Sublethal Pyriproxyfen Exposure Alters Anopheles arabiensis Fitness and Pyrethroid Susceptibility without Trans-Generational Carry-Over

1. Background

Malaria remains a major problem, with an estimated 263 million cases globally in 2023, an increase of 11 million from the previous year. This rise underscores the threat malaria poses, especially in regions with limited resources [1,2]. According to the World Malaria Report 2024, sub-Saharan Africa continues to bear a disproportionate share of the global malaria burden, accounting for approximately 95% of malaria cases and deaths worldwide [2]. Controlling mosquito-borne diseases remains vital for public health and the well-being of vulnerable communities in sub-Saharan African countries. Alarming statistics reveal that Tanzania is among the four countries in sub-Saharan Africa accounting for over half of the worldwide malaria-related deaths [2,3].

Long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have long been the cornerstone of malaria control in Africa [2,4]. However, the effectiveness of these indoor interventions is increasingly threatened by the emergence of insecticide resistance in targeted mosquito populations [2,5]. This growing resistance necessitates the exploration of alternative vector control strategies to sustain malaria control efforts.

To complement LLINs and IRS, many African countries are now adopting larval source management (LSM) as a supplementary malaria control strategy [2,6,7,8,9,10]. LSM involves applying chemical or biological products to mosquitoes breeding habitats to eliminate immature stages before they develop into adult vectors [2,11]. Unlike LLINs and IRS, which primarily target adult mosquitoes, LSM focuses on preventing the emergence of adult mosquitoes. By targeting breeding sites, LSM complement existing malaria control efforts and contributes towards elimination goal [2,7,10].

For effective larviciding, WHO has recommended its use in areas where mosquito breeding habitats are few, fixed and findable [1]. This presents a major challenge in rural and peri-urban areas, where mosquito breeding sites are often numerous, hidden in nature and difficult to access [12]. Additionally, the widespread adoption of LSM is hindered by the high operation costs. As an alternative, use of Unmanned Aerial Vehicles (UAVs) has improved the identification and treatment of breeding habitats in rural settings, surpassing the limitations of human-based larviciding efforts [12,13]. However, despite its potential, UAV-based larviciding remains costly and requires significant technical expertise, which limits its large-scale adoption [12,14].

Given these challenges, there is a pressing need for complementary cost-effective larviciding approaches. One promising solution is the autodissemination technique using PPF, an approach that baits mosquitoes themselves to transfer insecticides to their preferred breeding sites [15,16]. In this strategy, mosquitoes pick up a lethal dose of PPF from contaminated artificial resting sites and transfer it to their breeding habitats during oviposition, ultimately inhibiting the emergence of mosquitoes therein [15]. Several studies have demonstrated the effectiveness of PPF autodissemination in controlling Aedes, Culex and Anopheles mosquitoes in laboratory, semi field and field settings [15,16]. This method exploits mosquito behavior to achieve high coverage of breeding habitats. It requires minimal human effort, uses less insecticide, and incurs low associated labor costs [17,18].

Pyriproxyfen is a juvenile hormone analogue and insect growth regulator (IGR) that effectively disrupts the development of mosquitoes [19,20]. It mimics the action of natural juvenile hormones, interfering with mosquito development and preventing larvae from maturing into adults [21]. So far, there is no documented evidence of PPF resistance in malaria vectors. However, an in vitro study suggests that PPF can be metabolized by P450 enzymes, similar to pyrethroids [22]. A recent evidence by Opiyo et al., indicates that sublethal doses of PPF may amplify pyrethroid resistance in mosquitoes that already exhibit early signs of pyrethroid resistance [23]. However, the assessed effect of PPF exposure on pyrethroid susceptibility was in a single generation, with uncertain whether the observed resistance was temporary (tolerance) or actual resistance. To address this gap, this study assessed the effects of sublethal PPF exposure on An. arabiensis fitness parameters and susceptibility to pyrethroid insecticides over multiple generations.

2. Methods

2.1. Study Area

The study was conducted from March to November 2024 at Kinin’gina village, rural southeastern Tanzania (8.11417°S, 36.67484°E). Experiments were carried out in the semi field system (SFS) using laboratory reared An. arabiensis. Detailed description of the SFS at Ifakara Health Institute and its validation for entomological studies has been described elsewhere [24,25].

2.2. Mosquito Rearing

Third-instar larvae of pyrethroid-resistant An. arabiensis were obtained from the IHI insectary. This colony originated from field collections in Sakamaganga village, Ifakara, southern Tanzania, and has been maintained since 2011. The strain exhibits established resistance mechanisms, including kdr mutations and upregulation of cytochrome P450 enzymes. In June 2024, WHO susceptibility bioassays indicated 24% and 52% mortality when exposed to discriminating doses of permethrin and deltamethrin, respectively, confirming a high level of resistance. During all experiments, larvae were reared on TetraMin® fish food twice daily, and adults were maintained on a 10% sucrose solution ad libitum. For egg laying, female mosquitoes were starved for 12 hours prior to blood feeding, which was conducted by allowing them to feed on the arm of a consenting human volunteer for 30 minutes on two consecutive days to ensure full engorgement.

2.3. Tested Insecticide

PPF powder containing 50% active ingredient (a.i.) with a particle size of 20 to 63 µm, manufactured by Ban Field Bio Inc., USA, was used in all experiments.

2.4. Treatments with Pyriproxyfen

To establish test sublethal concentrations, a larval bioassay using twenty-five laboratory-reared third-instar larvae of An. arabiensis exposed in 200 ml of either untreated water (control) or PPF concentrations (0.00001, 0.0001, 0.0005, 0.0008, 0.001, 0.0015, 0.002, 0.01, 0.025, 0.05, 0.1, and 0.5 mg/L) were conducted. Each concentration was replicated four times. Larvae were fed with TetraMin® fish food twice daily. Using WHOPES guidelines, the number of live larvae, pupae, and emerged adults were recorded every 24 hours until all larvae had either died or emerged [27]. Test PPF concentrations of 0.0003, 0.0006, 0.0008, and 0.001 mg/L that resulted to 5%, 10%, 15%, and 20% adult emergence inhibition respectively, were selected for the follow-up experiment. In the main experiment, concentrations of 5% and 20% were used for rearing larvae used in the pyrethroid susceptibility bioassay experiment, while concentrations of 5%, 10%, 15%, and 20% were used for rearing larvae used in the fitness experiment. All test concentrations were prepared by serially diluting stock solution of 0.1 mg AI/L.

2.5. Effect of Pyriproxyfen Exposure on Insecticide Susceptibility

To assess the effect of larval exposure to sublethal doses of PPF on adult insecticide susceptibility, 3000 third-instar larvae of pyrethroid-resistant An. arabiensis were equally divided into six basins containing 2 L of water. Larvae were exposed to either 0.0003 mg/L or 0.001 mg/L PPF, or to untreated water. Adults emerging from PPF treated and untreated basins in the first generation were pooled and maintained in 35 × 35 × 35 cm net cages. Following blood feeding, the second and third generations larvae were not exposed to PPF. Emerged adults labelled as treatment and control groups, were kept separately, provided with 10% glucose, for WHO standard susceptibility assays. For each generation, 150 unfed mosquitoes of 3 – 5-day-old females from each treatment and control group were exposed to 0.75% permethrin, 0.05% deltamethrin, and 3.75% permethrin [28]. Six replicates of 25 mosquitoes per replicate were performed for each test insecticide, with respective controls of non-impregnated papers. To establish the effect of PPF, similar procedures were performed for adult mosquitoes reared in untreated water.

2.6. Effect of Pyriproxyfen Exposure on Fitness Parameters

To assess the effects of PPF on mosquito fitness exposed to sublethal doses, 2000 third-instar larvae in 2 L of water were exposed to each PPF concentrations of 0.0003, 0.0006, 0.0008, 0.001 mg/L, and untreated water (control). This setup was replicated four times. Emerged adults from each group in the first generation were kept in net cages and blood-fed to produce second and third generations that were reared without PPF. In each generation, 30 blood-fed An. arabiensis females (5–9 days old) from each treatment and control group were individually placed in oviposition cups lined with damp filter paper 72 hours’ post-blood feeding. Mosquitoes were maintained on 10% glucose solution ad libitum during oviposition monitoring. Oviposition cups were observed for up to seven days, and once eggs were detected, the filter paper was removed, eggs were counted under a stereomicroscope and transferred into basins containing 2 L of water. Eggs were monitored daily for three consecutive days to record hatching. Fecundity was determined by the number of eggs laid, while fertility was calculated as the proportion of eggs that hatched.

After oviposition, females were killed by freezing for 10 minutes, and one wing was removed, mounted on a slide, and measured (from the apical notch to the axillary margin) under a microscope using a micrometer to estimate body size [28].

2.7. Statistical Analysis and Presentation

The results were analyzed using open source software, R version 4.5.1 [29].

Experiment 1: Dose-response analysis

To evaluate emergence inhibition, a log dose–response analysis was performed. This analysis estimated the concentrations of PPF required to achieve 5%, 10%, 15%, and 20% emergence inhibition rates.

Experiment 2: Effect of sub-lethal PPF exposure on susceptibility to pyrethroids

The effect of sub-lethal PPF exposure on susceptibility to pyrethroids was evaluated using standard WHO bioassays with permethrin (0.75% and 3.75%) and deltamethrin (0.05%) across three mosquito generations. The outcomes assessed were knockdown at 60 minutes and 24-hour mortality, both modeled as binary responses. Binomial Generalized Linear Mixed Models (GLMMs) were fitted using the glmer function in the lme4 package [30], with PPF concentration, generation, and insecticide type as fixed effects, and replicate batches included as random intercepts. Separate models were fitted for each insecticide, and interactions between generation and PPF concentration were tested. Descriptive statistics (mean ± SD) and odds ratios with 95% confidence intervals were reported.

Experiment 3: Effect of sub-lethal PPF exposure on mosquito fitness

Fitness parameters assessed included fecundity, hatchability, and wing length across three generations. For fecundity, data showed zero inflation and skewness, confirmed by Shapiro–Wilk testing. Poisson GLMs were fitted separately for each generation, as well as an overall model testing the Generation × Concentration interaction. Predictions were generated with ggeffects.

Hatchability was analyzed using binomial GLMs fitted per generation, as well as an overall model that included the Concentration × Generation interaction. Overdispersion was tested and appropriately accounted for. Model results were expressed as odds ratios with 95% confidence intervals, and predicted proportions hatched (with 95% confidence intervals) were generated using ggeffects.

Wing length was treated as a continuous trait. Data were first tested for normality using the Shapiro–Wilk test, which showed mild skewness. Accordingly, Gamma GLMs with a log-link were fitted to model the effects of PPF exposure. Both separate and interaction models were fitted to assess PPF effects across generations. Predicted means with 95% confidence intervals.

3. Results

3.1. Initial Tests to Determine the Range of Sub-Lethal PPF Doses

Emergence inhibition of third-instar larvae exposed to various PPF doses are summarized in (Figure 1). Data from four replicates of the An. arabiensis colony were pooled per dose to estimate the concentrations inhibiting 5%, 10%, 15%, and 20% adult emergence, used in subsequent experiments. PPF doses between 3e-4 mg a.i/L and 0.003 mg a.i/L caused 5% and nearly 50% emergence inhibition. Therefore, subsequent experiments used doses in the range of 3e-4 mg a.i/L to 0.001 mg a.i/L.

3.2. Effect of Sub Lethal Dose of PPF on An. arabiensis Susceptibility to Pyrethroids

In the first generation, adult mosquitoes from larvae exposed to PPF showed significantly reduced mortality when exposed to permethrin and deltamethrin. For permethrin, mortality dropped significantly at 0.0003 mg a.i/L (p = 0.026) and at 0.001 mg a.i/L (p < 0.001). Similarly, deltamethrin showed significant reductions at both 0.0003 and 0.001 mg a.i/L (p < 0.001 for both). No significant differences in mortality were observed in the second and third generations (p > 0.05) (Table 1). However, exposure to 3.75% permethrin resulted in 100% knockdown and mortality across all PPF concentrations and controls in all generations.

A similar pattern was observed in knockdown at 60 minutes. Both insecticides showed significant knockdown reductions at 0.0003 and 0.001 mg a.i/L in the first generation (p < 0.001). No significant differences were found in second and third generations (p > 0.05) (Table 2).

3.3. Effect of Sublethal Dose of Pyriproxyfen on Body Size, Fecundity, and Fertility

Mosquitoes from PPF-exposed larvae in the first generation had significantly shorter mean wing lengths compared to the control group (3.07 mm). The mean wing lengths were 2.88 mm at 0.0003 mg a.i./L (p < 0.004), 2.82 mm at 0.0006 mg a.i./L, 2.77 mm at 0.0008 mg a.i./L, and 2.66 mm at 0.001 mg a.i./L (all p < 0.001). No significant differences in mean wing length were observed in the second and third generations (p > 0.05). (Table 3).

A similar trend was observed for mean egg production. In the first generation, the mean number of eggs per female significantly decreased in the PPF-exposed groups: 18.83 at 0.0003 mg a.i./L, 17.80 at 0.0006 mg a.i./L, 14.93 at 0.0008 mg a.i./L, and 13.90 at 0.001 mg a.i./L, all compared to a mean of 30.7 eggs in the control group (p < 0.001 for all). No significant differences in mean egg production were found in subsequent generations (p > 0.05). (Table 4).

Similarly, the mean proportion of hatched eggs per female was significantly lower in the first generation of PPF-exposed mosquitoes compared to the control group. The predicted mean proportion was 0.87 in controls, decreasing to 0.82, 0.82, 0.79, and 0.79 at 0.0003, 0.0006, 0.0008, and 0.001 mg a.i./L, respectively (all p ≤ 0.005). No significant differences were observed in the second and third generations, where proportions remained similar across all PPF concentrations (0.87–0.89, p > 0.005) (Table 5).

4. Discussion

Identifying new insecticides and innovative delivery methods is a public health priority due to the increasing spread of pyrethroid resistance among disease transmitting mosquitoes [11,19,31,32]. Pyriproxyfen a juvenile hormone analog, is one such insecticide known to induce sterilization and emergence inhibition in exposed mosquitoes [31,33,34]. Previous studies have examined the combined effects of PPF and pyrethroids to understand its interactions with conventional vector control tools like LLINs and IRS [22,23,35,36,37,38,39]. In this context, present study investigated the effects of sublethal PPF exposure on An. arabiensis pyrethroid susceptibility across three generations, and effect of such exposure on mosquito fecundity, fertility and body size. We examined these interactions in situations where the emerged adults were exposed to pyrethroid insecticide class (permethrin and deltamethrin) commonly used for adult mosquito control.

The findings show that PPF exposure does not lead to heritable insecticide resistance, but it induces a temporary increase in tolerance in the first generation. However, in the absence of continuous PPF exposure, the second and third generations exhibited susceptibility, fecundity, fertility and body size comparable to the control group. These results indicate that a single larval exposure to PPF produces transient effects that are not heritable, and that assessing three generations is sufficient to capture its transgenerational impact. As hypothesized, exposure to 3.75% permethrin resulted in 100% knockdown and mortality across all generations, regardless of PPF exposure. These results align with those of Opiyo et al., who reported increased resistance in An. arabiensis following sublethal PPF exposure [23]. This study suggests that pyrethroid resistance associated with aquatic exposure to PPF may be restricted to the first generation and is not carried over to subsequent mosquito generations. The observed response likely reflects temporary tolerance rather than true resistance, which arises from the selection of heritable traits within mosquito populations. Cross-resistance between PPF and other insecticide classes has been reported in housefly Musca domestica, against methoxyfenozide (diacylhydrazines), such as; cyromazine (triazines), and lufenuron (benzoylureas) [40]. Resistance to PPF in the greenhouse whitefly Trialeurodes vaporariorum has been linked to overexpression of the cytochrome P450 gene CYP4G61, highlighting the potential role of metabolic mechanisms. [37].

Some studies suggest that interactions between PPF and pyrethroids could upregulate detoxification enzymes, such as cytochrome P450s, which may metabolize both insecticide classes in An. arabiensis [22,23], raising concerns about cross-resistance. However, this effect might be dose-dependent with continuous exposure of mosquitoes to PPF. Prolonged contact with sublethal concentrations of insecticides and domestic pollutants such as hydrogen peroxide and soap detergent act as selective pressure that favors resistance traits. For instance, Shayo et al. reported increased tolerance in both susceptible An. gambiae and pyrethroid-resistant An. arabiensis following exposure to domestic pollutants such as hydrogen peroxide and soap detergent [41]. Similarly, Shilla et al. found enhanced insecticide tolerance in susceptible An. gambiae after exposure to microplastics combined with insecticides [41]. These findings highlight the importance of considering environmental co-factors when assessing resistance development.

Because mosquitoes contaminated with PPF through autodissemination become temporarily more tolerant to pyrethroids, this could reduce the short-term efficacy of pyrethroid-based interventions. A key strategy to address this is the rotation of insecticides with different modes of action, which helps maintain overall vector control effectiveness while minimizing potential negative interactions with PPF-based tools. When integrated into a broader management program, PPF interventions provide additional value by targeting immature mosquito stages and complementing existing control measures. Notably, when permethrin was tested at a high dose of 3.75%, all mosquitoes, whether pre-exposed to PPF or not, were fully susceptible across all three generations. This confirms that high-dose intensity assays are effective in resistant populations.

Nevertheless, exposure to sublethal doses of PPF during the larval stage significantly affected key reproductive and fitness parameters in the first generation of An. arabiensis mosquitoes. Fecundity and fertility were both markedly reduced, with egg-laying and hatching rates significantly lower in PPF-exposed groups compared to controls. Additionally, exposed mosquitoes had shorter wing lengths, indicating reduced body size, a known proxy for overall fitness and survival. These effects were not observed in the second and third generations, suggesting that the impact of PPF is transient and confined to direct exposure. Our findings are consistent with previous studies demonstrating PPF’s ability to impair mosquito reproduction and development [31,43,44,45,46,47,48,49]. The reduction on fecundity and fertility could be due to the fact that PFF mimics the action of JH at a time that it should not be present, affecting the vitellogenesis or some other step in the formation of eggs [31,34,44,45]. Similar effects have been reported in LLINs co-treated with PPF, where resistant mosquitoes were sterilized upon exposure to Olyset Duo [39,50,51]. These findings highlight the added value of PPF in vector control, as it can induce sterility even in resistant mosquitoes. The sterilizing effect of PPF either at larval or adult exposure supports its potential as a complementary tool for managing resistant mosquito populations and interrupting transmission.

The smaller body sizes observed in the exposed groups are likely a consequence of PPF induced stress during larval development, although other factors such as chemical exposure or larval competition are also known to produce smaller adults [46,48,52]. From a vector control perspective, PPF exposure is particularly valuable, as it can reduce mosquito population growth and vectorial capacity even without causing immediate mortality. By limiting reproductive output and reducing adult fitness, PPF contributes to an integrated approach that suppresses vector populations and complements existing interventions.

The limitations of this study include: 1) Only the first generation of mosquitoes was exposed to PPF, while subsequent generations were reared without further exposure. Future studies should extend across multiple generations with repeated PPF exposure to better capture long-term effects. 2) Further investigations are needed to assess delayed effects on mosquitoes beyond the 24-hour period following PPF pre-exposure. Additionally, it remains unclear whether similar sublethal effects particularly the temporary increase in pyrethroid tolerance would occur in Anopheles gambiae s.s. or Anopheles funestus. These species differ in larval ecology, behavior, and resistance mechanisms, which could influence their responses to PPF, emphasizing the need for species-specific assessments in future studies. [3] It should be noted that this study assessed phenotypic resistance, which reflects the observable response of mosquitoes to insecticide exposure. While the results suggest that metabolic resistance may play a role, it is likely that other underlying mechanisms also contribute to the observed tolerance. Therefore, further investigations, including molecular and gene expression analyses, are necessary to identify the specific genes and pathways responsible for the upregulation of detoxification enzymes and to clarify the mechanisms driving resistance in these mosquito populations. Moreover, studies should be conducted to examine whether field applications of PPF, either by larviciding or other means, exacerbate pyrethroid resistance in areas where such resistance already exists in wild vector populations. This study provides important insights into the transient nature of PPF-induced effects on insecticide susceptibility and mosquito fitness, supporting its potential role as a complementary tool in integrated vector management strategies.

5. Conclusions

This study demonstrates temporary effects of sublethal dose of PPF exposure on An. arabiensis susceptibility to pyrethroids and fitness across multiple generations. The findings support the strategic use of PPF in integrated vector management through cross-rotation with pyrethroids, helping to manage resistance while preserving the effectiveness of existing interventions.