Vector control is a key intervention against mosquito borne diseases. However, conventional methods have several limitations and alternate strategies are in urgent need. Vector control with endectocides such as ivermectin is emerging as a novel strategy. The short half-life of ivermectin is a limiting factor for its application as a mass therapy tool for vector control. Isoxazoline compounds like fluralaner, a class of veterinary acaricides with long half-life hold promise as an alternative. However, information about their mosquitocidal effect is limited. We explored the efficacy of fluralaner against laboratory reared vector mosquitoes—Aedes aegypti, Anopheles stephensi, and, Culex quinquefasciatus. 24 h post-blood feeding, fluralaner showed a significant mosquitocidal effect with LC50 values in the range of 24.04–49.82 ng/mL for the three different mosquito species tested. Effects on life history characteristics (fecundity, egg hatch success, etc.) were also observed and significant effects were noted at drug concentrations of 20, 25 and 45 ng/mL for Ae. aegypti, An. stephensi, and, Cx. quinquefasciatus respectively. At higher drug concentration of 250 ng/mL, significant mortality was observed within 1–2 h of post blood feeding. Potent mosquitocidal effect coupled with its long half-life makes fluralaner an excellent candidate for drug based vector control strategies.
Keywords: Fluralaner; Endectocide; Drug based vector control; Mosquitocidal effect; Life history characteristics
Vector borne diseases such as malaria, lymphatic filariasis, dengue and Zika virus pose significant public health threats globally[
Alternative endectocides with longer half-life which are proved to be safe or having only minimal side effects in animals and humans are highly desirable. Isoxazoline compounds like fluralaner (Bravecto®) and afoxolaner (NexGard®) used as acaricides in veterinary practice have an unusually long half-life (14.27 ± 2.53 days)[
With its excellent safety profile in animals, long half-life and the potent acaricidal effect, fluralaner can be an alternative choice for drug based vector control strategies. However, information on its mosquitocidal effect is very limited[
In the present study, the adulticidal effect of fluralaner was studied against three vector mosquito species from India: (
Based on the preliminary results of initial bioassays, experiments were performed with narrow range drug concentrations (with three technical replicates), as described in methodology section for each of the species and the 24 h LC
Table 1 Lethal concentration values.
Species Post blood feeding time (in hours) Slope (± SE) Chi2 goodness of fit (P- value) LC50 (95% CI) Heterogeneity factor 24 0.11 (0.004) 3.13 (0.08) 24.04 (14.74–45.27) 3.13 24 0.12 (0.005) 2.40 (0.12) 33.22 (24.30–51.56) 2.40 24 0.17 (0.01) 0.11 (0.90) 49.82 (49.35–50.29) 0.11
Probit regression analysis: SE, standard error; CI, confidence interval; LC, lethal concentration.
The risk of death was significantly higher in dosage 35 (HR = 150.45 (95% CI: 94.76–238.87), P-value < 0.001) when compared to control i.e., dosage 0 (Table 2) (Fig. 1A).
Table 2 Effect of fluralaner on survivorship of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus.
Dose Median survival duration (in hours) Cox-regression analysis HR (95% CI) P-value 0 (Control) – 1.00 – 5 ng/mL – 3.45 (2.07–5.74) < 0.001 10 ng/mL 96 17.19 (10.86–27.22) < 0.001 20 ng/mL 48 40.61 (25.77–64.01) < 0.001 35 ng/mL 24 150.45 (94.76–238.87) < 0.001 0 (Control) – 1.00 – 5 ng/mL – 3.48 (2.02–5.97) < 0.001 15 ng/mL – 17.21 (10.63–27.88) < 0.001 25 ng/mL 72 39.72 (24.73–63.80) < 0.001 40 ng/mL 24 231.94 (142.68–377.05) < 0.001 0 (Control) – 1.00 – 15 ng/mL – 3.72 (1.99–7.00) < 0.001 30 ng/mL – 20.82 (11.88–36.49) < 0.001 45 ng/mL 48 91.53 (52.45–159.75) < 0.001 60 ng/mL 18 516.41 (292.07–913.07) < 0.001
Cox-regression analysis: HR, hazard ratio; CI, confidence interval.
Graph: Figure 1Effect of fluralaner on survivorship of treated mosquitoes in comparison with control (dosage 0 ng/mL). (A) Aedes aegypti. (B) Anopheles stephensi. (C) Culex quinquefasciatus.
The risk of death was significantly higher in dosage 40 (HR = 231.94 (95% CI: 142.68–377.05), P-value < 0.001) when compared to control i.e., dosage 0 (Table 2) (Fig. 1B).
The risk of death was significantly higher in dosage 60 (HR = 516.41 (95% CI: 292.07–913.07), P-value < 0.001) when compared to control i.e., dosage 0 (Table 2) (Fig. 1C).
The overall fecundity (average number of eggs laid per female mosquito) of Ae. aegypti, An. stephensi and Cx. quinquefasciatus females treated with fluralaner at different drug concentrations are given in (Table 3). The mean fecundity differed significantly between treated and control groups at drug concentrations nearing 24 h LC
Table 3 Fecundity success.
Mosquito species Fluralaner (ng/mL) Number of eggs laid per mosquito Mean (SD) Mean difference P-value n Treated n Control 5 48 95.10 (1.70) 49 97.70 (0.60) 2.60 0.018 10 39 91.90 (1.30) 49 96.70 (0.60) 4.80 0.012 20 16 78.70 (1.00) 48 96.70 (1.10) 18.00 0.011 5 48 88.30 (0.50) 50 90.30 (0.60) 2.00 0.007 15 40 84.20 (1.50) 50 90.00 (0.00) 5.80 0.011 25 31 72.00 (2.10) 50 89.30 (0.60) 17.30 0.012 15 47 124.20 (2.60) 49 136.00 (3.60) 11.80 0.012 30 40 120.30 (2.10) 49 134.30 (2.50) 14.00 0.011 45 7 117.30 (3.00) 49 135.30 (1.50) 18.00 0.012
n = number of mosquitoes kept for fecundity.
The egg hatch success of Ae. aegypti, An. stephensi and Cx. quinquefasciatus mosquitoes treated with fluralaner at different drug concentrations are given in Table 4. The mean egg hatch success differed significantly between the treated and control groups (P-value < 0.05) at higher drug concentrations, compared to the effect observed at lower concentrations (Table 4).
Table 4 Egg hatch success.
Mosquito species Fluralaner (ng/mL) Number of eggs hatched Mean (SD) Mean difference P-value n Treated n Control 5 500 403.67 (4.74) 500 414.00 (6.24) 10.33 0.026 10 500 395.67 (5.29) 500 419.67 (4.04) 24.00 0.013 20 500 386.00 (5.17) 500 416.00 (3.60) 30.00 0.012 5 500 435.22 (6.96) 500 456.33 (4.51) 21.11 0.013 15 500 420.22 (7.05) 500 452.67 (3.05) 32.44 0.013 25 500 408.89 (4.94) 500 453.33 (12.86) 44.44 0.013 15 517 426.22 (9.97) 533 462.66 (8.14) 36.44 0.013 30 524 425.11 (11.88) 515 446.00 (18.68) 20.89 0.060 45 518 417.22 (4.82) 531 457.33 (24.68) 40.11 0.012
n = number of eggs kept for hatching.
The overall immature development of Ae. aegypti, An. stephensi and Cx. quinquefasciatus mosquitoes treated with fluralaner at different drug concentrations are given in Table 5. The mean immature development varied significantly between control and treatment groups at higher drug concentrations tested: for Ae. aegypti20 ng/mL; for An. stephensi25 ng/mL; for Cx. quinquefasciatus45 ng/mL (P-value < 0.05 for all species).
Table 5 Immature survival.
Mosquito species Fluralaner (ng/mL) Number of pupae developed Mean (SD) Mean difference P-value n Treated n Control 5 100 98.80 (0.70) 100 99.70 (0.60) 0.90 0.07 10 100 97.40 (1.10) 100 99.00 (1.00) 1.60 0.07 20 100 91.90 (2.60) 100 99.00 (0.00) 7.10 0.01 5 100 99.10 (0.80) 100 100.00 (0.00) 0.90 0.07 15 100 97.80 (1.40) 100 99.00 (0.00) 1.20 0.11 25 100 96.40 (1.30) 100 98.70 (0.60) 2.20 0.01 15 100 98.20 (0.70) 100 99.30 (1.10) 1.10 0.11 30 100 93.80 (1.90) 100 99.30 (0.60) 5.60 0.01 45 100 89.20 (3.00) 100 99.30 (0.60) 10.10 0.01
n = number of larvae kept for pupal development.
The overall adult emergence success of Ae. aegypti, An. stephensi and Cx. quinquefasciatus mosquitoes exposed to fluralaner at different drug concentrations are given in Table 6. Certain morphological abnormalities in a few drug exposed mosquitoes at concentration nearing 24 h LC
Table 6 Adult emergence success.
Mosquito species Fluralaner (ng/mL) Number of adults emerged Mean(SD) Mean difference P-value n Treated n Control 5 50 49.56 (0.53) 50 50.00 (0.00) 0.44 0.17 10 50 49.22 (0.67) 50 49.67 (0.58) 0.44 0.30 20 50 48.44 (0.88) 50 50.00 (0.00) 1.56 0.02 5 50 49.44 (0.53) 50 50.00 (0.00) 0.56 0.10 15 50 48.00 (0.71) 50 49.67 (0.58) 1.67 0.01 25 50 46.78 (1.30) 50 48.67 (0.58) 1.89 0.04 15 50 49.56 (0.53) 50 50.00 (0.00) 0.44 0.17 30 50 48.78 (0.44) 50 49.67 (0.58) 0.89 0.02 45 50 47.67 (1.32) 50 49.67 (0.58) 2.00 0.02
n = number of pupae kept for adult emergence.
Graph: Figure 2(A) Partially emerged dead adult Ae. aegypti mosquitoes. (B) Partially emerged dead adult An. stephensi mosquitoes. (C) Partially emerged dead adult Cx. quinquefasciatus mosquitoes. (D) Adult Cx. quinquefasciatus mosquito with legs attached to pupal exuviae.
Estimation of mortality at different time points on feeding with different concentrations of the drug for all the three species is depicted in supplementary graphs 1–3. Speed of killing was also studied at a concentration 4–5 folds higher (250 ng/mL) (Fig. 3). When treated with this concentration, An. stephensi showed 100.00% mortality at 3 h of post blood feeding. In the case of Ae. aegypti, 99.00% mortality was recorded at 6 h of post blood feeding. For Cx. quinquefasciatus, it took 7 h to produce 100.00% mortality post blood feeding with fluralaner. The LT
Graph: Figure 3Mean percent mortality at different time point observed with three species of vector mosquitoes treated with fluralaner at a concentration of 250 ng/mL.
Table 7 Lethal time (LT
Species Fluralaner (ng/mL) LT50 (h) (95% CI) 250 2.65 (2.58–2.70) 250 1.63 (1.55–1.70) 250 3.76 (3.65–3.87)
An ideal endectocide for administration as a mass therapy tool for vector control should have potent mosquitocidal effect along with long half-life property so that the drug effect lasts for weeks to months after one-time drug administration. In the present study, oral treatment with fluralaner resulted in significant mosquitocidal effect against the three vector species studied, with the estimated 24 h LC
Mosquitocidal effect of fluralaner observed in the current study was similar to that of in an earlier study, which reported 24 h LC
Fluralaner affected the fecundity, egg hatch success, immature development and adult emergence success in all the three species when they were exposed to drug concentrations nearing 24 h LC
Studies on susceptible and resistant strains of horn and house flies have shown high level of mortality at lower concentration with fluralaner when compared with imidacloprid[
In the current study, complete mortality was observed within 7 h of post blood feeding at 250 ng/mL drug concentration in all the three species tested. Blood concentration much above this level is expected during the initial weeks in animals treated with fluralaner. This is a notable advantage over ivermectin which takes 72 h to produce 70.00–100.00% morality in mosquitoes at a dose of 93 ng/mL in animal experiments[
Currently, ivermectin mass therapy is being investigated in peri-domestic animals and humans in field studies for its endectocidal effect and as a complementary tool for malaria vector control[
With several emerging and remerging vector borne and zoonotic diseases, there is a great interest in 'One-Health' efforts being enforced round the globe. This would be particularly relevant in Africa and Asia where humans and animals live in close proximity and the drug based vector control strategy holds promise in such settings[
There are some limitations of the current study. We used chicken blood, not human blood for carrying out the efficacy trials for convenience. Another limitation is that we have carried out the study only in laboratory maintained colonies and not on field caught mosquitoes. The number of survived mosquitoes assessed for life history characteristics were not comparable between treatment and control groups. The lab obtained results may not be exactly replicable in field conditions. Another limitation is that we did not perform direct blood feeding experiments with mosquitoes fed upon animals treated with fluralaner. This experiment is necessary to understand the effect of the drug on mosquitoes in the context of its metabolism in the treated animals.
In conclusion, our study showed significant oral toxic effect of fluralaner in adult vector mosquitoes with additional effect on life history characteristics (reduced fecundity, egg hatch success, larval development and adult emergence). Fluralaner may be a suitable candidate for future drug based mosquito control strategies. It is required to carry out future safety studies of fluralaner in human beings.
Fluralaner was extracted from the commercially available tablets for veterinary use (Bravecto®-purchased from local veterinary drug shop; manufactured by Merck Animal Health, Madison, Vienna, Austria) using the method described by Miglianico et al.[
Laboratory reared mosquito species of Ae. aegypti, An. stephensi and Cx. quinquefasciatus were obtained from the rearing and colonization facility at the Indian Council of Medical Research—Vector Control Research Centre (ICMR-VCRC), Puducherry, India. These strains were originally collected from the Union Territory of Puducherry, India and the colonies have been maintained in the insectary since 1975. Cx. quinquefasciatus were collected in 1975 and Ae. aegypti and An. stephensi were collected in 1976. They were maintained at a constant temperature of 27 ± 2 °C and relative humidity of 80 ± 10% in one-foot Barraud cages (30 cm L × 30 cm W × 30 cm H) and provided with 10% sucrose solution.
To determine the activity range of fluralaner, initially, bioassays were performed to assess the mortality effect at 24 h using wide range concentrations (20–120 ng/mL). Based on the results of these experiments, a narrow range of four drug concentrations were selected for bioassays to determine LC
Five days old, non-blood fed female mosquitoes from same batch were used for blood feeding experiments. Prior to blood feeding, cotton pads soaked with 10% sucrose solution were removed and the adult mosquitoes were allowed to starve at least for 12 h to improve the feeding rate. Five mL of heparinized chicken blood containing desired concentration of fluralaner was used for feeding the mosquitoes in the treatment group via artificial blood feeding apparatus made of glass fitted with blood mixing rotor and parafilm for about 60–90 min. Treatment group consists of four drug concentrations and each concentration constituted of three technical replicate cages (each with 50 females). 50 adult females from the same batch of mosquitoes fed on chicken blood with only solvent were used as controls. After blood feeding, mosquitoes (fully engorged) were transferred to clean cages, provided with 10% sucrose solution and the mortality effect was observed at 4, 8, 18, 24, 48, 72 and 96 h of post feeding. 48 h (Ae. aegypti, An. stephensi) and 72 h (Cx. quinquefasciatus) of post blood feeding, survived mosquitoes were observed for fecundity. The experiment was repeated three times on different days using fresh batches of mosquitoes and freshly prepared fluralaner concentration as biological replicates.
In continuation to the bioassay experiments for mortality effect of fluralaner, life history characteristics of survived mosquitoes in each drug treatment groups were assessed in comparison to the control groups. Number of survived mosquitoes in each replicate cage varied with the species and also with the drug concentration.
For fecundity assessment of survived Ae. aegypti and An. stephensi, oviposition cups filled with 75–90 mL water and lined with filter paper were introduced into the cages at 48 h of post blood feeding. For Cx. quinquefasciatus, oviposition cups without filter paper were introduced into the cages at 72 h as it takes 3 days to become fully gravid. All the oviposition cups were placed in the center of the cage. To assess the effect of fluralaner in egg hatch success, eggs laid by survived mosquitoes in treatment group were compared with that of eggs laid by mosquitoes in the control group. For hatching experiment of Ae. aegypti and An. stephensi, five hundred eggs counted under microscope were floated in trays (45 cm L × 30 cm W × 10 cm H) filled with water using a fine tip brush. In case of Cx. quinquefasciatus, as eggs were laid in rafts, numbers were slightly more than five hundred. The rafts were carefully transferred to tray using brush without damaging it. Hundred first instar larvae hatched from eggs laid by survived mosquitoes were observed for development up to pupal stage by providing mosquito larval food [yeast (40%) and dog biscuit (60%)] once in 2 days and were compared with the control group. Rearing water was changed every 2 days. Daily mortality was recorded (if any) in both treatment and control group larvae. Similarly fifty pupae from survived mosquitoes were observed for adult emergence by transferring them into containers filled with 3/4
We performed a set of experiments to assess the speed of killing of the mosquitoes when they were fed with a high concentration of fluralaner. In the initial weeks of drug administration in animals, the blood concentration is expected to be higher which may result in rapid killing of mosquitoes within hours of blood feeding. Consequently, a drug concentration level, specifically five times the highest LC
Mean and standard deviation value from three replicates of different concentration were taken to obtain the data, which was further used for various statistical analysis. Mortality data over time were subjected to Probit regression analysis to estimate LC
We gratefully acknowledge the technical assistance of Mr. Shrihari Murmu, Senior Technician-2 and Mr. K. Manimaran, Technical Officer-A, ICMR-VCRC for maintenance of mosquito colonies and blood feeding. We thank Mr. M. Sundharesan, Technical Assistant, ICMR-VCRC for his assistance in extraction of active compound from the commercially available tablet.
V.S.K., S.C. and H.K.S. contributed in study design. H.K.S., V.S. and S.V. executed the work, wrote the initial draft of the manuscript. N.M. facilitated extraction of active compound. V.B. worked out statistical analysis for the data. V.S.K. and A.K. critically reviewed the final version of manuscript. All authors read and approved the final version of the manuscript. All authors approved and gave their consent to publish the manuscript.
We acknowledge the institutional funding (ICMR-Vector Control Research Centre, Puducherry) provided to carry out the study.
The datasets generated and/or analyzed during the current study are available from the corresponding author on request.
The authors declare no competing interests.
Graph: Supplementary Figures.
The online version contains supplementary material available at https://doi.org/10.1038/s41598-024-56053-x.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
By Harish Kumar Shah; Vaishnavi Srinivasan; Shakila Venkatesan; Vijayakumar Balakrishnan; Sadanandane Candasamy; Nisha Mathew; Ashwani Kumar and Vijesh Sreedhar Kuttiatt
Reported by Author; Author; Author; Author; Author; Author; Author; Author