers were 98% and 100% respectively. With the wild An gambiae sl from Cove, knockdown and mortality were 11% and 8% respectively (Table 1) showing that the wild strain had a high frequency of resistance to deltamethrin.
The overall experimental hut results obtained over 12 months of the trial are presented in Table 2. The primary experimental hut data are provided in S1 Table (1–6 months) and S2 Table (7–12 months).
The average total number of mosquitoes collected per hut treatment over the course of the 12-month trial was 7,876 (Table 2). A significantly higher number of mosquitoes entered the control hut compared to the treated huts (Table 2; P<0.005). The proportion exiting the control hut was 43%. With all hut wall substrates, exiting rates with deltamethrin (67–77%) and the clothianidin + deltamethrin mixtures (67–76%), were consistently and significantly higher than the control hut (43%; P<0.05) and the hut treated with clothianidin alone (39%, P<0.05). Exiting rates did not differ considerably between the deltamethrin treated huts and the mixture huts for all three types of hut wall substrates. These results showed that treatment induced exiting from the mixture-treated huts was largely due to the deltamethrin component.
Delayed mortality: Fig 1 presents the overall mortality rates recorded after 24, 48, 72, 96 and 120 hours in huts with cement, mud and plywood walls. Mortality with the control was <5% after 24h and increased to only 12% after 120h. For all hut wall substrates, clothianidin whether applied alone or as a mixture with deltamethrin demonstrated a delayed expression in mortality with a full effect showing only after 5 days of observation. For example, mortality in cement walls treated with clothianidin alone increased steadily from 38% after 24h to 91% after 120 hours (Fig 1). This trend of delayed mortality was not strongly expressed in huts treated with deltamethrin alone; mortality did not exceed 29% after 120h.
Overall mortality after 12 months: The overall mortality rates (recorded after 120 hours) over the 12-month trial are presented in Fig 2. Overall mortality was 12% in the control hut and < 30% in deltamethrin treated huts (24% with cement walled hut and 29% each with mud and plywood walled huts). Mortality rates were generally higher with the mixtures relative to deltamethrin alone (51%–82% vs. 24–29%; P<0.05). For each hut wall substrate, proportions dead were also generally higher in the hut with the higher dose mixture (CT200+DM25) relative to the hut with the lower dose mixture (CT100+DM25). In the cement-walled huts for example, mortality was 67% with the CT100+DM25 mixture and 82% with the cloth200+delta25 mixture (P<0.001). The mixtures killed higher proportions of mosquitoes in mud walled huts (75% with CT100+DM25 and 87% with CT200+DM25) relative to cement walled huts (67% with CT100+DM25 and 82% with CT200+DM25; P<0.05) and plywood walled huts (53% with CM100+DM25 and 61% with CT200+DM25; P<0.05). The highest overall mortality rate was achieved in the cement-walled hut treated with clothianidin alone (91%).
The monthly mortality rates of wild free flying An gambiae sl which entered the experimental huts during the course of the 12-month trial are presented in Figs 3, 4 and 5 for cement, mud and wooden wall huts respectively.
The quarterly residual mortality rates (120h post-exposure) of susceptible An gambiae Kisumu laboratory strain exposed in monthly in situ cone bioassays to the different hut treatments are presented in Fig 6A, 6B and 6C for cement, mud and plywood walls respectively. Cone bioassay mortality on the control hut walls did not exceed 10% in any quarter of the trial. With the mixtures and clothianidin treatments, cone bioassay mortality was very high (>90%) through all 4 quarters of the trial irrespective of the wall substrate (Fig 6). Cone bioassay mortality with deltamethrin on cement walls was >80% for the first 2 quarters but declined very steeply to <40% thereafter (Fig 6A). A similar trend was observed with deltamethrin treated mud walls, however, mortality increased to 75% in the last 3 months of the trial (Fig 6B). Deltamethrin induced more persistent mortality on plywood walls; cone bioassay mortality was >80% in all quarters of the trial (Fig 6C).
The mortality rates recorded with clothianidin whether alone or in the mixture clearly demonstrated a delayed effect lasting up to 5 days. Neonicotinoids by acting on the insect nicotinic actetylcholine receptor exhibit both acute contact toxicity and stomach poison [20] and the latter could be responsible for the delayed effect on mortality of mosquitoes. Previous studies on fruit flies also demonstrated a significant increase in mortality with clothianidin when observation time increased from 24h to 72h [21]. This delayed effect has also been observed in a similar parallel study evaluating the efficacy of clothianidin and deltamethrin IRS mixture against multiple insecticide resistant malaria vectors in Cote D’Ivoire (unpublished data) thus confirming our findings. The results therefore demonstrate that failure to assess delayed mortality could lead to an underestimation of the efficacy of neonicotinoids in evaluation studies. This could explain the low toxicity reported with the neonicotinoid dinotefuran, in a previous study testing its efficacy against various mosquito strains [22] where adult mortality was recorded only for 24hours.
Since pyrethroids have been the most widely used insecticides for malaria vector control for decades, WHOPES guidelines were tailored towards the evaluation of fast-acting insecticides (like pyrethroids), hence the guidelines state that mortality rates should be recorded within 24 hours post-exposure [19]. The identification of new active ingredients for malaria vector control poses a major challenge to chemical companies, it is therefore important that the guidelines used for their evaluation are adapted to the mode of action of the insecticide being tested.
Because the current study was designed as a proof of concept to demonstrate the efficacy of an IRS mixture of clothianidin and deltamethrin, the mixture was prepared as a tank mix of both formulations prior to the hut applications. The WHO however recommends against the use of ad hoc mixtures for IRS; for maximum impact, mixtures should ideally be co-formulated into a single product to facilitate application by field operators and ensure that both active ingredients decay at the same rate [4]. Based on the positive results from the current preliminary study, Bayer has formulated the mixture into a single wettable powder product in water soluble sachets containing 500g/kg of clothianidin and 62.5g/kg of deltamethrin also known as Fludora®. Studies are underway to assess the efficacy of Fludora® against pyrethroid resistant malaria vectors in WHOPES Phase II experimental hut studies and Phase III community randomized controlled trials in several countries across East and West Africa.
Although neonicotinoids have not been used in public health, they are widely applied in agricultural operations in sub-Saharan Africa. There is increasing evidence of the link between agricultural use of insecticides and selection of resistance to these insecticides in mosquitoes in the region [23]. Modelling studies have suggested that slow/late life acting insecticides like clothianidin could impose less selection pressure for resistance compared to faster acting insecticides [24]. Based on simulation studies [14], it is also expected that the clothianidin and deltamethrin mixture if deployed in areas where resistance to either active ingredient is not yet established, will slow down the development of resistance to clothianidin in malaria vectors and better delay the spread of pyrethroid resistance compared to clothianidin alone or deltamethrin alone. Further studies in areas with low to moderate levels of pyrethroid resistance should be performed to assess the resistance management potential of this new mixture for IRS. Modelling studies have indicated that for mixtures to be effective it is important that both components are applied at the recommended operational dose [14, 25]. The results showed greater persistence in mortality with the higher dose mixture relative to the lower dose mixture thus suggesting that the clothianidin 200 + deltamethrin 25 mg/m2 mixture would be a more appropriate dose to achieve effective and prolonged control of pyrethroid resistant mosquitoes and for resistance management.
While the mixture induced considerably high (60–80%) and long residual mortality against wild pyrethroid resistant mosquitoes on all wall substrates, this effect lasted longest on mud walls (9 months) compared to cement and wooden walls (6–8 months). Most IRS insecticides tested on a variety of wall substrates have shown significantly longer residual activity on cement walls than on mud walls [26–28]. The results therefore demonstrate that the clothianidin and deltamethrin WP mixture may be a more suitable insecticide for IRS campaigns in most rural endemic areas in sub-Saharan Africa where most houses have mud walls. The reasons behind this longer residual mortality of clothianidin on mud substrates is however unclear; this requires further investigations into the characteristics of the different substrates that could affect the efficacy of different insecticides and formulations.

Sections

"[{\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref001\", \"pone.0189575.ref001\", \"pone.0189575.ref002\"], \"section\": \"Background\", \"text\": \"With increased funding and impetus from the Global Fund and the US President\\u2019s Malaria Initiative (PMI), the use of indoor residual spraying (IRS) for malaria vector control in Africa has increased significantly in recent years [1]. This together with a massive scale up in the distribution of LLINs has contributed significantly to the substantial reductions in malaria morbidity and mortality seen today [1]. Twelve insecticides belonging to four classes of chemistry can be used for IRS [2]. Apart from DDT which lasts over 6 months most WHO-recommended IRS insecticides have a short residual life (2\\u20136 months) on local wall substrates and thus would require multiple application rounds in holoendemic areas where transmission occurs year-round. This poses a major challenge to the effective use of IRS in most of sub-Saharan Africa given the operational resources required for the implementation of re-current IRS campaigns.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref003\", \"pone.0189575.ref003\", \"pone.0189575.ref004\", \"pone.0189575.ref005\", \"pone.0189575.ref006\", \"pone.0189575.ref007\", \"pone.0189575.ref010\"], \"section\": \"Background\", \"text\": \"Of the four classes of insecticides approved for IRS, pyrethoids have been the most widely used owing to their low cost, low mammalian toxicity and efficacy [3]. However, resistance to pyrethroids and DDT is now widespread across sub-Saharan Africa [3, 4] and this has led to an increased interest in the use of carbamate and organophosphate insecticides within the existing portfolio of WHO approved insecticides for IRS [5, 6]. Nevertheless, resistance to organophosphates and carbamates especially due to the insensitive acetyl cholinesterase gene (Ace1R) has been reported in several areas in West Africa and is spreading [7\\u201310]. The identification of novel long-lasting IRS insecticides which do not show any cross resistance to current insecticides is thus vital.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref011\", \"pone.0189575.ref012\", \"pone.0189575.ref013\"], \"section\": \"Background\", \"text\": \"Clothiandin is a neonicotinoid insecticide which represents a new class of chemistry for public health. Neonicotinoids are widely used in crop protection against piercing/sucking insects and in veterinary medicine against cat and dog fleas [11]. They exhibit great biological efficacy against a broad spectrum of insect pests among the Hemiptera, Coleoptera, Thysanoptera, Lepidoptera and Diptera. Clothianidin acts as an agonist on nicotinic actetylcholine receptors thus demonstrating a different mode of action to pyrethroids, organophosphates and carbamates. Because neonicotinoids have very low affinity for vertebrate nicotinic receptors relative to insect nicotinic receptors, they generally show very low toxicity to mammals [12, 13] and are thus promising for public health use. Considering the novel mode of action, they show potential to provide improved control of vector populations that have developed resistance to existing public health insecticides.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref004\", \"pone.0189575.ref010\", \"pone.0189575.ref014\"], \"section\": \"Background\", \"text\": \"The use of mixtures of unrelated insecticides in co-formulations for IRS has the potential to improve malaria vector control through the differential effects of the insecticides involved and manage insecticide resistance in areas where resistance to either active ingredient is not yet established [4]. The latter is based on the concept that insect vectors which are resistant to one component of the mixture can be killed by the other; thus, preventing selection of insecticide resistant genotypes [10, 14]. Because IRS targets the insect vector at a stage when it is more likely to make longer contact with the insecticide (fed and resting on the wall), the use of mixtures for IRS could be very effective.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref015\", \"pone.0189575.ref017\", \"pone.0189575.ref016\"], \"section\": \"Study site and experimental huts\", \"text\": \"The study was performed in a newly constructed experimental hut station in Cove, Central Benin belonging to the LSHTM/CREC Collaborative Research Programme which is being used for the evaluation of several indoor vector control tools to WHOPES Phase 2 standards [15\\u201317]. The station is situated at the center of a large rice growing field. The rice paddies provide extensive breeding sites for An gambiae sl throughout the year. The vector population consists of both An coluzzi and An gambiae ss with the latter occurring at much lower frequencies (~23%) and mostly in the drier seasons. The rainy season extends from March to October and the dry season from November to February. The local vector population is highly resistant to pyrethroids. Molecular genotyping and microarray studies showed that pyrethroid resistance in the Cove population is mediated by high levels of L1014F gene (90%) and overexpression of metabolic genes including CYP6P3, a P450 which metabolizes pyrethroids [16].\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref018\"], \"section\": \"Study site and experimental huts\", \"text\": \"The trial was carried out over 12 months between December 2013 and November 2014 in 11 experimental huts of the WHO-approved West African design [18]. The experimental huts are built on concrete plinths surrounded by water-filled moats to prevent entry of scavenging ants, and have veranda traps to capture the exiting mosquitoes. They are made of brick plastered with cement on the inside, with a corrugated iron roof. The huts have a ceiling of palm thatch and four window slits (1cm gap) on the walls through which mosquitoes enter.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref019\"], \"section\": \"Hut trial procedure\", \"text\": \"The trial followed WHOPES guidelines [19]. Treatments were randomly allocated to the experimental huts. Eleven consenting adult human volunteers slept in the huts from 20:00 to 05:00 each night of the study to attract mosquitoes into the hut and were rotated between huts on successive nights to adjust for any variation in individual attractiveness to mosquitoes. In the morning of each day of the trial, mosquitoes were collected from the rooms and verandahs of the hut and brought to the laboratory where they were identified and scored for blood feeding status and mortality. Mortality was recorded for up to 120 hours across all the hut treatments including the control.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref019\"], \"section\": \"Residual activity of insecticide treatments\", \"text\": \"To assess residual activity of the insecticide treatments on the different substrates, 2\\u20135 days old mosquitoes of the susceptible An. gambiae Kisumu laboratory strain were tested in WHO cone bioassays in situ on the interior treated walls of each hut 3 days after application of the treatment and subsequently every month of the trial. A total of 50 mosquitoes were tested per hut in batches of 10 mosquitoes per cone and one cone was tested per wall/ceiling surface. Mosquitoes were exposed for 30 min according to WHO IRS guidelines [19]. Knockdown was recorded after 60 minutes and mortality after a 120-hour holding period.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.t001\"], \"section\": \"Susceptibility tests\", \"text\": \"Knock down and mortality rates of the susceptible laboratory An gambiae Kisumu strain after exposure to deltamethrin 0.05% treated papers were 98% and 100% respectively. With the wild An gambiae sl from Cove, knockdown and mortality were 11% and 8% respectively (Table 1) showing that the wild strain had a high frequency of resistance to deltamethrin.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.t002\", \"pone.0189575.s001\", \"pone.0189575.s002\"], \"section\": \"Experimental hut results\", \"text\": \"The overall experimental hut results obtained over 12 months of the trial are presented in Table 2. The primary experimental hut data are provided in S1 Table (1\\u20136 months) and S2 Table (7\\u201312 months).\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.t002\", \"pone.0189575.t002\"], \"section\": \"Overall hut entry and exiting\", \"text\": \"The average total number of mosquitoes collected per hut treatment over the course of the 12-month trial was 7,876 (Table 2). A significantly higher number of mosquitoes entered the control hut compared to the treated huts (Table 2; P<0.005). The proportion exiting the control hut was 43%. With all hut wall substrates, exiting rates with deltamethrin (67\\u201377%) and the clothianidin + deltamethrin mixtures (67\\u201376%), were consistently and significantly higher than the control hut (43%; P<0.05) and the hut treated with clothianidin alone (39%, P<0.05). Exiting rates did not differ considerably between the deltamethrin treated huts and the mixture huts for all three types of hut wall substrates. These results showed that treatment induced exiting from the mixture-treated huts was largely due to the deltamethrin component.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.g001\", \"pone.0189575.g001\"], \"section\": \"Mortality rates\", \"text\": \"Delayed mortality: Fig 1 presents the overall mortality rates recorded after 24, 48, 72, 96 and 120 hours in huts with cement, mud and plywood walls. Mortality with the control was <5% after 24h and increased to only 12% after 120h. For all hut wall substrates, clothianidin whether applied alone or as a mixture with deltamethrin demonstrated a delayed expression in mortality with a full effect showing only after 5 days of observation. For example, mortality in cement walls treated with clothianidin alone increased steadily from 38% after 24h to 91% after 120 hours (Fig 1). This trend of delayed mortality was not strongly expressed in huts treated with deltamethrin alone; mortality did not exceed 29% after 120h.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.g002\"], \"section\": \"Mortality rates\", \"text\": \"Overall mortality after 12 months: The overall mortality rates (recorded after 120 hours) over the 12-month trial are presented in Fig 2. Overall mortality was 12% in the control hut and < 30% in deltamethrin treated huts (24% with cement walled hut and 29% each with mud and plywood walled huts). Mortality rates were generally higher with the mixtures relative to deltamethrin alone (51%\\u201382% vs. 24\\u201329%; P<0.05). For each hut wall substrate, proportions dead were also generally higher in the hut with the higher dose mixture (CT200+DM25) relative to the hut with the lower dose mixture (CT100+DM25). In the cement-walled huts for example, mortality was 67% with the CT100+DM25 mixture and 82% with the cloth200+delta25 mixture (P<0.001). The mixtures killed higher proportions of mosquitoes in mud walled huts (75% with CT100+DM25 and 87% with CT200+DM25) relative to cement walled huts (67% with CT100+DM25 and 82% with CT200+DM25; P<0.05) and plywood walled huts (53% with CM100+DM25 and 61% with CT200+DM25; P<0.05). The highest overall mortality rate was achieved in the cement-walled hut treated with clothianidin alone (91%).\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.g003\", \"pone.0189575.g004\", \"pone.0189575.g005\"], \"section\": \"Residual efficacy over 12 months\", \"text\": \"The monthly mortality rates of wild free flying An gambiae sl which entered the experimental huts during the course of the 12-month trial are presented in Figs 3, 4 and 5 for cement, mud and wooden wall huts respectively.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.g006\", \"pone.0189575.g006\", \"pone.0189575.g006\", \"pone.0189575.g006\", \"pone.0189575.g006\"], \"section\": \"Residual bioassay activity\", \"text\": \"The quarterly residual mortality rates (120h post-exposure) of susceptible An gambiae Kisumu laboratory strain exposed in monthly in situ cone bioassays to the different hut treatments are presented in Fig 6A, 6B and 6C for cement, mud and plywood walls respectively. Cone bioassay mortality on the control hut walls did not exceed 10% in any quarter of the trial. With the mixtures and clothianidin treatments, cone bioassay mortality was very high (>90%) through all 4 quarters of the trial irrespective of the wall substrate (Fig 6). Cone bioassay mortality with deltamethrin on cement walls was >80% for the first 2 quarters but declined very steeply to <40% thereafter (Fig 6A). A similar trend was observed with deltamethrin treated mud walls, however, mortality increased to 75% in the last 3 months of the trial (Fig 6B). Deltamethrin induced more persistent mortality on plywood walls; cone bioassay mortality was >80% in all quarters of the trial (Fig 6C).\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref020\", \"pone.0189575.ref021\", \"pone.0189575.ref022\"], \"section\": \"Discussion\", \"text\": \"The mortality rates recorded with clothianidin whether alone or in the mixture clearly demonstrated a delayed effect lasting up to 5 days. Neonicotinoids by acting on the insect nicotinic actetylcholine receptor exhibit both acute contact toxicity and stomach poison [20] and the latter could be responsible for the delayed effect on mortality of mosquitoes. Previous studies on fruit flies also demonstrated a significant increase in mortality with clothianidin when observation time increased from 24h to 72h [21]. This delayed effect has also been observed in a similar parallel study evaluating the efficacy of clothianidin and deltamethrin IRS mixture against multiple insecticide resistant malaria vectors in Cote D\\u2019Ivoire (unpublished data) thus confirming our findings. The results therefore demonstrate that failure to assess delayed mortality could lead to an underestimation of the efficacy of neonicotinoids in evaluation studies. This could explain the low toxicity reported with the neonicotinoid dinotefuran, in a previous study testing its efficacy against various mosquito strains [22] where adult mortality was recorded only for 24hours.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref019\"], \"section\": \"Discussion\", \"text\": \"Since pyrethroids have been the most widely used insecticides for malaria vector control for decades, WHOPES guidelines were tailored towards the evaluation of fast-acting insecticides (like pyrethroids), hence the guidelines state that mortality rates should be recorded within 24 hours post-exposure [19]. The identification of new active ingredients for malaria vector control poses a major challenge to chemical companies, it is therefore important that the guidelines used for their evaluation are adapted to the mode of action of the insecticide being tested.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref004\"], \"section\": \"Discussion\", \"text\": \"Because the current study was designed as a proof of concept to demonstrate the efficacy of an IRS mixture of clothianidin and deltamethrin, the mixture was prepared as a tank mix of both formulations prior to the hut applications. The WHO however recommends against the use of ad hoc mixtures for IRS; for maximum impact, mixtures should ideally be co-formulated into a single product to facilitate application by field operators and ensure that both active ingredients decay at the same rate [4]. Based on the positive results from the current preliminary study, Bayer has formulated the mixture into a single wettable powder product in water soluble sachets containing 500g/kg of clothianidin and 62.5g/kg of deltamethrin also known as Fludora\\u00ae. Studies are underway to assess the efficacy of Fludora\\u00ae against pyrethroid resistant malaria vectors in WHOPES Phase II experimental hut studies and Phase III community randomized controlled trials in several countries across East and West Africa.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref023\", \"pone.0189575.ref024\", \"pone.0189575.ref014\", \"pone.0189575.ref014\", \"pone.0189575.ref025\"], \"section\": \"Discussion\", \"text\": \"Although neonicotinoids have not been used in public health, they are widely applied in agricultural operations in sub-Saharan Africa. There is increasing evidence of the link between agricultural use of insecticides and selection of resistance to these insecticides in mosquitoes in the region [23]. Modelling studies have suggested that slow/late life acting insecticides like clothianidin could impose less selection pressure for resistance compared to faster acting insecticides [24]. Based on simulation studies [14], it is also expected that the clothianidin and deltamethrin mixture if deployed in areas where resistance to either active ingredient is not yet established, will slow down the development of resistance to clothianidin in malaria vectors and better delay the spread of pyrethroid resistance compared to clothianidin alone or deltamethrin alone. Further studies in areas with low to moderate levels of pyrethroid resistance should be performed to assess the resistance management potential of this new mixture for IRS. Modelling studies have indicated that for mixtures to be effective it is important that both components are applied at the recommended operational dose [14, 25]. The results showed greater persistence in mortality with the higher dose mixture relative to the lower dose mixture thus suggesting that the clothianidin 200 + deltamethrin 25 mg/m2 mixture would be a more appropriate dose to achieve effective and prolonged control of pyrethroid resistant mosquitoes and for resistance management.\"}, {\"pmc\": \"PMC5734732\", \"pmid\": \"29252986\", \"reference_ids\": [\"pone.0189575.ref026\", \"pone.0189575.ref028\"], \"section\": \"Discussion\", \"text\": \"While the mixture induced considerably high (60\\u201380%) and long residual mortality against wild pyrethroid resistant mosquitoes on all wall substrates, this effect lasted longest on mud walls (9 months) compared to cement and wooden walls (6\\u20138 months). Most IRS insecticides tested on a variety of wall substrates have shown significantly longer residual activity on cement walls than on mud walls [26\\u201328]. The results therefore demonstrate that the clothianidin and deltamethrin WP mixture may be a more suitable insecticide for IRS campaigns in most rural endemic areas in sub-Saharan Africa where most houses have mud walls. The reasons behind this longer residual mortality of clothianidin on mud substrates is however unclear; this requires further investigations into the characteristics of the different substrates that could affect the efficacy of different insecticides and formulations.\"}]"

Metadata

"{\"Data Availability\": \"All relevant data are within the paper and its Supporting Information files.\"}"