Comparative susceptibility of wild and laboratory-reared Aedes and Anopheles larvae to Ivermectin®

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OUEDRAOGO, Fabrice A. SOME, Andre B. SAGNA, Emmanuel SOUGUE, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5408919/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jul, 2025 Read the published version in Malaria Journal → Version 1 posted 8 You are reading this latest preprint version Abstract Background Administering ivermectin to humans and livestock renders their blood toxic for mosquitoes like Anopheles and Aedes , offering a promising approach for controlling these vectors. However, the impact of such treatment on larval stages exposed to the drug through contaminated breeding sites is not fully understood. This study looked at how ivermectin affects the development of Aedes and Anopheles larvae. Methods We exposed 4 instars laboratory-reared ( An. gambiae Kisumu and Ae. aegypti Bora Bora) and wild-derived ( An. coluzzii VK5 and Ae. aegypti Bobo) larvae to ivermectin-medium containing the molecule at concentrations ranging from 0 to 100 ng/ml for 24h, then transferred surviving larvae into ivermectin-free medium to monitor development until adult stage and female fecundity. Parameters measured were: larval survival, pupation dynamics, teneral emergence rates, and fecundity of the adult females in terms of numbers of eggs developed and laid. Two independent experiments were performed, each with four biological replicates. Data obtained for each life history parameter were compared between treatments to characterize ivermectin effects. Results Data indicated that highest ivermectin concentrations (100, 75, and 50 ng/ml) reduced larval survival by over 50% within 24 to 48 hours post-exposure, with varying effects across different strains. Wild-derived larvae showed lower susceptibility to ivermectin compared to laboratory larvae for both Anopheles and Aedes species. The concentrations leading to 50% larval mortality (4-day-LC50) were 3.65 and 1.86 ng/ml for Anopheles VK5 and Kisumu strains, and 15.60 and 2.56 ng/ml for Aedes Bobo and Bora Bora strains, respectively. Notably, while high concentrations severely impacted larval development, low concentration (1 ng/ml) appear to be a sublethal concentration and allowed for adult emergence. No significant effects on the number of laid eggs were observed across the different strains. Conclusion Overall, these data showed how development parameters of lab-raised and wild-derived Anopheles and Aedes larvae are affected differently by ivermectin, highlighting potential implications for vector control strategies and ecological concerns regarding non-target organisms and environment persistence. Further investigations are planned to understand existing mechanisms allowing wild-derived larvae to better survive than laboratory ones despite the presence of ivermectin in their breeding environment. Ivermectin larvae Aedes Anopheles sub-lethal concentrations tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Ivermectin is a member of the avermectin family of drugs that is widely used in veterinary medicine as an anthelmintic (dewormer) medication to treat multiple species of internal (endo-) and external (ecto-) parasites [ 1 ]. The drug works by interfering with the glutamate-gated chloride (GluCl) channels in nerve and muscle cells that are needed for signal transmission. This causes the channels to remain in an "open" position leading to the flaccid paralysis and death of the invertebrates [ 2 ]. Lice, mites, and grubs are among ectoparasites that can be treated with ivermectin in animals [ 3 , 4 ]. In humans, ivermectin is primarily used to treat river blindness (onchocerciasis) and lymphatic filariasis [ 5 – 7 ], but may also be used to treat lice and mites responsible of impetigo infestations [ 8 – 10 ]. Owing to its wide use in livestock and its broad spectrum of activity [ 11 ], the fate of ivermectin in the environment and its potential effects on non-target aquatic and terrestrial invertebrates have been increasingly reported. Indeed, ivermectin is poorly metabolized in the human and animal body and the large proportion (80–98%) of the administered dose of ivermectin and/or its metabolites are excreted almost exclusively in the feces [ 12 ]. Fecal residues of ivermectin in cattle dung have been shown to affect components of the pasture ecosystem including dung-dwelling insects and the process of dung degradation [ 1 ]. These threats to coprophagic insect activity can occur in dung deposited by animals treated weeks or even months earlier, depending upon ivermectin formulation and insect species. Additionally, ivermectin has a low water solubility and a high adsorption coefficient to sediment [ 13 ], suggesting that the risk of exposure to ivermectin for aquatic organisms living or feeding in various water body strata may vary. Entomological field studies in areas where humans were treated with ivermectin during mass drug administration (MDA) to control onchocerciasis and lymphatic filariasis have reported a decline in female Anopheles ( An. ) gambiae s.l. survival after blood feeding on ivermectin-treated humans [ 14 – 17 ]. This effect was confirmed by several laboratory studies showing that ivermectin impacted not only the survival of adult mosquitoes but also their fecundity [ 18 – 20 ]. These results have sparked enthusiasm of actors involved in the fight against malaria whom are seeing ivermectin as a complementary tool to boost malaria control, which has been stalling for almost a decade. An. coluzzii and Aedes ( Ae. ) aegypti are, respectively, important vectors of Plasmodium , the causative pathogen of malaria, and of arboviruses, responsible of diseases such as dengue and chikungunya in sub-Saharan Africa. Vector control through insecticide-based tools remains the main strategy to combat these mosquito-borne diseases. Although MDA of ivermectin to humans or livestock could be an effective and complementary strategy for controlling adult mosquito populations, there remains a gap in knowledge regarding the additional impact of such treatment on larval stages. Anopheles coluzzii larval habitats include man-made and natural breeding sites, such as dug-out wells, furrows from irrigated canals, clean, sunlit water from rains and larger breeding sites mostly consisting of rice paddles [ 21 ]. This mosquito species, one member of An. gambiae s.l. complex, is widely known for its ability to adapt to various aquatic environments, which facilitates its proliferation. Similarly, An. gambiae , another key species in the same complex, also colonizes a variety of aquatic habitats for its larvae notably temporary pools of water and rain-filled breeding sites [ 21 ]. Potential environmental contamination with ivermectin from MDA could potentially pollute An. coluzzii and An. gambiae breeding sites and expose the larvae to the drug. Aedes aegypti larvae on the contrary are mainly found in abandoned tires, artificial and domestic containers [ 22 , 23 ]. However, larvae of Ae. aegypti are increasingly reported in drains containing polluted water in urban areas [ 24 ], suggesting that larvae of this species could also be exposed to ivermectin from MDA. This difference in breeding site may lead to differential ivermectin exposure-risks between the two mosquito species and associated toxicity. Indeed, related to adult stages, Aedes mosquitoes tends to be more tolerant than Anopheles mosquitoes [ 25 , 26 ]. This study aimed to examine the effects of ivermectin on larval survival, larval development and carried-over-effects on fecundity of resulting adult females. Potential tolerance to ivermectin in wild mosquitoes has been examined through the comparison of life history traits between laboratory-reared strains and progeny of wild females from both Anopheles and Aedes mosquitoes. Impacts of indirect exposure of vectors larval stages to ivermectin may represent a yet unexplored added value to the promising approach of ivermectin-based MDAs. Methods Mosquito strains Larvae of two laboratory-reared mosquito colonies ( Anopheles gambiae "Kisumu" and Aedes aegypti "Bora Bora") known as laboratory reference strains for insecticide susceptibility testing and of two wild-derived mosquito strains ( Anopheles coluzzii "VK5" and Aedes aegypti "Bobo") were used for laboratory bioassays. Anopheles gambiae "Kisumu" is originating from Kisumu, Kenya and has been housed Since 2013 in the insectary of the Institut de Recherche en Science de la Santé, Bobo-Dioulasso, Burkina Faso. Aedes aegypti "Bora Bora" has been also hosted in our insectary since 2017 as part of the WHO “Dose-Diagnosis-Multicenter” project. The susceptibility status of both colonies to currently used insecticides is routinely checked using phenotypical and PCR assays as recommended by WHO [ 27 ]. Larvae of An. coluzzii "VK5"(first generation F1) were obtained from eggs laid by gravid females collected in December 2022 and February 2023 in Kou Valley, Bama, Burkina Faso (11° 23’ 59’’ N, 4° 25’ 46’’ W). Collected F0 females were transported to the insectary and placed in individual cups for oviposition. After eggs laying, molecular analysis by PCR [ 28 ] was performed to identify and group the F1 eggs by F0 species, specifically targeting An. coluzzii . This ensured that the larvae used in the subsequent bioassays were accurately identified, providing a reliable source of An. coluzzii larvae for the tests. Aedes aegypti "Bobo" is a wild-derived strain established in October 2022 by collecting larvae in breeding sites at Bobo-Dioulasso, Burkina Faso (11° 10’ 37’’ N, 4° 17’ 52’’ W). Adults from these larvae were morphologically identified using morphological determination key [ 29 ]. Mosquitoes have been maintained in separate rooms using rearing standard techniques under laboratory conditions (temperature: 27 ± 2°C, relative humidity: 70 ± 10%, photoperiod: 12h light followed by 12h dark). Larvae were fed daily with Tetramin® Baby Fish and adults were maintained in cages (30 cm X 30 cm) with cotton wool pads soaked in 10% glucose solution. Preparation of ivermectin test solutions A stock solution of ivermectin (10 mg/ml) was obtained by dissolving 200 mg of ivermectin powder (Sigma-Aldrich, St. Louis, USA) in 20 ml of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, USA). From this stock solution, three solutions (1 mg/ml, 0.1 mg/ml, and 0.01 mg/ml) were successively prepared by serial dilutions (2 ml in 18ml of distilled water). The solution at the concentration of 0.01 mg/ml was further diluted to obtain six more ivermectin solutions at concentrations of 1, 10, 25, 50, 75, and 100 ng/ml that were used for the bioassays ( Additional file 1 ). Two solutions without ivermectin were used as controls: the first was distilled water only and the second obtained by adding 2 ml of DMSO to 18 ml of distilled water. This was to control for any potential effect that the DMSO could induce on larval survival and development. Larval bioassays For each species, two experiments were performed on two separate dates, with four replicates per ivermectin test concentration. In each experiment and each strain, 4 tests cups with approximately 20–25 late third and early fourth instar larvae herein referred as L3/L4 were exposed to the 8 tests solutions (including the two negative controls). The larvae were provided with Tetramin ® ad libitum during the day before each experiment. Using Pasteur pipettes, larvae were transferred from larval rearing pans to labeled disposable plastic tests cups, which were filled until 99 ml using filtered dechlorinated water. Then, adequate volume of dechlorinated water and of the ivermectin solution at concentration of 0.01 mg/ml was added, giving the final concentrations of 1, 10, 25, 50, 75, and 100 ng/ml for a final volume of 100 ml for each container ( Additional file 2 ). Water was gently agitated manually to allow rapid and homogenous repartition of the ivermectin solution. The test cups containing larvae were maintained under standard rearing conditions at 27 ± 2°C, 70 ± 10% relative humidity and photoperiods of 12 h light followed by 12 h darkness. After 24h exposure, larval mortality was recorded, then larvae were rinsed with clean water to remove any remaining debris and ivermectin, transferred into new labeled cups containing dechlorinated tap water only, and provided with larval food (Tetramin ® ) until pupation. No food was provided during the 24h exposition to ivermectin. For each concentration and each technical and biological replicate, larvae mortality was monitored daily, as was the number of pupae, until all larvae died or pupated. Pupae were transferred into plastic cups filled with 5 ml of dechlorinated water and covered with mosquito net tiles for emergence. Emerging adults were counted each day and regrouped inside a same 15 cm X 15 cm cage, by ivermectin concentration used for their exposure; they were provided 10% glucose solution ad libitum on a soaked cotton ball. Three days after the last adult emerged, adults were blood-fed for fecundity assays. The larvae of An. coluzzii used in these experiments were obtained from eggs laid by gravid females collected at two different dates in field, Bama locality. Fecundity and fertility assays The fecundity and fertility assay were carried out to assess the reproductive fitness of emerging adults. Adult females that emerged after each larval exposure to ivermectin were allowed to blood feed on rabbits (25-2021/CEIRES). Oviposition containers (small petri dishes of 35 mm diameter containing moistened cotton with circular filter paper placed on the surface) were placed into labelled plastic cups labelled per ivermectin concentration and covered by an insecticide-free net. Engorged females were individually transferred into the oviposition containers through an opening made in the covered mosquito net. Females were provided 10% glucose solution and monitored daily for egg laying during 5 days. The filter papers were removed from the plastic cups and laid eggs were counted under a binocular (magnification 20X) using a hand-held counter. Females that had not laid eggs were dissected to check for the presence of developed eggs in the ovaries. The laid eggs were quickly put in separate plastic pans filled with dechlorinated tap water until hatching. The number of emerged larvae of each strain and per concentration was recorded at their third stage (L3) to facilitate counting. Statistical analysis Data were analyzed using Excel spreadsheets software (version 2021, Microsoft corporation) and R software (R version 4.1.3 (2021-09-02)). A linear mixed model was applied to analyze the data, incorporating treatment as a fixed effect and replicate as a random effect. Kaplan Meier curves were plotted to visualize larval survival for each strain and each experimental ivermectin concentration. To assess the effect of ivermectin analysis of variance (ANOVA) was performed to compare survival rates at each time point according to ivermectin concentration and post-hoc comparisons were made using student’s t-tests. A non-linear regression analysis was then performed, using the drc package (v3.0-1; [ 30 ]) to study the relationship between ivermectin concentration and larval responses, and estimate the lethal concentrations (LC) for each species and strain. The effect of ivermectin concentration on the total number of eggs laid by each emerging female was assessed using generalized linear regression. This statistical method allowed us to model the relationship between the continuous independent variable (ivermectin concentration) and the dependent variable (total number of eggs laid). A correlation analysis was also performed to examine the relationship between the concentrations of ivermectin tested and the number of females that laid eggs, as well as the mean number of developed and/or laid eggs. For all tests and models, p values < 0.05 were considered statistically significant. Results Our analysis revealed no difference between the two controls, Water and DMSO (LRT χ 2 = 0.26, df = 1, p = 0.6) Effect of ivermectin on larval survival In total, 5005 larvae from the four mosquito strains were exposed to ivermectin: An. coluzzii VK5 (1,340), An. gambiae Kisumu (1,207), Ae. aegypti Bobo (1,178), and Ae. aegypti Bora Bora (1,280). The mean survival time of the four larval strains at different ivermectin concentrations from 24h to 168h post-exposure is shown in Fig. 1 . Survival of the four larval mosquito strains was high in the control group (distilled water and DMSO, 0.0 ng/mL) and was 100% at 24h, with no significant difference between the two controls. It declined slightly and steadily over time, but remained above 90% for An. coluzzii VK5, Ae. aegypti Bobo, and Ae. aegypti Bora Bora at 168h. Only An. gambiae Kisumu displayed a survival rate below 75% at 168h. In the ivermectin-exposed group, larval survival declined markedly with increased ivermectin concentrations, with a magnitude depending on the mosquito strain and species (Fig. 1 ). The survival to high ivermectin concentrations (namely 50, 75, and 100 ng/mL) after the 24h exposure period was 91, 83, and 82% for An. coluzzii , 56; 50, and 7% for An. gambiae Kisumu, 96, 94, and 92% for Ae. aegypti Bobo, and 93, 80, and 68% for Ae. aegypti Bora Bora, respectively. The survival fell below 50% for all mosquito strains at 72h post-exposure, being 32, 14, and 9.7% for An. coluzzii , 2.8, 2.8 and 0% for An. gambiae Kisumu, 46.9, 35.8, and 41.1% for Ae. aegypti Bobo, and 6.5, 5.0, and 4.8% for Ae. aegypti Bora Bora, in 50, 75, and 100 ng/mL ivermectin concentrations, respectively. No An. gambiae Kisumu and Ae. aegypti Bora Bora larvae (laboratory-reared strains) survived after 120h post-exposure and onward in 50, 75 and 100 ng/mL ivermectin concentrations. An. gambiae Kisumu exhibited the highest susceptibility to ivermectin with no larval survival at 48h post exposure in 100 ng/mL. In low to moderate ivermectin concentrations (1, 10, and 25 ng/mL), larval survival at 24h post-exposure was high and remained above 90% for all mosquito strains. The survival in 10 and 25 ng/mL ivermectin fell below 50% at 72h post-exposure for An. coluzzii VK5, An. gambiae Kisumu, and Ae. aegypti Bora Bora, but not for Ae. aegypti Bobo where it remained above 75%. At 168h post-exposure, less than 10% of An. coluzzii VK5, An. gambiae Kisumu, and Ae. aegypti Bora Bora larvae survived in 10 and 25 ng/mL ivermectin concentrations, while almost 40% of Ae. aegypti Bobo larvae survived in the same concentrations. Larval survival remained high throughout the study in 1 ng/mL ivermectin concentration, being 72% for An. coluzzii VK5, 90% for Ae. aegypti Bobo, and 84% for Ae. aegypti Bora Bora at 168h post-exposure. Only An. gambiae Kisumu displayed larval survival below 50% at 168h post-exposure. Comparison between mosquito strains revealed significant differences in larval susceptibility to ivermectin. Comparison of larval survival between An. coluzzii VK5 (the wild-derived strain) and An. gambiae Kisumu (the laboratory-reared strain) after exposure to various concentrations of ivermectin indicated that An. coluzzii VK5 larvae exhibited higher survival rates compared to An. gambiae Kisumu across all concentrations of ivermectin (LRT χ 2 = 102.1, df = 1, p < 0.001). Similarly, when comparing the survival rates of Ae. aegypti Bobo (the wild-derived strain) and Ae. aegypti Bora Bora (the laboratory-reared strain) larvae after exposure to the same ivermectin concentrations, Ae. aegypti Bobo larvae displayed a greater tolerance to ivermectin with higher survival rates compared to Ae. aegypti Bora Bora across the range of ivermectin concentrations tested (LRT χ 2 = 70.99, df = 1, p < 0.001). The laboratory-reared Aedes mosquito showed a marked decline in survival rates with more than 85% reduction in larval survival at 72h post-exposure in concentrations ≥ 25 ng/mL compared to the wild-derived Aedes mosquito with only a 60% reduction at the same period ( p < 0.001). At 120h, all Ae. aegypti Bora Bora larvae died while between 6 to 45% of Ae. aegypti Bobo larvae survived at these concentrations. Furthermore, comparison between the two wild-derived mosquito species ( An. coluzzii VK5 and Ae. aegypti Bobo) revealed that An. coluzzii VK5 larvae were more susceptible to ivermectin than those of Ae. aegypti Bobo (Fig. 1 ). Ae. aegypti Bobo larvae displayed higher mean survival across all concentrations compared to those of An. coluzzii VK5 (LRT χ 2 = 111.1, df = 1, p < 0.001). The survival of An. coluzzii VK5 larvae fell below 50% after 72 hours post-exposure while it took 96 to 144h to have the same drop for Ae. aegypti Bobo larvae. The ivermectin concentrations leading to 50% larval mortality (4-day LC50) varied significantly between the four species (Table 1 ), being 1.86 ng/mL for An. gambiae Kisumu, 3.65 ng/mL for An. coluzzii VK5, 2.56 ng/mL for Ae. aegypti Bora Bora, and 15.6 ng/mL for Ae. aegypti Bobo. Laboratory-reared Ae. aegypti Bora Bora and wild-derived An. coluzzii VK5 larvae displayed a slightly similar 4-day LC50 (Table 1 ). Laboratory-reared An. gambiae Kisumu larvae had the lowest 4-day LC50 with 1.86 ng/mL, while wild-derived Ae. aegypti Bobo larvae had the highest one with 15.6 ng/mL. Table 1 Lethal ivermectin concentrations (LC) provoking 20, 50 and 70% cumulated larval mortalities of the different mosquitoes’ strains after 4 days following the ivermectin exposure (4-day LC) Larval strain LC20 (ng/mL) LC50 (ng/mL) LC70 (ng/mL) An. coluzzii VK5 1.03 [0.70–1.36] 3.65 [2.85–4.44] 7.90 [6.41–9.38] An. gambiae Kisumu 0.59 [0.29–0.90] 1.86 [1.18–2.53] 3.74 [2.57–4.90] Ae. aegypti Bobo 5.27 [3.07–7.47] 15.60 [11.91–19.28] 30.28 [25.24–35.31] Ae. aegypti Bora Bora 1.35 [1.08–1.63] 2.56 [2.09–3.02] 3.76 [3.04–4.49] LC = Lethal concentration Effects of larval exposure to ivermectin on post-larval stages Effect on pupal development The effect of ivermectin on pupation rates were monitored in ivermectin-exposed larvae. In control groups (distilled water and DMSO, 0.0 ng/mL), pupation rates were high, with no differences between both controls. In ivermectin-exposed larvae, high ivermectin concentrations (100, 75, and 50 ng/mL) did not lead to any pupation in all mosquito strains. While wild-derived An. coluzzii VK5 and Ae. aegypti Bobo showed a marginal percentage of pupae at concentration 25 ng/mL, 0.6% and 4.0%, respectively, the two laboratory-reared strains showed no pupation. The wild-derived Ae. aegypti Bobo was the only strain to develop substantial number of pupae (n = 25, 20.7%) at concentration 10 ng/ml compared to the other strains which only developed marginal numbers of pupae (4 for An. coluzzii VK5, 2 for An. gambiae Kisumu and 6 for Ae. aegypti Bora-Bora) (Fig. 2 ). The concentration of 1 ng/mL seemed to have no significant effects on pupal development for wild-derived An. coluzzii VK5 ( t = 10.27, df = 1, p-value = 0.061) and Ae. aegypti Bobo ( t = 2.80, df = 1, p-value = 0.21), and for the laboratory-reared Ae. aegypti Bora Bora ( t = 7.75, df = 1, p-value = 0.08). However, a significant effect was observed for An. gambiae Kisumu with a 63% decrease in pupation rate as compared to the control groups ( t = 13.66, df = 1, p-value = 0.04). Furthermore, pupation dynamics was analyzed for larvae exposed to the 1 ng/mL ivermectin concentration (Fig. 3 ). Our results showed no difference in pupal development dynamics at any time point between exposed and non-exposed larvae for both Ae. aegypti strains (Bobo and Bora Bora). For An. coluzzii VK5 (F = 5.97, p = 0.011) and An. gambiae Kisumu (F = 8.26, p = 0.004), a lower pupation rate was obtained at a concentration of 1 ng/mL starting at 144h post-exposure compared to the control groups. Effect on the emergence of adult mosquitoes The effects of 24h larval exposure to ivermectin on the emergence of adult mosquitoes were also monitored for each strain. As seen for larval mortality and pupal development, high ivermectin concentrations did not favor adult emergence. The ivermectin concentration of 1 ng/mL was the only one leading to adult emergence (Fig. 4 ). Overall, larval exposure to ivermectin significantly reduced the proportion of adults for both laboratory and wild-derived strains. However, the effects were more marked for An. gambiae Kisumu with about 77% reduction of adults’ emergence (Fig. 4 , F = 24.34, df = 2, p = 0.001). In addition, exposure to ivermectin slightly skewed the sex ratio in favor of females (more than 50%) for all strains except for Kisumu (Fig. 5 ). However, the difference was not statistically significant (F = 1.51, df = 2, p = 0.23). Effect on the fecundity of adult females The fecundity of adult females from larvae exposed to ivermectin was also evaluated by assessing both the average number of laid eggs and the average number of developed eggs in the abdomen. For An. coluzzii VK5, a decreasing trend in the number of laid eggs was observed in females from exposed larvae to 1 ng/ml ivermectin concentration compared to the control groups, but the difference was not statistically significant (F = 3.002, df = 2, p = 0.06). There was also no significant correlation between the number of developed eggs in the abdomen and treatment (r = -0.056), and between the number of laid eggs and treatment (r = -0.216). Overall, there is no strong relationship between these two variables and exposure to ivermectin; both the number of laid eggs and the number observed eggs after ovarian dissection did not vary in any predictable way between the exposed and control groups. The same trend was observed for Ae. aegypti Bobo and Ae. aegypti Bora Bora strains (Table 2 ). Larval exposure to ivermectin did not have any impact on females’ fecundity. Unfortunately, we were unable to assess the effect on emerging females of An. gambiae Kisumu due to low numbers, accentuated by difficulties in obtaining blood meals during our multiple gorging attempts. Table 2 Fecundity of the females’ mosquitoes from the different colonies that emerged from surviving larvae exposed 24h to ivermectin concentration of 1ng/ml. Larvae Strain Mean developed eggs (number of females) Mean laid eggs (number of females) C6 (1ng/ml) DMSO (0ng/ml) WATER (0ng/ml) C6 (1ng/ml) DMSO (0ng/ml) WATER (0ng/ml) An. coluzzii VK5 71.0(n = 11 ; N = 38) 92.53(n = 15 ; N = 48) 83.2(n = 10 ; N = 39) 51.08(n = 12 ; N = 38) 62.52(n = 25 ; N = 48) 62.19(n = 21 ; N = 39) Ae. aegypti Bobo 79.33(n = 6 ; N = 19) 62.0(n = 9 ; N = 19) 67.43(n = 7 ; N = 25) 20.2(n = 5 ; N = 19) 26.83(n = 6 ; N = 19) 41.55(n = 11 ; N = 25) Ae. aegypti Bora Bora 0(n = 0 ; N = 22) 0(n = 0 ; N = 18) 45(n = 1 ; N = 24) 34.1(n = 13 ; N = 22) 21.75(n = 12 ; N = 18) 34.54(n = 13 ; N = 24) n = number of females that developed eggs in ovaries or number of females that laid eggs N = total number of females for fecundity assay Discussion Ivermectin is a systemic insecticide that can help control malaria in a near future [ 31 ]. The efficacy of MDA with ivermectin to humans or livestock in managing adult mosquito populations in the field is under investigation in many malaria-endemic areas [ 32 – 34 ]. However, its mode of excretion and its broad spectrum of activity against a wide range of invertebrates raised concerns about its possible impact on non-target organisms. In Burkina Faso and other parts of sub-Saharan Africa, cattle are often treated with ivermectin. An ecotoxicological study has shown that after cattle receive a long-acting depot injection of ivermectin, high concentrations of ivermectin can be found in their dung, with mean levels reaching up to 530 ± 327 ng/g dry weight on the seventh day [ 35 ]. Another case study has highlighted that ivermectin excreted in cattle dung can leach into surrounding water surfaces [ 36 ]. Depending on mosquitoes’ ecology, mosquito larvae could be exposed to high ivermectin concentration through a contamination of their breeding sites like cattle hoof prints, as suggested by Imbahale [ 37 ]. The impact of exposure to ivermectin of larval stages on mosquito development is not well understood. The present study investigated the effect of various concentrations of ivermectin on the larval survival, their development, and the fecundity of adult females from exposed larvae of four mosquito strains: An. coluzzii VK5, An. gambiae Kisumu, Ae. aegypti Bobo, and Ae. aegypti Bora Bora. Our findings pointed out significant species-specific responses to ivermectin exposure, indicating that both genetic background and environment factors from which strains are derived (wild type and laboratory-reared) significantly influence their sensitivity to the drug. Indeed, larvae survival varied significantly depending on the strain with ivermectin concentration ≥ 50 ng/mL leading to more larvae mortality compared to lower concentrations. This is in line with other studies that have also reported differential susceptibility to ivermectin between different mosquito species and populations [ 38 – 41 ]. Larvae from laboratory-reared strains ( An. gambiae Kisumu and Ae. aegypti Bora Bora) exhibited higher susceptibility to ivermectin compared to those from wild-derived strains ( An. coluzzii VK5 and Ae. aegypti Bobo). A same trend was previously reported when larvae of these strains were exposed to different essential oils [ 42 ]. Both An. gambiae Kisumu and Ae. aegypti Bora Bora are known as reference strains established and maintained over several years in insectaries for insecticide evaluations. In addition, the domestication and controlled rearing conditions might have contributed to reduce tolerance to ivermectin exposure, possibly due to a reduced genetic diversity compared to wild-derived larvae populations. On the contrary, the higher tolerance observed in wild-derived larvae could be indicative of a broader genetic variability and/or suggest pre-existing resistance mechanisms that can mitigate the lethal effects of ivermectin as seen for resistance to public health insecticides in Aedes and Anopheles populations [ 43 , 44 ]. The study also revealed a significant difference in susceptibility between wild-derived Ae. aegypti Bobo and An. coluzzii VK5. Ae. aegypti Bobo larvae displayed a stronger tolerance to ivermectin with a 4-fold higher LC50 than that of An. coluzzii VK5. This high tolerance of Ae. aegypti Bobo larvae to ivermectin is similar to what was observed with adult stages where high concentrations of ivermectin are required to reach lethal impact [ 39 , 45 ]. Apart from differential susceptibility observed, ivermectin has been shown to reduce larval survival. This means that fewer larvae reach adulthood, reducing the overall mosquito population. The effects of ivermectin extended beyond larval survival to influence subsequent developmental stages crucial for mosquito population dynamics. This is reflected in the resulting pupation and emergence rates across all strains following larval exposure to ivermectin. These results are consistent with the expected outcomes of exposure to a larvicide as demonstrated by several studies [ 46 – 50 ]. Interestingly, an ivermectin concentration as low as 1 ng/mL favored the emergence of adults, highlighting a critical threshold where developmental progression was still possible. High concentrations (50 ng/mL and above) almost completely suppressed the transition larvae-to-adult stages across both laboratory-reared and wild-derived species, indicating the potential of ivermectin as a good larvicidal agent. However, the observed ability of some larvae, particularly those derived from wild strains to withstand even intermediate concentrations (25 ng/ml) and continue their development could signal potential issues with resistance development. This is particularly critical as the continued emergence of adults from exposed larvae could allow for the maintenance of a breeding population capable of passing on resistant genes to their offspring. Regarding fecundity assays, our study found no significant effects on the fecundity of adult females from ivermectin-exposed larvae. Similar results have been found with Anopheles gambiae larvae exposed to ivermectin [ 51 ]. However, exposure of Culex quinquefasciatus larvae to ivermectin seemed to disrupt egg development, resulting in a reduction of the number of laid eggs [ 52 ]. This disruption could be explained by changes in larval fat cells, which play an important role in egg production in female mosquitoes. These results highlight implications for vector control management, as females from larvae that survive to ivermectin exposure may retain their reproductive capacity and still contribute to population recovery over time, facilitating continued disease transmission. The intensive use of ivermectin through mass distribution campaigns could have an indirect beneficial effect by affecting pre-imaginal stages, but could also lead to persistence of the product and therefore bioaccumulation of residues in the environment, raising environmental concerns such as the impact on non-target fauna and the aquatic ecosystem [ 53 ]. So, it would be necessary to assess ivermectin concentrations more accurately, through chemical analyses of water and soil samples from breeding sites. A limitation of this study was that the wild species used were from limited geographic area which does not necessarily mimic the same environmental and ecological interactions experienced by mosquito of other localities. Expanding collections zones from multiple geographic settings for future experiments could be more informative. Additionally, we exposed only third/fourth instars larvae. However, it is possible that the earlier larval stages such as first/second instar larvae are more susceptible to ivermectin potentially due to their smaller size, less developed cuticle, and less efficient metabolic systems. It would also be interesting to prolong larval contact to ivermectin above to 24 hours because in environment, larvae could be exposed continually in contaminated breeding sites. Conclusion The use of ivermectin through mass drug administration (MDA) in vector control might seem a bit more complex. Although it shows promise in targeting adult mosquitoes, its environmental impact, especially on larval stages in various breeding habitats, must be carefully considered. This study focused on the impact of ivermectin on the life history traits of larvae from wild-derived and laboratory-reared Aedes and Anopheles which might be exposed to the drug through treated humans or animals waste after MDA campaigns. The findings revealed different levels of susceptibility to the drug between wild and laboratory larvae, as well as between the different genera, emphasizing the need of considering both genetic background and environmental origins. Additionally, the pupation and adult emergence rates observed in high ivermectin concentrations demonstrate that larval exposure to ivermectin can substantially impact mosquito populations by limiting their developmental progression. However, there was no clear effect of larval exposure on the fecundity of adults, indicating that while larval development is impaired, those that manage to emerge as adults retain their reproductive capabilities. These results highlight the potential challenges posed by ivermectin-based MDA for controlling vector borne diseases. Further investigations are required to understand why wild-type larvae survive better, and to explore the long-term implications of larval exposure to ivermectin for vector control. Abbreviations Ae Aedes An Anopheles DMSO Dimethyl sulfoxide GluCl Glutamate-gated chloride LC Lethal Concentration MDA Mass Drug Administration PCR Polymerase Chain reaction s.l sensus lato VK5 Vallée du Kou, secteur 5 WHO World Health Organization Declarations Acknowledgements We would like to thank the IRSS-DRO technical team for their assistance. We would also like to thank the institutions and donors for their support. Funding This work is supported by funding from Unitaid under the IMPACT grant Availability of data and materials Data are fully available from the corresponding author upon request. Authors’ contributions COWO, FAS, ABS, KM and RKD conceived and designed the study. COWO, ES collected the data. COWO and DDS analyzed the data. COWO drafted the original manuscript. FAS, ABS, ES, DDS, MN, FHC, SHP, LZ, CR, EHAN, KM, RKD reviewed the manuscript. All authors read and approved the final manuscript. Ethics approval This study is part of IMPACT project that got approval from the Institutional Ethics Committee for Health Sciences Research under reference number 25-2021/CEIRES of June 15 th 2021. The verbal consent of heads of households was requested before any mosquito collection in dwellings. 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A: survival of \u003cem\u003eAn. coluzzii \u003c/em\u003eVK5; B: survival of \u003cem\u003eAn. gambiae \u003c/em\u003eKisumu; C: survival of \u003cem\u003eAe. aegypti \u003c/em\u003eBobo; D: survival of \u003cem\u003eAe. aegypti \u003c/em\u003eBora Bora\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5408919/v1/1883fc17e0f4c75694dc1c07.png"},{"id":69838565,"identity":"dc4dae14-3af1-481c-84ea-66ecd6b1c244","added_by":"auto","created_at":"2024-11-25 16:46:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":655577,"visible":true,"origin":"","legend":"\u003cp\u003ePupation rate of the different mosquitoes’ strains after 24h exposure to the different ivermectin concentrations. A: pupation rate of \u003cem\u003eAn. coluzzii \u003c/em\u003eVK5; B: pupation rate of \u003cem\u003eAn. gambiae \u003c/em\u003eKisumu; C: pupation rate of \u003cem\u003eAe. aegypti \u003c/em\u003eBobo; D: pupation rate of \u003cem\u003eAe. aegypti \u003c/em\u003eBora Bora\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5408919/v1/fba5ee8aa3eedcaa03a5bf76.png"},{"id":69838566,"identity":"b6b92839-18d1-44d1-8607-a32ff10a7e1c","added_by":"auto","created_at":"2024-11-25 16:46:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1209624,"visible":true,"origin":"","legend":"\u003cp\u003ePupation dynamics of the different mosquitoes’ strains after 24h exposure to ivermectin at the concentration of 1ng/ml. For each time-point, dots represent the average proportion of pupae formed. A: pupation dynamics of \u003cem\u003eAn. coluzzii \u003c/em\u003eVK5; B: pupation dynamics of \u003cem\u003eAn. gambiae \u003c/em\u003eKisumu; C: pupation dynamics of \u003cem\u003eAe. aegypti \u003c/em\u003eBobo; D: pupation dynamics of \u003cem\u003eAe. aegypti \u003c/em\u003eBora Bora\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5408919/v1/8503002bb74753b04f2c6881.png"},{"id":69838568,"identity":"c5c5dcbe-5101-4181-b6a5-c9c1f5f62ddb","added_by":"auto","created_at":"2024-11-25 16:46:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":704640,"visible":true,"origin":"","legend":"\u003cp\u003eAdult emergence rates: adult emergence based on the total number of formed nymphs (bars in light gray) \u003cstrong\u003evs \u003c/strong\u003eadult emergence based on the initial number of larvae (bars in dark gray)\u003c/p\u003e\n\u003cp\u003eA: emergence rate of \u003cem\u003eAn. coluzzii \u003c/em\u003eVK5; B: emergence rate of \u003cem\u003eAn. gambiae \u003c/em\u003eKisumu; C: emergence rate of \u003cem\u003eAe. aegypti \u003c/em\u003eBobo; D: emergence rate of \u003cem\u003eAe. aegypti \u003c/em\u003eBora Bora\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5408919/v1/292cc6f2aa8e4d3f68efc929.png"},{"id":69839159,"identity":"a93716c6-1c94-4533-b4b9-67349082eb0b","added_by":"auto","created_at":"2024-11-25 16:54:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":685723,"visible":true,"origin":"","legend":"\u003cp\u003eFemales proportion at emergence of the different mosquitoes’ strains when exposed to ivermectin concentration of 1ng/ml: adult emergence (bars in light gray) \u003cstrong\u003evs \u003c/strong\u003efemale emergence (bars in light blue). A: \u003cem\u003eAn. coluzzii \u003c/em\u003eVK5; B: \u003cem\u003eAn. gambiae \u003c/em\u003eKisumu; C: \u003cem\u003eAe. aegypti \u003c/em\u003eBobo; D: \u003cem\u003eAe. aegypti \u003c/em\u003eBora Bora\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5408919/v1/1c833f3d72f59c70cbc3a706.png"},{"id":86700046,"identity":"d2b84122-450b-4a03-83a9-2e9c4b489f5e","added_by":"auto","created_at":"2025-07-14 16:11:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5204255,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5408919/v1/c4acda3d-bd4a-49c0-8f8f-823848e030e8.pdf"},{"id":69838570,"identity":"944d6ba6-3ded-49c6-938d-a4e3a5f03da3","added_by":"auto","created_at":"2024-11-25 16:46:21","extension":"tiff","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":478648,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5408919/v1/864a45b5f14dbfbcf1bb7bdf.tiff"},{"id":69838569,"identity":"44ed5f32-0789-41e0-8fd8-8c9e17ee5b4b","added_by":"auto","created_at":"2024-11-25 16:46:21","extension":"tiff","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":805150,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5408919/v1/135919a71829969d92c5a4f2.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative susceptibility of wild and laboratory-reared Aedes and Anopheles larvae to Ivermectin®","fulltext":[{"header":"Background","content":"\u003cp\u003eIvermectin is a member of the avermectin family of drugs that is widely used in veterinary medicine as an anthelmintic (dewormer) medication to treat multiple species of internal (endo-) and external (ecto-) parasites [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The drug works by interfering with the glutamate-gated chloride (GluCl) channels in nerve and muscle cells that are needed for signal transmission. This causes the channels to remain in an \"open\" position leading to the flaccid paralysis and death of the invertebrates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Lice, mites, and grubs are among ectoparasites that can be treated with ivermectin in animals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In humans, ivermectin is primarily used to treat river blindness (onchocerciasis) and lymphatic filariasis [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], but may also be used to treat lice and mites responsible of impetigo infestations [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOwing to its wide use in livestock and its broad spectrum of activity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], the fate of ivermectin in the environment and its potential effects on non-target aquatic and terrestrial invertebrates have been increasingly reported. Indeed, ivermectin is poorly metabolized in the human and animal body and the large proportion (80\u0026ndash;98%) of the administered dose of ivermectin and/or its metabolites are excreted almost exclusively in the feces [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Fecal residues of ivermectin in cattle dung have been shown to affect components of the pasture ecosystem including dung-dwelling insects and the process of dung degradation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese threats to coprophagic insect activity can occur in dung deposited by animals treated weeks or even months earlier, depending upon ivermectin formulation and insect species. Additionally, ivermectin has a low water solubility and a high adsorption coefficient to sediment [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], suggesting that the risk of exposure to ivermectin for aquatic organisms living or feeding in various water body strata may vary.\u003c/p\u003e \u003cp\u003eEntomological field studies in areas where humans were treated with ivermectin during mass drug administration (MDA) to control onchocerciasis and lymphatic filariasis have reported a decline in female \u003cem\u003eAnopheles\u003c/em\u003e (\u003cem\u003eAn.\u003c/em\u003e) \u003cem\u003egambiae s.l.\u003c/em\u003e survival after blood feeding on ivermectin-treated humans [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This effect was confirmed by several laboratory studies showing that ivermectin impacted not only the survival of adult mosquitoes but also their fecundity [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These results have sparked enthusiasm of actors involved in the fight against malaria whom are seeing ivermectin as a complementary tool to boost malaria control, which has been stalling for almost a decade.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAn. coluzzii\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e (\u003cem\u003eAe.\u003c/em\u003e) \u003cem\u003eaegypti\u003c/em\u003e are, respectively, important vectors of \u003cem\u003ePlasmodium\u003c/em\u003e, the causative pathogen of malaria, and of arboviruses, responsible of diseases such as dengue and chikungunya in sub-Saharan Africa. Vector control through insecticide-based tools remains the main strategy to combat these mosquito-borne diseases. Although MDA of ivermectin to humans or livestock could be an effective and complementary strategy for controlling adult mosquito populations, there remains a gap in knowledge regarding the additional impact of such treatment on larval stages. \u003cem\u003eAnopheles coluzzii\u003c/em\u003e larval habitats include man-made and natural breeding sites, such as dug-out wells, furrows from irrigated canals, clean, sunlit water from rains and larger breeding sites mostly consisting of rice paddles [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This mosquito species, one member of \u003cem\u003eAn. gambiae s.l.\u003c/em\u003e complex, is widely known for its ability to adapt to various aquatic environments, which facilitates its proliferation. Similarly, \u003cem\u003eAn. gambiae\u003c/em\u003e, another key species in the same complex, also colonizes a variety of aquatic habitats for its larvae notably temporary pools of water and rain-filled breeding sites [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Potential environmental contamination with ivermectin from MDA could potentially pollute \u003cem\u003eAn. coluzzii\u003c/em\u003e and \u003cem\u003eAn. gambiae\u003c/em\u003e breeding sites and expose the larvae to the drug. \u003cem\u003eAedes aegypti\u003c/em\u003e larvae on the contrary are mainly found in abandoned tires, artificial and domestic containers [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, larvae of \u003cem\u003eAe. aegypti\u003c/em\u003e are increasingly reported in drains containing polluted water in urban areas [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], suggesting that larvae of this species could also be exposed to ivermectin from MDA. This difference in breeding site may lead to differential ivermectin exposure-risks between the two mosquito species and associated toxicity. Indeed, related to adult stages, \u003cem\u003eAedes\u003c/em\u003e mosquitoes tends to be more tolerant than \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to examine the effects of ivermectin on larval survival, larval development and carried-over-effects on fecundity of resulting adult females. Potential tolerance to ivermectin in wild mosquitoes has been examined through the comparison of life history traits between laboratory-reared strains and progeny of wild females from both \u003cem\u003eAnopheles\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e mosquitoes. Impacts of indirect exposure of vectors larval stages to ivermectin may represent a yet unexplored added value to the promising approach of ivermectin-based MDAs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMosquito strains\u003c/h2\u003e \u003cp\u003eLarvae of two laboratory-reared mosquito colonies (\u003cem\u003eAnopheles gambiae\u003c/em\u003e \"Kisumu\" and \u003cem\u003eAedes aegypti\u003c/em\u003e \"Bora Bora\") known as laboratory reference strains for insecticide susceptibility testing and of two wild-derived mosquito strains (\u003cem\u003eAnopheles coluzzii\u003c/em\u003e \"VK5\" and \u003cem\u003eAedes aegypti\u003c/em\u003e \"Bobo\") were used for laboratory bioassays. \u003cem\u003eAnopheles gambiae\u003c/em\u003e \"Kisumu\" is originating from Kisumu, Kenya and has been housed Since 2013 in the insectary of the Institut de Recherche en Science de la Sant\u0026eacute;, Bobo-Dioulasso, Burkina Faso. \u003cem\u003eAedes aegypti\u003c/em\u003e \"Bora Bora\" has been also hosted in our insectary since 2017 as part of the WHO \u0026ldquo;Dose-Diagnosis-Multicenter\u0026rdquo; project. The susceptibility status of both colonies to currently used insecticides is routinely checked using phenotypical and PCR assays as recommended by WHO [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Larvae of \u003cem\u003eAn. coluzzii\u003c/em\u003e \"VK5\"(first generation F1) were obtained from eggs laid by gravid females collected in December 2022 and February 2023 in Kou Valley, Bama, Burkina Faso (11\u0026deg; 23\u0026rsquo; 59\u0026rsquo;\u0026rsquo; N, 4\u0026deg; 25\u0026rsquo; 46\u0026rsquo;\u0026rsquo; W). Collected F0 females were transported to the insectary and placed in individual cups for oviposition. After eggs laying, molecular analysis by PCR [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] was performed to identify and group the F1 eggs by F0 species, specifically targeting \u003cem\u003eAn. coluzzii\u003c/em\u003e. This ensured that the larvae used in the subsequent bioassays were accurately identified, providing a reliable source of \u003cem\u003eAn. coluzzii\u003c/em\u003e larvae for the tests. \u003cem\u003eAedes aegypti\u003c/em\u003e \"Bobo\" is a wild-derived strain established in October 2022 by collecting larvae in breeding sites at Bobo-Dioulasso, Burkina Faso (11\u0026deg; 10\u0026rsquo; 37\u0026rsquo;\u0026rsquo; N, 4\u0026deg; 17\u0026rsquo; 52\u0026rsquo;\u0026rsquo; W). Adults from these larvae were morphologically identified using morphological determination key [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Mosquitoes have been maintained in separate rooms using rearing standard techniques under laboratory conditions (temperature: 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity: 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, photoperiod: 12h light followed by 12h dark). Larvae were fed daily with Tetramin\u0026reg; Baby Fish and adults were maintained in cages (30 cm X 30 cm) with cotton wool pads soaked in 10% glucose solution.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of ivermectin test solutions\u003c/h3\u003e\n\u003cp\u003eA stock solution of ivermectin (10 mg/ml) was obtained by dissolving 200 mg of ivermectin powder (Sigma-Aldrich, St. Louis, USA) in 20 ml of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, USA). From this stock solution, three solutions (1 mg/ml, 0.1 mg/ml, and 0.01 mg/ml) were successively prepared by serial dilutions (2 ml in 18ml of distilled water). The solution at the concentration of 0.01 mg/ml was further diluted to obtain six more ivermectin solutions at concentrations of 1, 10, 25, 50, 75, and 100 ng/ml that were used for the bioassays (\u003cb\u003eAdditional file 1\u003c/b\u003e). Two solutions without ivermectin were used as controls: the first was distilled water only and the second obtained by adding 2 ml of DMSO to 18 ml of distilled water. This was to control for any potential effect that the DMSO could induce on larval survival and development.\u003c/p\u003e\n\u003ch3\u003eLarval bioassays\u003c/h3\u003e\n\u003cp\u003eFor each species, two experiments were performed on two separate dates, with four replicates per ivermectin test concentration. In each experiment and each strain, 4 tests cups with approximately 20\u0026ndash;25 late third and early fourth instar larvae herein referred as L3/L4 were exposed to the 8 tests solutions (including the two negative controls). The larvae were provided with Tetramin\u003csup\u003e\u0026reg;\u003c/sup\u003e \u003cem\u003ead libitum\u003c/em\u003e during the day before each experiment. Using Pasteur pipettes, larvae were transferred from larval rearing pans to labeled disposable plastic tests cups, which were filled until 99 ml using filtered dechlorinated water. Then, adequate volume of dechlorinated water and of the ivermectin solution at concentration of 0.01 mg/ml was added, giving the final concentrations of 1, 10, 25, 50, 75, and 100 ng/ml for a final volume of 100 ml for each container (\u003cb\u003eAdditional file 2\u003c/b\u003e). Water was gently agitated manually to allow rapid and homogenous repartition of the ivermectin solution. The test cups containing larvae were maintained under standard rearing conditions at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity and photoperiods of 12 h light followed by 12 h darkness. After 24h exposure, larval mortality was recorded, then larvae were rinsed with clean water to remove any remaining debris and ivermectin, transferred into new labeled cups containing dechlorinated tap water only, and provided with larval food (Tetramin\u003csup\u003e\u0026reg;\u003c/sup\u003e) until pupation. No food was provided during the 24h exposition to ivermectin. For each concentration and each technical and biological replicate, larvae mortality was monitored daily, as was the number of pupae, until all larvae died or pupated. Pupae were transferred into plastic cups filled with 5 ml of dechlorinated water and covered with mosquito net tiles for emergence. Emerging adults were counted each day and regrouped inside a same 15 cm X 15 cm cage, by ivermectin concentration used for their exposure; they were provided 10% glucose solution \u003cem\u003ead libitum\u003c/em\u003e on a soaked cotton ball. Three days after the last adult emerged, adults were blood-fed for fecundity assays. The larvae of \u003cem\u003eAn. coluzzii\u003c/em\u003e used in these experiments were obtained from eggs laid by gravid females collected at two different dates in field, Bama locality.\u003c/p\u003e\n\u003ch3\u003eFecundity and fertility assays\u003c/h3\u003e\n\u003cp\u003eThe fecundity and fertility assay were carried out to assess the reproductive fitness of emerging adults. Adult females that emerged after each larval exposure to ivermectin were allowed to blood feed on rabbits (25-2021/CEIRES). Oviposition containers (small petri dishes of 35 mm diameter containing moistened cotton with circular filter paper placed on the surface) were placed into labelled plastic cups labelled per ivermectin concentration and covered by an insecticide-free net. Engorged females were individually transferred into the oviposition containers through an opening made in the covered mosquito net. Females were provided 10% glucose solution and monitored daily for egg laying during 5 days. The filter papers were removed from the plastic cups and laid eggs were counted under a binocular (magnification 20X) using a hand-held counter. Females that had not laid eggs were dissected to check for the presence of developed eggs in the ovaries. The laid eggs were quickly put in separate plastic pans filled with dechlorinated tap water until hatching. The number of emerged larvae of each strain and per concentration was recorded at their third stage (L3) to facilitate counting.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using Excel spreadsheets software (version 2021, Microsoft corporation) and R software (R version 4.1.3 (2021-09-02)). A linear mixed model was applied to analyze the data, incorporating treatment as a fixed effect and replicate as a random effect. Kaplan Meier curves were plotted to visualize larval survival for each strain and each experimental ivermectin concentration. To assess the effect of ivermectin analysis of variance (ANOVA) was performed to compare survival rates at each time point according to ivermectin concentration and post-hoc comparisons were made using student\u0026rsquo;s t-tests. A non-linear regression analysis was then performed, using the \u003cem\u003edrc\u003c/em\u003e package (v3.0-1; [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]) to study the relationship between ivermectin concentration and larval responses, and estimate the lethal concentrations (LC) for each species and strain. The effect of ivermectin concentration on the total number of eggs laid by each emerging female was assessed using generalized linear regression. This statistical method allowed us to model the relationship between the continuous independent variable (ivermectin concentration) and the dependent variable (total number of eggs laid). A correlation analysis was also performed to examine the relationship between the concentrations of ivermectin tested and the number of females that laid eggs, as well as the mean number of developed and/or laid eggs. For all tests and models, \u003cem\u003ep\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eOur analysis revealed no difference between the two controls, Water and DMSO (LRT χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.26, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.6)\u003c/p\u003e\n\u003ch3\u003eEffect of ivermectin on larval survival\u003c/h3\u003e\n\u003cp\u003eIn total, 5005 larvae from the four mosquito strains were exposed to ivermectin: \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 (1,340), \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu (1,207), \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo (1,178), and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora (1,280). The mean survival time of the four larval strains at different ivermectin concentrations from 24h to 168h post-exposure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Survival of the four larval mosquito strains was high in the control group (distilled water and DMSO, 0.0 ng/mL) and was 100% at 24h, with no significant difference between the two controls. It declined slightly and steadily over time, but remained above 90% for \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5, \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo, and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora at 168h. Only \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu displayed a survival rate below 75% at 168h. In the ivermectin-exposed group, larval survival declined markedly with increased ivermectin concentrations, with a magnitude depending on the mosquito strain and species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The survival to high ivermectin concentrations (namely 50, 75, and 100 ng/mL) after the 24h exposure period was 91, 83, and 82% for \u003cem\u003eAn. coluzzii\u003c/em\u003e, 56; 50, and 7% for \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu, 96, 94, and 92% for \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo, and 93, 80, and 68% for \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora, respectively. The survival fell below 50% for all mosquito strains at 72h post-exposure, being 32, 14, and 9.7% for \u003cem\u003eAn. coluzzii\u003c/em\u003e, 2.8, 2.8 and 0% for \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu, 46.9, 35.8, and 41.1% for \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo, and 6.5, 5.0, and 4.8% for \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora, in 50, 75, and 100 ng/mL ivermectin concentrations, respectively. No \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora larvae (laboratory-reared strains) survived after 120h post-exposure and onward in 50, 75 and 100 ng/mL ivermectin concentrations. \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu exhibited the highest susceptibility to ivermectin with no larval survival at 48h post exposure in 100 ng/mL. In low to moderate ivermectin concentrations (1, 10, and 25 ng/mL), larval survival at 24h post-exposure was high and remained above 90% for all mosquito strains. The survival in 10 and 25 ng/mL ivermectin fell below 50% at 72h post-exposure for \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5, \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu, and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora, but not for \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo where it remained above 75%. At 168h post-exposure, less than 10% of \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5, \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu, and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora larvae survived in 10 and 25 ng/mL ivermectin concentrations, while almost 40% of \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo larvae survived in the same concentrations. Larval survival remained high throughout the study in 1 ng/mL ivermectin concentration, being 72% for \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5, 90% for \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo, and 84% for \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora at 168h post-exposure. Only \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu displayed larval survival below 50% at 168h post-exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eComparison between mosquito strains revealed significant differences in larval susceptibility to ivermectin. Comparison of larval survival between \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 (the wild-derived strain) and \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu (the laboratory-reared strain) after exposure to various concentrations of ivermectin indicated that \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 larvae exhibited higher survival rates compared to \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu across all concentrations of ivermectin (LRT χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;102.1, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). Similarly, when comparing the survival rates of \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo (the wild-derived strain) and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora (the laboratory-reared strain) larvae after exposure to the same ivermectin concentrations, \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo larvae displayed a greater tolerance to ivermectin with higher survival rates compared to \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora across the range of ivermectin concentrations tested (LRT χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;70.99, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). The laboratory-reared \u003cem\u003eAedes\u003c/em\u003e mosquito showed a marked decline in survival rates with more than 85% reduction in larval survival at 72h post-exposure in concentrations\u0026thinsp;\u0026ge;\u0026thinsp;25 ng/mL compared to the wild-derived \u003cem\u003eAedes\u003c/em\u003e mosquito with only a 60% reduction at the same period (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). At 120h, all \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora larvae died while between 6 to 45% of \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo larvae survived at these concentrations. Furthermore, comparison between the two wild-derived mosquito species (\u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 and \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo) revealed that \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 larvae were more susceptible to ivermectin than those of \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo larvae displayed higher mean survival across all concentrations compared to those of \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 (LRT χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;111.1, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001). The survival of \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 larvae fell below 50% after 72 hours post-exposure while it took 96 to 144h to have the same drop for \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo larvae. The ivermectin concentrations leading to 50% larval mortality (4-day LC50) varied significantly between the four species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), being 1.86 ng/mL for \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu, 3.65 ng/mL for \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5, 2.56 ng/mL for \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora, and 15.6 ng/mL for \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo. Laboratory-reared \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora and wild-derived \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 larvae displayed a slightly similar 4-day LC50 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Laboratory-reared \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu larvae had the lowest 4-day LC50 with 1.86 ng/mL, while wild-derived \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo larvae had the highest one with 15.6 ng/mL.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLethal ivermectin concentrations (LC) provoking 20, 50 and 70% cumulated larval mortalities of the different mosquitoes\u0026rsquo; strains after 4 days following the ivermectin exposure (4-day LC)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarval strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLC20 (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC50 (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLC70 (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e VK5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.03 [0.70\u0026ndash;1.36]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.65 [2.85\u0026ndash;4.44]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.90 [6.41\u0026ndash;9.38]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.59 [0.29\u0026ndash;0.90]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.86 [1.18\u0026ndash;2.53]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.74 [2.57\u0026ndash;4.90]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e Bobo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.27 [3.07\u0026ndash;7.47]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.60 [11.91\u0026ndash;19.28]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.28 [25.24\u0026ndash;35.31]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.35 [1.08\u0026ndash;1.63]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.56 [2.09\u0026ndash;3.02]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.76 [3.04\u0026ndash;4.49]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cb\u003eLC\u0026thinsp;=\u0026thinsp;Lethal concentration\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eEffects of larval exposure to ivermectin on post-larval stages\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect on pupal development\u003c/h2\u003e \u003cp\u003eThe effect of ivermectin on pupation rates were monitored in ivermectin-exposed larvae. In control groups (distilled water and DMSO, 0.0 ng/mL), pupation rates were high, with no differences between both controls. In ivermectin-exposed larvae, high ivermectin concentrations (100, 75, and 50 ng/mL) did not lead to any pupation in all mosquito strains. While wild-derived \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 and \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo showed a marginal percentage of pupae at concentration 25 ng/mL, 0.6% and 4.0%, respectively, the two laboratory-reared strains showed no pupation. The wild-derived \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo was the only strain to develop substantial number of pupae (n\u0026thinsp;=\u0026thinsp;25, 20.7%) at concentration 10 ng/ml compared to the other strains which only developed marginal numbers of pupae (4 for \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5, 2 for \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu and 6 for \u003cem\u003eAe. aegypti\u003c/em\u003e Bora-Bora) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The concentration of 1 ng/mL seemed to have no significant effects on pupal development for wild-derived \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.27, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.061) and \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.80, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.21), and for the laboratory-reared \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.75, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08). However, a significant effect was observed for \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu with a 63% decrease in pupation rate as compared to the control groups (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.66, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). Furthermore, pupation dynamics was analyzed for larvae exposed to the 1 ng/mL ivermectin concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Our results showed no difference in pupal development dynamics at any time point between exposed and non-exposed larvae for both \u003cem\u003eAe. aegypti\u003c/em\u003e strains (Bobo and Bora Bora). For \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 (F\u0026thinsp;=\u0026thinsp;5.97, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011) and \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu (F\u0026thinsp;=\u0026thinsp;8.26, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), a lower pupation rate was obtained at a concentration of 1 ng/mL starting at 144h post-exposure compared to the control groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect on the emergence of adult mosquitoes\u003c/h2\u003e \u003cp\u003eThe effects of 24h larval exposure to ivermectin on the emergence of adult mosquitoes were also monitored for each strain. As seen for larval mortality and pupal development, high ivermectin concentrations did not favor adult emergence. The ivermectin concentration of 1 ng/mL was the only one leading to adult emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall, larval exposure to ivermectin significantly reduced the proportion of adults for both laboratory and wild-derived strains. However, the effects were more marked for \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu with about 77% reduction of adults\u0026rsquo; emergence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, F\u0026thinsp;=\u0026thinsp;24.34, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). In addition, exposure to ivermectin slightly skewed the sex ratio in favor of females (more than 50%) for all strains except for Kisumu (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the difference was not statistically significant (F\u0026thinsp;=\u0026thinsp;1.51, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.23).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect on the fecundity of adult females\u003c/h2\u003e \u003cp\u003eThe fecundity of adult females from larvae exposed to ivermectin was also evaluated by assessing both the average number of laid eggs and the average number of developed eggs in the abdomen. For \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5, a decreasing trend in the number of laid eggs was observed in females from exposed larvae to 1 ng/ml ivermectin concentration compared to the control groups, but the difference was not statistically significant (F\u0026thinsp;=\u0026thinsp;3.002, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06). There was also no significant correlation between the number of developed eggs in the abdomen and treatment (r = -0.056), and between the number of laid eggs and treatment (r = -0.216). Overall, there is no strong relationship between these two variables and exposure to ivermectin; both the number of laid eggs and the number observed eggs after ovarian dissection did not vary in any predictable way between the exposed and control groups. The same trend was observed for \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Larval exposure to ivermectin did not have any impact on females\u0026rsquo; fecundity. Unfortunately, we were unable to assess the effect on emerging females of \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu due to low numbers, accentuated by difficulties in obtaining blood meals during our multiple gorging attempts.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFecundity of the females\u0026rsquo; mosquitoes from the different colonies that emerged from surviving larvae exposed 24h to ivermectin concentration of 1ng/ml.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLarvae Strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMean developed eggs (number of females)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eMean laid eggs (number of females)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eC6\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eDMSO\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eWATER\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eC6\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(1ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eDMSO\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eWATER\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e \u003cp\u003eVK5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.0(n\u0026thinsp;=\u0026thinsp;11\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92.53(n\u0026thinsp;=\u0026thinsp;15\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.2(n\u0026thinsp;=\u0026thinsp;10\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.08(n\u0026thinsp;=\u0026thinsp;12\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62.52(n\u0026thinsp;=\u0026thinsp;25\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e62.19(n\u0026thinsp;=\u0026thinsp;21\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;39)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e\u003c/p\u003e \u003cp\u003eBobo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.33(n\u0026thinsp;=\u0026thinsp;6\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.0(n\u0026thinsp;=\u0026thinsp;9\u0026nbsp;;\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.43(n\u0026thinsp;=\u0026thinsp;7\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.2(n\u0026thinsp;=\u0026thinsp;5\u0026nbsp;;\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.83(n\u0026thinsp;=\u0026thinsp;6\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e41.55(n\u0026thinsp;=\u0026thinsp;11\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e\u003c/p\u003e \u003cp\u003eBora Bora\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(n\u0026thinsp;=\u0026thinsp;0\u0026nbsp;;\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0(n\u0026thinsp;=\u0026thinsp;0\u0026nbsp;;\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45(n\u0026thinsp;=\u0026thinsp;1\u0026nbsp;;\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.1(n\u0026thinsp;=\u0026thinsp;13\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.75(n\u0026thinsp;=\u0026thinsp;12\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.54(n\u0026thinsp;=\u0026thinsp;13\u0026nbsp;; N\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003en\u0026thinsp;=\u0026thinsp;number of females that developed eggs in ovaries or number of females that laid eggs\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003eN\u0026thinsp;=\u0026thinsp;total number of females for fecundity assay\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIvermectin is a systemic insecticide that can help control malaria in a near future [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The efficacy of MDA with ivermectin to humans or livestock in managing adult mosquito populations in the field is under investigation in many malaria-endemic areas [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, its mode of excretion and its broad spectrum of activity against a wide range of invertebrates raised concerns about its possible impact on non-target organisms. In Burkina Faso and other parts of sub-Saharan Africa, cattle are often treated with ivermectin. An ecotoxicological study has shown that after cattle receive a long-acting depot injection of ivermectin, high concentrations of ivermectin can be found in their dung, with mean levels reaching up to 530\u0026thinsp;\u0026plusmn;\u0026thinsp;327 ng/g dry weight on the seventh day [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Another case study has highlighted that ivermectin excreted in cattle dung can leach into surrounding water surfaces [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Depending on mosquitoes\u0026rsquo; ecology, mosquito larvae could be exposed to high ivermectin concentration through a contamination of their breeding sites like cattle hoof prints, as suggested by Imbahale [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The impact of exposure to ivermectin of larval stages on mosquito development is not well understood. The present study investigated the effect of various concentrations of ivermectin on the larval survival, their development, and the fecundity of adult females from exposed larvae of four mosquito strains: \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5, \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu, \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo, and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora. Our findings pointed out significant species-specific responses to ivermectin exposure, indicating that both genetic background and environment factors from which strains are derived (wild type and laboratory-reared) significantly influence their sensitivity to the drug. Indeed, larvae survival varied significantly depending on the strain with ivermectin concentration\u0026thinsp;\u0026ge;\u0026thinsp;50 ng/mL leading to more larvae mortality compared to lower concentrations. This is in line with other studies that have also reported differential susceptibility to ivermectin between different mosquito species and populations [\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Larvae from laboratory-reared strains (\u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora) exhibited higher susceptibility to ivermectin compared to those from wild-derived strains (\u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 and \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo). A same trend was previously reported when larvae of these strains were exposed to different essential oils [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Both \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora are known as reference strains established and maintained over several years in insectaries for insecticide evaluations. In addition, the domestication and controlled rearing conditions might have contributed to reduce tolerance to ivermectin exposure, possibly due to a reduced genetic diversity compared to wild-derived larvae populations. On the contrary, the higher tolerance observed in wild-derived larvae could be indicative of a broader genetic variability and/or suggest pre-existing resistance mechanisms that can mitigate the lethal effects of ivermectin as seen for resistance to public health insecticides in \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eAnopheles\u003c/em\u003e populations [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe study also revealed a significant difference in susceptibility between wild-derived \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo and \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5. \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo larvae displayed a stronger tolerance to ivermectin with a 4-fold higher LC50 than that of \u003cem\u003eAn. coluzzii\u003c/em\u003e VK5. This high tolerance of \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo larvae to ivermectin is similar to what was observed with adult stages where high concentrations of ivermectin are required to reach lethal impact [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Apart from differential susceptibility observed, ivermectin has been shown to reduce larval survival. This means that fewer larvae reach adulthood, reducing the overall mosquito population.\u003c/p\u003e \u003cp\u003eThe effects of ivermectin extended beyond larval survival to influence subsequent developmental stages crucial for mosquito population dynamics. This is reflected in the resulting pupation and emergence rates across all strains following larval exposure to ivermectin. These results are consistent with the expected outcomes of exposure to a larvicide as demonstrated by several studies [\u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Interestingly, an ivermectin concentration as low as 1 ng/mL favored the emergence of adults, highlighting a critical threshold where developmental progression was still possible. High concentrations (50 ng/mL and above) almost completely suppressed the transition larvae-to-adult stages across both laboratory-reared and wild-derived species, indicating the potential of ivermectin as a good larvicidal agent. However, the observed ability of some larvae, particularly those derived from wild strains to withstand even intermediate concentrations (25 ng/ml) and continue their development could signal potential issues with resistance development. This is particularly critical as the continued emergence of adults from exposed larvae could allow for the maintenance of a breeding population capable of passing on resistant genes to their offspring.\u003c/p\u003e \u003cp\u003eRegarding fecundity assays, our study found no significant effects on the fecundity of adult females from ivermectin-exposed larvae. Similar results have been found with \u003cem\u003eAnopheles gambiae\u003c/em\u003e larvae exposed to ivermectin [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, exposure of \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e larvae to ivermectin seemed to disrupt egg development, resulting in a reduction of the number of laid eggs [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This disruption could be explained by changes in larval fat cells, which play an important role in egg production in female mosquitoes. These results highlight implications for vector control management, as females from larvae that survive to ivermectin exposure may retain their reproductive capacity and still contribute to population recovery over time, facilitating continued disease transmission.\u003c/p\u003e \u003cp\u003eThe intensive use of ivermectin through mass distribution campaigns could have an indirect beneficial effect by affecting pre-imaginal stages, but could also lead to persistence of the product and therefore bioaccumulation of residues in the environment, raising environmental concerns such as the impact on non-target fauna and the aquatic ecosystem [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. So, it would be necessary to assess ivermectin concentrations more accurately, through chemical analyses of water and soil samples from breeding sites.\u003c/p\u003e \u003cp\u003eA limitation of this study was that the wild species used were from limited geographic area which does not necessarily mimic the same environmental and ecological interactions experienced by mosquito of other localities. Expanding collections zones from multiple geographic settings for future experiments could be more informative. Additionally, we exposed only third/fourth instars larvae. However, it is possible that the earlier larval stages such as first/second instar larvae are more susceptible to ivermectin potentially due to their smaller size, less developed cuticle, and less efficient metabolic systems. It would also be interesting to prolong larval contact to ivermectin above to 24 hours because in environment, larvae could be exposed continually in contaminated breeding sites.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe use of ivermectin through mass drug administration (MDA) in vector control might seem a bit more complex. Although it shows promise in targeting adult mosquitoes, its environmental impact, especially on larval stages in various breeding habitats, must be carefully considered. This study focused on the impact of ivermectin on the life history traits of larvae from wild-derived and laboratory-reared \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eAnopheles\u003c/em\u003e which might be exposed to the drug through treated humans or animals waste after MDA campaigns. The findings revealed different levels of susceptibility to the drug between wild and laboratory larvae, as well as between the different genera, emphasizing the need of considering both genetic background and environmental origins. Additionally, the pupation and adult emergence rates observed in high ivermectin concentrations demonstrate that larval exposure to ivermectin can substantially impact mosquito populations by limiting their developmental progression. However, there was no clear effect of larval exposure on the fecundity of adults, indicating that while larval development is impaired, those that manage to emerge as adults retain their reproductive capabilities. These results highlight the potential challenges posed by ivermectin-based MDA for controlling vector borne diseases. Further investigations are required to understand why wild-type larvae survive better, and to explore the long-term implications of larval exposure to ivermectin for vector control.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAe\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eAedes\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAn\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eAnopheles\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDimethyl sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGluCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutamate-gated chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLethal Concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMass Drug Administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase Chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003es.l\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esensus lato\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVK5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVall\u0026eacute;e du Kou, secteur 5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the IRSS-DRO technical team for their assistance. We would also like to thank the institutions and donors for their support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by funding from Unitaid under the IMPACT grant\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are fully available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCOWO, FAS, ABS, KM and RKD conceived and designed the study. COWO, ES collected the data. COWO and DDS analyzed the data. COWO drafted the original manuscript. FAS, ABS, ES, DDS, MN, FHC, SHP, LZ, CR, EHAN, KM, RKD reviewed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is part of IMPACT project that got approval from the Institutional Ethics Committee for Health Sciences Research under reference number 25-2021/CEIRES of June 15\u003csup\u003eth\u003c/sup\u003e 2021. The verbal consent of heads of households was requested before any mosquito collection in dwellings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFloate KD. 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South Asian J Parasitol. 2022;6(4):30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOchola JB, Mutero CM, Marubu RM, Haller BF, Hassanali A, Lwande W. Mosquitoes larvicidal activity of \u003cem\u003eOcimum kilimandscharicum\u003c/em\u003e oil formulation under laboratory and field-simulated conditions. Insects. 2022;13(2):203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkumu FO, Knols BG, Fillinger U. Larvicidal effects of a neem (Azadirachta indica) oil formulation on the malaria vector \u003cem\u003eAnopheles gambiae\u003c/em\u003e. Malar J. 2007;6:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiuru C, Ominde K, Muturi M, Babu L, Wanjiku C, Chaccour C, et al. Effects of larval exposure to sublethal doses of ivermectin on adult fitness and susceptibility to ivermectin in \u003cem\u003eAnopheles gambiae\u003c/em\u003e ss. Parasites Vectors. 2023;16(1):293.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlves SN, Serr\u0026atilde;o JE, Mocelin G, Melo AL. Effect of ivermectin on the life cycle and larval fat body of \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e. Brazilian archives biology Technol. 2004;47:433\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForbes A. Ecotoxicology in malaria vector control. Nat Sustain. 2024;7(6):694\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ivermectin, larvae, Aedes, Anopheles, sub-lethal concentrations, tolerance","lastPublishedDoi":"10.21203/rs.3.rs-5408919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5408919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAdministering ivermectin to humans and livestock renders their blood toxic for mosquitoes like \u003cem\u003eAnopheles\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e, offering a promising approach for controlling these vectors. However, the impact of such treatment on larval stages exposed to the drug through contaminated breeding sites is not fully understood. This study looked at how ivermectin affects the development of \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eAnopheles\u003c/em\u003e larvae.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe exposed 4 instars laboratory-reared (\u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu and \u003cem\u003eAe. aegypti\u003c/em\u003e Bora Bora) and wild-derived (\u003cem\u003eAn. coluzzii\u003c/em\u003e VK5 and \u003cem\u003eAe. aegypti\u003c/em\u003e Bobo) larvae to ivermectin-medium containing the molecule at concentrations ranging from 0 to 100 ng/ml for 24h, then transferred surviving larvae into ivermectin-free medium to monitor development until adult stage and female fecundity. Parameters measured were: larval survival, pupation dynamics, teneral emergence rates, and fecundity of the adult females in terms of numbers of eggs developed and laid. Two independent experiments were performed, each with four biological replicates. Data obtained for each life history parameter were compared between treatments to characterize ivermectin effects.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eData indicated that highest ivermectin concentrations (100, 75, and 50 ng/ml) reduced larval survival by over 50% within 24 to 48 hours post-exposure, with varying effects across different strains. Wild-derived larvae showed lower susceptibility to ivermectin compared to laboratory larvae for both \u003cem\u003eAnopheles\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e species. The concentrations leading to 50% larval mortality (4-day-LC50) were 3.65 and 1.86 ng/ml for \u003cem\u003eAnopheles\u003c/em\u003e VK5 and Kisumu strains, and 15.60 and 2.56 ng/ml for \u003cem\u003eAedes\u003c/em\u003e Bobo and Bora Bora strains, respectively. Notably, while high concentrations severely impacted larval development, low concentration (1 ng/ml) appear to be a sublethal concentration and allowed for adult emergence. No significant effects on the number of laid eggs were observed across the different strains.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOverall, these data showed how development parameters of lab-raised and wild-derived \u003cem\u003eAnopheles\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e larvae are affected differently by ivermectin, highlighting potential implications for vector control strategies and ecological concerns regarding non-target organisms and environment persistence. Further investigations are planned to understand existing mechanisms allowing wild-derived larvae to better survive than laboratory ones despite the presence of ivermectin in their breeding environment.\u003c/p\u003e","manuscriptTitle":"Comparative susceptibility of wild and laboratory-reared Aedes and Anopheles larvae to Ivermectin®","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 16:46:16","doi":"10.21203/rs.3.rs-5408919/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-23T14:05:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-13T09:43:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100974817687142321623724508729571954735","date":"2025-03-03T15:19:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230076631050107530438414388892494206529","date":"2025-01-09T08:45:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-08T15:14:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-07T13:19:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-07T13:18:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2024-11-07T10:00:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b79029c2-5a46-4a93-8142-aa714a5859c9","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:09:14+00:00","versionOfRecord":{"articleIdentity":"rs-5408919","link":"https://doi.org/10.1186/s12936-025-05470-y","journal":{"identity":"malaria-journal","isVorOnly":false,"title":"Malaria Journal"},"publishedOn":"2025-07-08 15:57:44","publishedOnDateReadable":"July 8th, 2025"},"versionCreatedAt":"2024-11-25 16:46:16","video":"","vorDoi":"10.1186/s12936-025-05470-y","vorDoiUrl":"https://doi.org/10.1186/s12936-025-05470-y","workflowStages":[]},"version":"v1","identity":"rs-5408919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5408919","identity":"rs-5408919","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}