Background
Long-lasting insecticidal nets (LLINs) and indoor spray with residual insecticides (IRS) have remained the core interventions for malaria control [
1,
2]. However, the gains achieved with these indoor-based interventions are threatened by the on-going development of insecticide resistance within targeted malaria vector populations [
3,
4]. Worryingly, increased outdoor biting as a result of mosquito behavioural adaptation, and change in human behaviour by spending more time outdoors altogether makes these tools less effective in sustaining the gains [
5‐
9]. Despite the urgent call for additional vector control tools to complement LLINs and IRS to accelerate the efforts toward malaria elimination, the additional tools need to align with the local context of the specific malaria endemic countries [
10].
Larval source management (LSM) particularly chemical or microbial larviciding, is one of the promising tools that can be used in conjunction with adult-based interventions for effective control of malaria vectors [
11‐
13]. Larviciding has additional benefits because it targets the aquatic stage of vectors and thus controls the population of exophilic, endophagic, and resistant mosquitoes that are associated with malaria transmission [
13,
14]. It is recommended by the World Health Organization (WHO) to be implemented as a supplementary intervention in areas where breeding habitats are only few, findable, and easy to map and treat [
15].
The potential of using larviciding for malaria control in urban settings was first demonstrated in Tanzania as part of urban malaria control programme, resulting to a 32% reduction in annual mean vector densities and sporozoite prevalence in three malaria vectors:
Anopheles funestus,
Anopheles coustani and
Anopheles gambiae [
16]. The government of Tanzania, leveraged these successes, and established the Tanzania Biotech Products Limited (TBPL), a bio-larvicide plant. This initiative has enabled the piloting of the larviciding intervention to 11 municipals in 2016 and gradually scaled up to all municipal councils in 2020 [
17‐
19].
Effective implementation of larviciding depends on the accurate identification and targeting of productive mosquito breeding habitats [
20]. This necessitates a sustainable surveillance system for monitoring the availability and distribution of breeding habitats before implementation [
21‐
23]. Recently, the application of geospatial technology and deployment of unmanned aerial vehicles (UAV) have proven effective in identifying and targeting water bodies in a wide space, which would not have been possible by only relying on personnel [
24‐
26]. On the other hand, both UAV and geospatial technology are resource demanding, and require high operational and analysis skills. In addition, they are both limited in distinguishing the mosquito productive breeding habitats from merely water bodies [
25,
26]. On this basis, the high cost and limitations associated with both the conventional larviciding and deployment of UAV highlight the need for alternative larviciding strategies that are cost-effective and complementary to LLINs and IRS, such as the autodissemination technique [
27‐
29].
The autodissemination technique, also known as “Mosquito-assisted larviciding” is a technique that exploit females mosquito oviposition behaviour, to transfer insecticides from a contaminated resting station to its breeding habitat and results in mortality or prevents emergence of the immature mosquito therein [
28]. Several studies have demonstrated the effectiveness of the autodissemination technique, mainly with pyriproxyfen, an insect growth regulator, in controlling
Aedes,
Culex, and
Anopheles mosquitoes under controlled and field settings [
28‐
34]. The autodissemination technique interrupts malaria transmission by preventing the emergence of adult vectors from contaminated breeding habitats resulting in the reduction of malaria vector density [
35,
36]. By relying on female mosquitoes that know suitable places to breed, this technique can precisely enhance high coverage of targeted breeding habitat during field application and overcome the need for widespread application of insecticide and excessive use of labour [
32,
37,
38].
Pyriproxyfen a juvenile hormone mimic is an insect growth regulator (IGR) that has been demonstrated to effectively control disease-carrying mosquitoes of different species [
29,
39‐
41]. Pyriproxyfen works by mimicking the action of a naturally occurring juvenile hormone by interfering with the growth and development of the target insect resulting in either sterilizing the contaminated mosquitoes [
42,
43] or inhibiting adult emergence [
44]. In addition, the compound is highly specific and effective at a ultralow concentration [
45]. To date, there is no evidence of pyriproxyfen resistance in malaria vectors with practical implications, with exception of animal model experiments that suggest pyriproxyfen can be metabolized in the same way as pyrethroid [
46]. Another study highlighted a partial increase in the level of mosquito tolerance to pyrethroids when used in sub-lethal doses [
47].
Of importance, pyriproxyfen is the safe compound, with allowable amount of 300 parts per billion in human drinking water, which is 6 times higher than amount recommended by the WHO for effective mosquito control [
45]. While the autodissemination technique and sterilization impact of pyriproxyfen has been demonstrated with
An. gambiae and
An. arabiensis [
29,
34], the ability of
An. funestus, a dominant malaria vector, to perform autodissemination of pyriproxyfen remains unknown. Therefore, this study was conducted in a semi-field setting in rural Tanzania to evaluate the efficacy of pyriproxyfen to control the
An. funestus via autodissemination and sterilization effects.
Discussion
The current study has proven that sterilized female
An. funestus exposed one day post blood meal, can transfer a lethal dose of pyriproxyfen to the breeding habitat located 5 m from a contaminated clay pot. Overall, forced contaminated
An. funestus with pyriproxyfen resulted to 78% and 81% adult emergence inhibition of its filial at 30 min and 48 h of exposure respectively. These findings are corroborated by previous studies in
Anopheles that documented successful autodissemination events by
An. arabiensis,
An. gambiae and
Anopheles quadrimaculatus, via either self or forceful mosquito contamination [
29,
31,
33,
34,
56]. The recorded similarity in emergence inhibition at 30 min and 48 h might be due to loss of the picked pyriproxyfen particles because of their grooming behaviour when mosquitoes are exposed longer, its absorption to mosquito cuticle, and during flight to breeding habitat [
57‐
59].
In this study, the females were held in presence of pyriproxyfen for 30 min and 48 h to mimic possible minimum and maximum resting time for rest seeking blood fed mosquitoes in the field environment [
60‐
64]. In a situation where mosquitoes are transiting the contamination stations, the success of autodissemination events might be impaired [
28,
57,
65].
Of importance, this study documented
An. funestus vulnerability to pyriproxyfen sterilization after being exposed one day post blood meal, and confirmed significant reduction in eggs laid (fecundity). Overall, at 30 min and 48 h of pyriproxyfen exposure, the mean number of eggs laid by the exposed group was reduced by 85.9% and 80.1% respectively compared to the control group. Similarly, negative effect of pyriproxyfen on mosquito fecundity has been also shown in several studies [
34,
42,
54,
66,
67]. Consistent with previous study [
54], the effect of pyriproxyfen on fecundity and fertility (eggs hatchability) in exposed
An. funestus was observed up to third gonotrophic cycle, suggesting that this effect might be irreversible during mosquito lifespan.
Previous studies have reported that pyriproxyfen interferes with the balance of hormones levels between juvenile hormone and ecdysone hormone, and disrupt the hormonal pathways responsible for egg’s maturation [
44,
54,
66,
68]. Similarly, in this currently study, the dissection of PPF exposed female mosquito revealed that pyriproxyfen sterilization effect was via retention of under developed (unmatured) eggs. Longer exposure time resulted to high proportion of mosquitoes that retain underdeveloped eggs compared to shorter exposure time. Many underdeveloped eggs were arrested at Christopher stage IV, a proxy indication for sterilization effect [
68]. It has been documented in other studies that the sterilization effect interferes with the desire of contaminated female to find a place for oviposition. [
34,
58,
66]. This depends on the time of pyriproxyfen exposure relative to when the female obtains a blood meal. While Mbare and others reported unlikelihood of contaminated female mosquito to visit the oviposition habitat after being exposed to pyriproxyfen within 24 h before and after the blood meal [
34], Itoh et al., reported the frequency of visiting the oviposition habitat to be lower for female exposed to pyriproxyfen before blood meal and higher for female exposed to pyriproxyfen after blood meal [
58].
Furthermore, Yadav et al. [
66], when assessing surface treated with a range of pyriproxyfen concentrations, reported a lower frequency of visiting oviposition habitat to a female exposed to a lower concentration of pyriproxyfen at 24 h before blood meal and higher to the females exposed at 24 h after blood. But the frequency of visiting the oviposition habitat was the same only for the female exposed to higher concentration [
66]. In this current study, female mosquitoes exposed 24 h post blood meal were capable of visiting oviposition habitat. The difference in oviposition behaviour for contaminated female mosquitoes across different studies might be due to differences in pyriproxyfen exposure methods, pyriproxyfen formulation (e.g., powder or suspension), pyriproxyfen doses and environments under which the study was conducted.
It has been reported in previous studies that environmental factors, such as wind speed, temperature, and relative humidity are responsible for triggering oviposition flights of gravid female mosquitoes [
69]. Because the current study was conducted in a semi-field environment, the observed oviposition behaviour in sterilized
An. funestus is more representative to what might happen under actual field settings compared to similar studies that were conducted under laboratory conditions [
34,
58,
66].
Overall, the findings of this study further support the potential of autodissemination of pyriproxyfen in controlling primary susceptible and resistant malaria vectors. More striking, is the fact that sterilized mosquitoes were capable to autodisseminate pyriproxyfen enough to cause adult emergence inhibition at the breeding habitats. Therefore, its potential use could be aligned with the current recommended integrated vectors control approach, which focuses on controlling and eliminating outdoor and residual malaria transmission [
15,
70,
71]. Furthermore, this presents an opportunity of scaling up this technique along recently recommended next generation bed nets co-treated with pyriproxyfen and pyrethroid [
71]. It was envisaged that, host-seeking resistant mosquitoes sterilized by pyriproxyfen nets might transfer pyriproxyfen upon successful access to a bloodmeal and resting in a contaminated station. In addition, the combined effect of these two modes of actions of pyriproxyfen can be mathematically modelled to assess its additive or synergistic effect on malaria transmission interruption.
The appropriate time for deploying autodissemination of pyriproxyfen is mainly during the dry season [
72,
73] characterized by few but stable breeding habitats. This season provides ideal condition to attain optimal doses to prevent adult emergence in the breeding habitats. On the contrary, implementing autodissemination of pyriproxyfen during rainy season, associated with flooding, hence dilution of PPF in the breeding habitats, might amplify resistance levels of the already pyrethroid resistant mosquito population as the results of the sub-lethal doses in the habitats [
47].
Despite achieving the main objective of this study, some limitations were observed. The effect of autodissemination of pyriproxyfen was not directly monitored at the provided breeding habitat but through larval bioassays. All experiment were conducted in presence of small water volumes (1 L), which was important to prove the principle, but not representative of actual habitats found in the field environment [
74]. Lastly, the resistance status of the exposed mosquitoes was not assessed, instead the supposition that they were resistant was based on the most recent reports from the same study area [
75‐
78].
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