Pyrethroid and organophosphate resistance in Ae. aegypti

In the Caribbean, insecticide resistance is widely developed in Aedes aegypti and represents a serious obstacle for dengue vector control. The efficacy of pyrethroid and organophosphate ultra-low volume space sprays was investigated in Martinique where Ae. aegypti has been shown to be resistant to conventional insecticides. In the laboratory, a wild-field caught population showed high levels of resistance to deltamethrin, organophosphate (naled), and pyrethrum. Simulated-field trials showed that this resistance can strongly reduce the knock-down effect and mortality of deltamethrin and synergized pyrethrins when applied by thermal-fogging. Conversely, the efficacy of naled was high against insecticide-resistant mosquitoes. Chemical analyses of nettings exposed to the treatments showed a decrease in residues over distance from release for the pyrethroids, and naled was not detected. This finding has important implications for dengue vector control and emphasizes the need to develop innovative strategies to maintain effective control of resistant Ae. aegypti populations. 746 MARCOMBE AND OTHERS mosquitoes. This purpose of this study was to provide mosquito control services with practical information to implement more effective vector control and resistance management strategies in the future. MATERIALS AND METHODS Biological material. Two strains of Ae. aegypti were used in this study. The susceptible reference Bora strain, originating from Bora-Bora in French Polynesia, has been colonized for many years and is free of any detectable insecticide resistance mechanisms. It is checked regularly for resistance mechanisms (e.g., kdr mutation and detoxification enzyme activity) as part of our laboratory routine. The Vauclin strain, which was our resistant strain, is a colony of Ae. aegypti established from wild field-caught mosquito larvae collected from individual houses in the locality of Vauclin, Martinique. Adults obtained from the F 1 progeny were used for bioassays (phase I) and field experiments (phase II). Insecticides and formulations. Laboratory bioassays were carried out by using technical grades of deltamethrin (100% [w/w]; AgrEVO, Herts, United Kingdom), pyrethrum (25.44% [w/w]; Pyrethrum Board of Kenya, Nakuru, Kenya), and naled (97.2% [w/w]; Sigma-Aldrich, Seelze, Germany). For the field experiment, formulations of pyrethrum (a mixture of six pyrethrins with the synergist piperonyl butoxide [PBO]; Pynet ® , 5% EC [w/v] plus 20% PBO [w/v]; Pyrethrum Board of Kenya), synergized pyrethrins (AquaPy ® , 3% [EW] [w/v] plus 13.5% PBO [w/v]; Bayer Environmental Science, Lyon, France), and naled (Dibrom ® 14 Concentrate, SL 87.4% [w/v] + dichlorvos < 2% [w/v]; AMVAC Chemical Corporation, Los Angeles, CA) were evaluated in comparison with two formulations of deltamethrin mixed with water (Aqua K-Othrine ® , EW 2% [w/v]) or gasoil (K-Othrine ® 15/5 ULV, UL 15% [w/v] + 0.5% esbiothrine [w/v], both from Bayer Environmental Science). K-Othrine ® 15/5 ULV is the reference formulation that has been used for many years in Martinique for the control of Ae. aegypti populations. Application rates were 1 g ai/ hectare for deltamethrin, 10 g ai/hectare for pyrethrins (Pynet ® and AquaPy ® ) and 114 g ai/hectare for naled. K-Othrine ® 15/5 ULV, Dibrom ® 14 Concentrate and Pynet ® were mixed with gasoil, and Aqua K-Othrine ® and AquaPy ® were mixed with water according to the manufacturers’ recommendations. Each insecticide and their formulations have been reported in the European Directive 98/8/EC of 16 February 1998 concerning the placing of biocidal products on the market. Tarsal contact with treated filter paper. Tarsal contact tests were run using filter papers treated with a technical grade of each insecticide. Filter papers were treated following a WHO protocol using acetone solutions of insecticide and silicone oil as the carrier. 23 Impregnation was conducted by dripping evenly onto paper 2 mL of technical grade chemical dissolved in acetone and silicone oil. Concentrations were expressed in w/w percentage of the active ingredient in silicone oil. The paper was dried for 24 hours before the test. Mortality resulting from tarsal contact with treated filter papers was measured using WHO test kits against adult mosquitoes of the Bora and Vauclin strains. Five batches of 20 non-blood fed females (2–5 days of age) were introduced into holding tubes and maintained for 60 minutes at 27 ± 2°C and a relative humidity of 80 ± 10%. Insects were then transferred into the exposure tube and placed vertically for 60 minutes under subdued light. Mortality was recorded 24 hours after exposure. Each test was replicated twice (n = 200 per dose). Field experiment. The efficacy of synergized pyrethrins, deltamethrin and naled was evaluated against Bora and Vauclin strains according to the WHO cage bioassay method. 22 The efficacy of each insecticide was measured by performing space spray applications using a 4 × 4 vehicle-mounted with a MaxiPro4 thermal fogger (Curtiss-Dynafog Ltd., Westfield, IN). Trials were conducted early in the morning (7:00 am to 9:00 am) in central southwestern Martinique in the locality of Ducos at Pays-Noyer in an open field setting. Before each treatment, the spraying system was calibrated (i.e., flow rates were 580 mL water/minute and 587 mL gasoil/minute). During application, the speed of the vehicle was 10 km/hour, and the volume of mixture applied was 700 mL/hectare. Cylindrical steel frame cages (90 mm diameter × 153 mm height) covered with a mosquito net (1-mm mesh) was used to house groups of 20 adult female mosquitoes. Cages were hung on steel poles 1 meter above the ground 15 minutes before spraying treatments began and were placed at increasing distances from the point of treatment (10, 20, 30, and 50 meters) and in five transects separated by 10 meters along the path of the vehicle releasing the insecticide ( Figure 1 ). This configuration and position of the cages has been shown to enable maximum penetration of aerosol into the cages. 24 A typical trial involved 40 cages being exposed to a one insecticide. Two cages, one containing Bora females and the other containing Vauclin females, were placed at each of the four distances from insecticide release for each of the five transects along the path of spray release ( Figure 1 ). Individual trials were conducted on separate days. However, there were exceptions, in that the first trials with K-Othrine ® and Aqua K-Othrine ® were carried out separately for the two strains and these trials only involved 20 cages. Twelve trials were performed, eight involving both strains and four involving only one strain. In each trial, the knock-down 25 effect was measured by counting the number of knocked-down females and/or dead females 20 minutes post-treatment. All mosquitoes from a particular cage were transferred into cages (20 cm × 20 cm × 20 cm) Figure 1. Layout of distance test experimental set-up with cages showing path of treatment from 10 meters to 50 meters using thermal fogger equipment. A total volume of 297 mL was applied over an area of 4,250 meters 2 where 40 cages of adults mosquitoes were placed on 5 transects. Each plot represents 2 cages (each cage containing 20 females of either the Bora or Vauclin strain) fixed on poles. 747 REDUCED EFFICACY OF SPACE SPRAYS AGAINST AE. AEGYPTI provided with sugar-soaked cotton (sugar diluted to a concentration of 10% in tap water) and brought back to the laboratory for assessment of post-treatment mortality 24 hours later. In each trial and for each strain, 5 cages containing 20 females were placed as controls 30 meters from the insecticide application area and in the opposite direction of the nozzle and wind direction. These cages were also assessed for the knockdown effect 20 minutes post-treatment and for mortality 24 hours later, and their values were used to correct for mortality observed in treatment cages. During each trial, wind velocity, direction, temperature, and relative humidity were recorded using an anemometer (Sylva ® ) and a portable meteorological station (Testo175 ® ). Trials were carried out when the wind direction was in the direction of the nozzle. No assays were carried out when the wind velocity exceed 5 meters/second. 26 The people spraying and those involved in recording the knock-down effect on mosquitoes were instructed about safety precautions. The people involved in spraying used protective clothing, shoes, and facemasks to reduce the risk of exposure to insecticide. Chemical analysis of pesticide residues on nettings. One netting sample from each distance of the transect L3 ( Figure 1 ) that had been exposed to each insecticide treatment was sent to the WHO collaborating center for the quality control of pesticides in Gembloux, Belgium, to determine the content of active substances. Two sub-samples of four pieces of 5 cm × 5 cm were cut randomly from each net to obtain two composite samples representative of the treatment. The Analytical method MEREPYRE of the Pesticides Research Department of the Walloon Agricultural Research Center was used. Detection of pyrethroids and pyrethrins was conducted by capillary gas chromatography with 63 Ni electron capture detection, and detection of naled was conducted by capillary gas chromatography with mass spectrometry detection using the external standard calibration. Statistical analysis. Mortality recorded in laboratory bioassay (WHO test kits) was corrected for control mortality by the formula of Abbott 27 (in case of control mortality > 5%) such that corrected mortality = [(X − Y)/X] × 100 where X = % survival in control cages and Y = % survival in treated cages. The data were then subjected to log-probit analysis 28 to determine 50% lethal dose (LD 50 ) and LD 95 values and their 95% confidence intervals. Bora and Vauclin strains were considered as having different susceptibility to a given pesticide when the ratio between their LD 50 (resistance ratio [RR] 50 ) values or LD 95 (RR 95 ) values had confidence intervals (CIs) excluding the value 1. Data from the field experiment were analyzed as a split-plot analysis of variance in a repeated measures design following the procedure of Milliken and Johnson. 29 The repeated measure was the knock-down effect on mosquitoes in each cage at 20 minutes post-treatment and mosquito mortality 24 hours post-treatment. In each case, the observed effect was corrected for mortality in control cages. The largest experiment unit, or whole-plot, involved 12 separate trials of insecticide application. Individual trials were nested within a particular insecticide (product [P]). The next experimental units were the groups of cages at a particular distance from the point of product release (distance [D]). The next units were cages of mosquitoes classed by the strain of mosquito they held (strain [S]). The smallest experimental units were the individual cages. The whole experiment involved a total of 400 cages, with two measures of mortality being taken from each cage. Data were arcsine square-root transformed before analysis. The analysis was performed using JMP version 5.1.2. 30