Background
Insecticide-treated nets (ITN) are highly effective at reducing child mortality and incidence of uncomplicated and severe malaria [
1]. Universal coverage with long-lasting insecticidal nets (LLINs) or indoor residual spraying (IRS) is a fundamental target for the protection of all people at risk of contracting malaria [
2]. Between 2012 and 2014 (3 years), a cumulative total of 427 million LLINs were supplied for use in sub-Saharan Africa, mostly free of charge through mass distribution campaigns [
3]. The rapid scale up of LLIN distribution has resulted in an estimated 49 % (44–54 %) of households in sub-Saharan Africa owning at least one ITN in 2013 compared with only 3 % in 2004 [
3]. Since the launch of the US President’s Malaria Initiative in 2005, there has also been a substantial increase in IRS coverage in sub-Saharan Africa, with a peak of 77 million people (11 % of the at risk population) protected by IRS in 2011 [
4]. Recently there has been a decrease in IRS coverage by 29 %, with only 55 million people protected in 2013 [
3].
Pyrethroid insecticides are currently the only insecticides that are recommended by the WHO for use on LLINs [
5]. Pyrethroids have been the chemical of choice for malaria vector control in recent decades but use of pyrethroids in agriculture and scaling up of malaria vector control has resulted in the evolution and spread of pyrethroid resistance in
Anopheles gambiae sensu lato [
6‐
8]. Target site insensitivity and metabolic resistance mechanisms are now widespread across sub-Saharan Africa and the effectiveness of LLINs and IRS treatment of houses with pyrethroid formulations is under threat [
6,
9‐
11]. IRS formulations of insecticides with a different mode of action to pyrethroids are more costly, which has led to decreasing IRS coverage [
12]. The situation for LLIN is more perilous, with no alternative insecticides currently recommended by WHO Pesticide Evaluation Scheme (WHOPES) for use on mosquito nets [
5]. Cost-effective, safe insecticides with different modes of action to those currently used in public health are urgently needed to sustain the effectiveness of LLINs [
13] and Innovative Vector Control Consortium (IVCC) are working together with industry and research partners to develop new active ingredients for vector control [
14].
Indoxacarb was the first commercialized insecticide of a new class known as the oxadiazines and is highly efficacious against a wide range of agricultural pests [
15] through a novel mode of action [
16]. Indoxacarb was registered in 2000 by the United States Environmental Protection Agency (US EPA) in water dispersible granules (WG) (Avaunt
®) and emulsifiable concentrate (EC) (Steward
®) formulations, initially for foliar application targeting lepidopteran pests of cotton, rice, apples, pears, sweet corn, lettuce and fruiting vegetables [
16,
17]. Indoxacarb has more recently been shown to be effective in the control of cockroaches [
18], fire ants [
19], termites [
20], fleas [
21], and houseflies [
22] and has been commercialized as a gel bait (Advion
®).
Indoxacarb is a neurotoxic insecticide that blocks voltage-dependent sodium channels, resulting in insect paralysis and death [
15]. Despite the sodium channel being a well known target site for DDT and pyrethroids, crucially the mode of action for indoxacarb is distinct from other sodium channel targets [
23]. This is possible due to the sodium channel being structurally large and complex, with at least 9 independent target sites for a variety of neurotoxins [
24]. Indoxacarb is a pro-insecticide which is metabolized into the more active form after entering the insect host [
16]. Bioactivation of indoxacarb (DPX-JW062) through decarbomethoxylation to the more active metabolite (DCJW) is attributed to the action of esterase and amidase enzymes within the insect [
15,
24]. The active metabolite of indoxacarb exerts its effect by blocking the voltage-gated sodium ion channels in insects and is at least forty times more potent than parent indoxacarb in its ability to block sodium channel ion current [
15,
21,
24].
Indoxacarb has proven effective as a broad spectrum oral insecticide against a wide variety of agricultural pests [
17] but few studies have evaluated indoxacarb as a contact insecticide for vector control. Testing of indoxacarb treated polyester netting in Benin using WHO standard three minutes cone bioassay against
An. gambiae showed a positive mortality dose–response, with dosages >100 mg/m
2 producing mortality above the WHOPES threshold of 80 % [
25,
26]. Tunnel test simulators using host-seeking mosquitoes confirmed good efficacy in terms of mortality at dosages >100 mg/m
2, but there was no protection in terms of blood-feeding inhibition [
25]. Time to first take-off testing demonstrated that even at a high dosage of 500 mg/m
2 indoxacarb was only a mild irritant to
An. gambiae and probably explains the lack of blood-feeding inhibition in tunnel tests [
25]. Of critical importance was the lack of cross-resistance through mechanisms offering resistance to pyrethroids; no difference was found between the mortality rates for susceptible and pyrethroid resistant strains of
An. gambiae [
25]. The different target site on the sodium channel means that cross-resistance to existing pyrethroid or DDT target site-based resistance mechanisms is unlikely [
24]. Indeed, strains of important crop pests the fall armyworm,
Spodoptera frugiperda, and the diamondback moth,
Plutella xylostella, which exhibit high levels of resistance to pyrethroids, showed no cross-resistance to indoxacarb [
27,
28].
A LLIN that reduces the longevity of
Anopheles mosquitoes but does not protect from biting can be a successful strategy at high coverage rates through a mass insecticidal effect [
29]. An alternative strategy is to combine a non-repellent insecticide (to provide high levels of mortality) in a mixture with a pyrethroid insecticide (to provide protection against blood-feeding through repellency) [
30]. In this study, indoxacarb was tested in bioassays and tunnel simulators as a single treatment and in a mixture with the pyrethroid alphacypermethrin against
An. gambiae and
Culex quinquefasciatus (pyrethroid susceptible and resistant strains) to determine its performance in terms of mortality and blood-feeding inhibition.
Discussion
Pyrethroid resistance has become widespread in malaria vectors throughout sub-Saharan Africa and the lack of alternative insecticides for use on mosquito nets is a particularly serious threat to malaria vector control [
5,
6]. The Global Plan for Insecticide Resistance Management in Malaria Vectors (GPIRM) states that if pyrethroids were to lose most of their efficacy 55 % of the benefits of vector control would be lost, leading to approximately 120,000 deaths not averted [
36]. New insecticides for ITN should ideally reduce the mean life expectancy of
Anopheles mosquitoes through a mass killing, provide individual user protection through repellency, show no cross-resistance to existing insecticides used for malaria control and be safe for humans and non-target organisms [
14,
36]. Standard WHOPES 3 min ball-frame bioassay produced 100 % mortality (72 h) for dosages of indoxacarb >100 mg/m
2, which is well above the threshold of 80 % specified in WHO guidelines for evaluation of LLINs [
26]. Similarly, indoxacarb produced high levels of mortality in tunnel tests against both
An. gambiae and
Cx. quinquefasciatus at dosages >100 mg/m
2. Although the majority of mortality occurred within 24 h of testing, delayed mortality should routinely be recorded up to 72 h after exposure. Insect species metabolize indoxacarb rapidly to the more active metabolite DCJW after ingestion, but more slowly after topical treatment, thus explaining the delayed mortality [
15]. Indoxacarb is conventionally thought to produce relatively low levels of repellency against mosquitoes [
25] and agricultural pests [
20], however in these studies high levels of blood-feeding inhibition were achieved with
An. gambiae and
Cx. quinquefasciatus, particularly at dosages >100 mg/m
2. Reduced penetration of holed, indoxacarb treated netting may be indicative of indoxacarb-induced repellency. However, 96 % of
An. gambiae mosquitoes killed immediately after overnight exposure were unfed, indicating that these were killed rapidly before being able to penetrate the netting and blood-feed. Mixtures of indoxacarb and alphacypermethrin produced particularly impressive levels of blood-feeding inhibition, with an apparent additive effect.
As with any new insecticide, questions must be asked regarding whether cross-resistance might be conferred by mechanisms of insecticide resistance that have developed in field populations of the target species, how rapidly resistance might develop, and which genes might be involved [
36]. Crucially, no cross-resistance has been detected to indoxacarb in a pyrethroid resistant strain of
Cx. quinquefasciatus or in pyrethroid resistant strains of agricultural pests [
22,
27,
28]. In houseflies, two insectary strains 5900 and 18,000 fold resistant to permethrin showed no cross-resistance to indoxacarb despite the presence of kdr and increased oxidative metabolism mediated by cytochrome P450 CYP6D1 [
22]. The lack of cross-resistance is partly due to the distinctive target site on the sodium channel compared to other insecticides that target sodium channels such as pyrethroids and DDT [
24]. The only record of indoxacarb resistance in wild mosquito populations was reported for
Aedes albopictus in Pakistan and was attributed to intensive indoxacarb application for the control of cotton pests over several generations and not due to cross-resistance [
37]. Indoxacarb has a solubility in water of 0.20 mg/l at 25 °C which is similar to alphacyano-pyrethroids and pyrrole insecticides [
38]. As with these other classes of insecticide with low water solubility, development of wash resistant formulations for long lasting treatment of mosquito nets should be feasible.
Authors’ contributions
RMO participated in the study design, data collection, analysis and drafted the manuscript. RN, CN, PKT and JK participated in the study design, data collection and helped to draft parts of the manuscript. DM and FWM participated in study design and critically revised the manuscript. MWR participated in study design, structure, and multiple revisions of the manuscript. All authors read and approved the final manuscript.