Pyriproxyfen-treated bed nets reduce reproductive fitness and longevity of pyrethroid-resistant Anopheles gambiae under laboratory and field conditions

Abstract

Background The efficacy of insecticide-treated nets (ITNs) containing the insect growth regulator pyriproxyfen (PPF) and pyrethroid insecticides (PPF-ITNs) is being assessed in clinical trials to determine whether they provide greater protection from malaria than standard pyrethroid-treated ITNs in areas where mosquitoes are resistant to pyrethroids. Understanding the entomological mode of action of this new ITN class will aide interpretation of the results from these trials. Methods Anopheles gambiae sensu lato (s.l.) mosquitoes from a susceptible laboratory strain were exposed to PPF-treated netting 24 h, 6 h, and immediately prior to, or 24 h post blood feeding, and the impact on fecundity, fertility and longevity recorded. Pyrethroid-resistant populations were exposed to nets containing permethrin and PPF (PPF-ITNs) in cone bioassays and daily mortality recorded. Mosquitoes were also collected from inside houses pre- and post-distribution of PPF-ITNs in a clinical trial conduced in Burkina Faso; female An. gambiae s.l. were then assessed for fecundity and fertility. Results PPF exposure reduced the median adult lifespan of insecticide-susceptible mosquitoes by 4 to 5 days in all exposure times (p < 0.05) other than 6 h pre-blood meal and resulted in almost complete lifelong sterilization. The longevity of pyrethroid-resistant mosquitoes was also reduced by at least 5 days after exposure to PPF-ITNs compared to untreated nets, but was unaffected by exposure to standard pyrethroid only ITNs. A total of 386 blood-fed or gravid An. gambiae s.l. females were collected from five villages between 1 and 12 months before distribution of PPF-ITNs. Of these mosquitoes, 75% laid eggs and the remaining 25% appeared to have normal ovaries upon dissection. In contrast, only 8.6% of the 631 blood-fed or gravid An. gambiae s.l. collected post PPF-ITN distribution successfully oviposited; 276 (43.7%) did not oviposit but had apparently normal ovaries upon dissection, and 301 (47.7%) did not oviposit and had abnormal eggs upon dissection. Egg numbers were also significantly lower (average of 138/female prior distribution vs 85 post distribution, p < 0.05). Conclusion Exposure to a mixture of PPF and pyrethroids on netting shortens the lifespan of mosquitoes and reduces reproductive output. Sterilization of vectors lasted at least one year under operational conditions. These findings suggest a longer effective lifespan of PPF-pyrethroid nets than reported previously.

The Automatic Classification of Pyriproxyfen-Affected Mosquito Ovaries

“…Such a model is useful when assessing the efficacy of PPF-based tools through measurements of induced sterility in laboratory reared and field-collected populations of mosquitoes [12], and it could be particularly useful for large-volume bioassays done for durability monitoring of bio-efficacy of PPF-treated ITNs distributed in disease-endemic communities over time. It can also be used in bioassays performed during resistance monitoring, whereby field-collected females are exposed to a discriminating concentration of PPF to measure induced sterility [8].…”

“…ITNs treated with a mixture of pyriproxyfen (PPF) and pyrethroids offer an alternative to standard pyrethroidtreated ITNs in areas where pyrethroid-resistant malaria vectors are prevalent [5][6][7][8]. The mode of action of PPF affects the fertility, longevity, and lifetime fecundity of malaria vectors [9,10], and PPF-treated ITNs have been shown to sterilise Anopheles mosquitos under both laboratory and field conditions [11,12]. As vector ovary development is inhibited by exposure to PPF [8], and females that fail to develop morphologically normal eggs have been shown to not oviposit [13,14], a means of measuring efficacy and monitoring the durability of PPF and PPF-treated tools is through the assessment of eggs for signs of abnormal or inhibited development [8,12].…”

“…The mode of action of PPF affects the fertility, longevity, and lifetime fecundity of malaria vectors [9,10], and PPF-treated ITNs have been shown to sterilise Anopheles mosquitos under both laboratory and field conditions [11,12]. As vector ovary development is inhibited by exposure to PPF [8], and females that fail to develop morphologically normal eggs have been shown to not oviposit [13,14], a means of measuring efficacy and monitoring the durability of PPF and PPF-treated tools is through the assessment of eggs for signs of abnormal or inhibited development [8,12]. Although different means of scoring sterility exist (e.g., by looking for the ability to prevent egg laying or oviposition inhibition), another method to determine fertility status is based on trained experts manually dissecting ovaries and classifying egg development according to Christopher's stages [15].…”

Design and methods for a quasi-experimental pilot study to evaluate the impact of dual active ingredient insecticide-treated nets on malaria burden in five regions in sub-Saharan Africa

“…Second, individual-based stochastic models that capture the mechanisms of malaria transmission [ 47 , 49 ] can be parameterized with the pilot study data including estimates for insecticide resistance (approximated as the proportion of mosquitoes surviving exposure to the discriminatory dose of pyrethroid at bioassay testing), treatment-seeking behaviour, timing of intervention deployment and subsequent adherence to net use by communities, mosquito species and related bionomics. The entomological efficacy of IG2, PBO, and standard pyrethroid-only ITNs have been separately determined using systematic review of experimental hut data [ 43 , 52 ] (with that for RG ITNs to follow using additional bioassays and experimental huts allowing the inclusion of the impact of pyriproxyfen on oviposition and adult emergence rates [ 53 ]). These inform the transmission model to allow predictions to be made about epidemiological efficacy as resistance, approximated using the percentage survival of mosquitoes testing in the discriminatory bioassay, changes.…”

Comparison of cone bioassay estimates at two laboratories with different Anopheles mosquitoes for quality assurance of pyrethroid insecticide-treated nets

“…The age [65,66] and nutritional status (blood [65] and sugar [67]) of mosquitoes alters their susceptibility to insecticides. The time that a mosquito received a blood meal relative to contact with an insect growth regulator or juvenile hormone analogue can impact the results of the bioassay [68]. Careful transport of mosquitoes from the insectary to the test room in sealed containers and allowing mosquitoes to acclimate to the test room before bioassay will minimize physiological stress and its effects on metabolic and physiological status and so avoid possible bias in observed mortality.…”