Efficacy of ULV and thermal aerosols of deltamethrin for control of Aedes albopictus in Nice, France
© The Author(s). 2016
Received: 3 August 2016
Accepted: 9 November 2016
Published: 23 November 2016
Ultra-low volume (ULV) insecticidal aerosols dispensed from vehicle-mounted cold-foggers are widely considered the method of choice for control of Aedes aegypti and Ae. albopictus during outbreaks of dengue and chikungunya and, more recently, Zika. Nevertheless, their effectiveness has been poorly studied, particularly in Europe. Nearly all published studies of ULV efficacy are bio-assays based on the mortality of caged mosquitoes. In our study we preferred to monitor the direct impact of treatments on the wild mosquito populations. This study was undertaken to evaluate the efficiency of the two widely used space spraying methods to control Ae. albopictus and Ae. aegypti.
We determined the susceptibility of local Ae. albopictus to deltamethrin by two methods: topical application and the “WHO Tube Test”. We used ovitraps baited with hay infusion and adult traps (B-G Sentinel) baited with a patented attractant to monitor the mosquitoes in four residential areas in Nice, southern France. The impact of deltamethrin applied from vehicle-mounted ULV fogging-machines was assessed by comparing trap results in treated vs untreated areas for 5 days before and 5 days after treatment. Four trials were conducted at the maximum permitted application rate (1 g.ha-1). We also made two small-scale tests of the impact of the same insecticide dispensed from a hand-held thermal fogger.
Susceptibility to the insecticide was high but there was no discernable change in the oviposition rate or the catch of adult female mosquitoes, nor was there any change in the parous rate. In contrast, hand-held thermal foggers were highly effective, with more than 90% reduction of both laid eggs and females.
We believe that direct monitoring of the wild mosquito populations gives a realistic assessment of the impact of treatments and suggest that the lack of efficacy is due to lack of interaction between the target mosquitoes and the ULV aerosol. We discuss the factors that influence the effectiveness of both methods of spraying in the context of epidemic situations.
Dengue is transmitted by two species of mosquito, Aedes aegypti and Ae. albopictus, that thrive in the urban environment. For this reason it is a predominantly urban disease. In the past 50 years, the incidence and prevalence of dengue have risen dramatically; it is now by far the most important arboviral disease and a major public health problem throughout most tropical and some sub-tropical regions worldwide. According to the most recent estimates, c.390 million people are infected each year and 96 million manifest with clinically apparent illness . Two other arboviral diseases, chikungunya and Zika, are transmitted by the same vectors and have achieved high profile in the past decade after major urban outbreaks in the Americas, Africa and Polynesia .
The tiger mosquito Ae. albopictus, native to Asia, was first detected in Europe in the 1970s  and is now widespread, often common in at least 18 countries. An outbreak of chikungunya (292 clinical cases) in northeastern Italy  confirmed that epidemic transmission of this virus is possible. Sporadic autochthonous cases of dengue and chikungunya associated with infected travellers continue to occur at various sites in Mediterranean Europe, particularly in France [5–7], a forewarning of future outbreaks . It is likely that the northward progression of the vector will expand the geographic range of such events. Moreover, Zika virus epidemics occurring currently in the Americas raise concerns, though this virus is adapted not only to Ae. aegypti and Ae. albopictus but to several species of mosquito [9, 10].
In the absence of a vaccine, vector control is the only option for suppression of transmission. In many countries, ultra-low volume (ULV) insecticidal aerosols dispensed from vehicle-mounted cold-foggers, widely used to combat nuisance species, are considered the method of choice during public health emergencies . Nevertheless, field trials have failed to demonstrate any significant impact on urban Ae. aegypti populations and there is no evidence that such treatments have any marked impact on epidemic transmission . Moreover, even if ULV were to achieve a major reduction of adult mosquitoes, the effect would probably be too transient for any marked reduction of transmission .
Aedes aegypti is an endophilic species that spends much of its time sequestered in sheltered sites indoors, typically among clothes in closets. The disappointing impact of ULV treatments on Ae. aegypti may be attributable to this behavior: once launched from the machine, aerosol particles are at the mercy of air movements to deliver them to the target, yet that target is cloistered in sites that are devoid of air movement. By contrast, because Ae. albopictus is markedly exophilic, we were optimistic that this species would be more vulnerable to outdoor treatments. In this article we report on a series of six field trials in which we used ovitraps and B-G Sentinel traps to monitor the impact of ULV deltamethrin on wild populations of Ae. albopictus in residential areas in Nice, France. We also present results of small-scale treatments by hand-held thermal fogger.
Adult Ae. albopictus were obtained by rearing eggs collected in ovitraps baited with seven day-old hay infusion . Larvae (200 per liter) were fed ‘Tetramin’ fish food (Tetramin Tropical Flakes-Spectrum Brands, Inc). F0 females were fed on cattle blood through a pig intestine membrane with the Hemotek membrane feeding system (Hemotek®). A 10% honey solution was available at all times except for 24 h before the blood meal. Three-five day-old F1 females were used in all assays.
Susceptibility of F1 Ae. albopictus females was determined by the standard WHO Bioassay  and by topical application. For the WHO test, technical grade (TG) deltamethrin 99.8% (Sigma-Aldrich, France) was diluted in acetone with silicone oil as the carrier. Eight concentrations of deltamethrin ranging from 0.0005 to 0.05%, active ingredient were used. For each replicate, four batches of 25 non-blood-fed females (2–4 day-old) were held in the exposure tubes for 30 min. Knockdown (Kd) was recorded every 5 min. Recovery tubes were maintained at 27 ± 2 °C and 80 ± 10% relative humidity with a small pad saturated with 10% honey solution. Mortality was recorded 24 h after exposure. For each concentration, a batch of 25 mosquitoes of a susceptible strain originating in French Polynesia (Bora Bora) was used as a control.
For tests by topical application, eight doses of deltamethrin (0.0013753 ng/mg to 0.0880281 ng/mg) diluted in acetone were used to provide a range of mortality from 0 to 100%. Two-five day-old non blood-fed females were anaesthetized with carbon dioxide for 60 s and transferred to a refrigerating plate at 4 °C. Insecticide solution (0.1 μl at the required concentration) was deposited on the upper pronotum by microcapillary. Mosquitoes were then transferred to plastic cups and maintained at (27 ± 2 °C) and humidity (80 ± 10%). Mortality was recorded 60 min after dosing and again after 24 h.
The ULV trials were conducted in gated communities in Nice, southern France, each with a close network of roads linking approximately 200 houses. Vegetation, largely a wide range of evergreen shrubs and trees, was abundant and meticulously maintained. In the dry Mediterranean summer, mosquito breeding sites were very hard to find, yet Ae. albopictus was plentiful, attacking in large numbers in many shaded sites. The two most frequent breeding sites of Ae. albopictus were man-made containers, particularlyflower pot saucers and catch basins.
Aqua K-Othrine® (Bayer SAS), containing 2% deltamethrin was used at maximum permitted dose (1 g active ingredient per hectare). The product is a patented formulation (Film Forming Aqueous Spray Technology) that forms a protective film around the spray droplets, thereby suppressing evaporation. Our trials were performed with Aqua K-Othrine®, aqueous emulsionnable FFAST formulation, this new formulation (at a dose of 1 g AI/ha) were reviewed by WHOPES which mentioned a mortality of 86% on Ae. albopictus in caged bioassays up to 50 m downwind from the point of spray discharge, which demonstrated the good adulticidal efficacy of Aqua K-Othrine .
Local regulations require that all residents receive a letter outlining the objectives of the trial, the date and time of the treatment, as well as safety measures (e.g. closed windows) that should be respected. On the eve of the treatment, further information was distributed in flyers and stickers, and by the management of the residence.
Cold fogging was by a vehicle-mounted London Fogger Model 18-20 (London Foggers, Long Lake, MN, USA) ULV aerosol generator with nozzle horizontal, parallel to the road. Liquid flow-rate was 0.5 l/min, 80% of droplets below 20 μm and vehicle speed was held below 12 km/h. The solution was applied at 2 l per hectare, equivalent to 1 g active ingredient with the maximum permitted dosage. Additional file 1: Figure S1, Additional file 2: Figure S2 and Additional file 3: Figure S3 show the route of spraying in the two different residential areas. All treatments were made punctually in late dawn, one hour after astronomic sunrise.
Trial dates and treatment times
1 h pre-sunrisea
2 h post-sunriseb
2 h post-sunrise
2 h post-sunrise
2 h post-sunrise
2 h post-sunrise
2 h post-sunrise
Monitoring the mosquito population
Mosquito populations were monitored with 25 ovitraps baited with hay infusion and seven adult traps baited with a patented attractant (BG-Sentinel® traps, BioGents, Regensburg, Germany). Traps were exchanged every 24-h. The thermal fog tests were on a much smaller scale, with 1 BG-Sentinel® trap and 6 ovitraps in each of the four private houses in the treatment and the control clusters.
The dose-mortality response was assessed by the R-script BioRssay . This computes the doses of insecticides killing 50% and 95% of the tested colony or strain. After developing a protocol for data exploration , a Generalized Linear Mixed Model (GLMM) was applied with negative binomial distribution or zero-inflated negative binomial (as the data were over-dispersed) using the glmm ADMB package . The response variables were Ae. albopictus female and egg abundance and parity rates. Explanatory variables are Control/Treatment and Pre/Post treatment and the interaction of both, while random variable was trap. Significant values were corrected by False Discovery Rate. Statistical analysis was performed in R version 2.14.2 . The interpretation of the variables used in our statistical analysis is summarized in Additional file 5: Table S1.
Deltamethrin susceptibility status of Aedes albopictus populations from Nice, southeastern France
WHO test kit assay
LD50 (ng/mg) (95% CI)
LD95 (ng/mg) (95% CI)
LD50 (%) (95% CI)
LD95 (%) (95% CI)
T50 (min) (95% CI)
KT95 (min) (95% CI)
Aedes aegypti (Bora)
By contrast, in treatment by thermal fog, oviposition rate and adult capture were reduced by about 95% (Fig.5) in test 1 and 61% in Test 2; “Treatment*Pre/Post” was negative and highly significant (P < 10-5; Additional file 6: Table S2; Additional file 7: Table S3). Therefore, the thermal fogging was effective to reduce the egg and the female abundance.
Intriguingly, even if the number of eggs and adults dropped drastically there was no effect of the spraying on the female’s parity rates as shown by the GLM analysis (Additional file 4: Figure S4; Additional file 8: Table S4). The statistical power of the analysis is illustrated by the explained variance in Additional file 9: Table S5.
Temperature and air velocity measurement inside and outside the vegetation in five points chosen randomly in the sprayed zone showed that atmospheric conditions were completely different from inside than outside bushes. Results emphasize the fact that the air velocity inside the vegetation is approximately 10-fold lower that in outside (outside vegetation: 1.13–2.01 m/s; inside vegetation: 0.15–0.5 m/s).
In contrast to previous evaluations, nearly all of which have relied on the mortality of caged mosquitoes, we assessed the impact of treatments by monitoring the wild mosquito population on a daily (24-h) basis. As reported in other temperate regions [21–23], the local strain of Ae. albopictus was fully susceptible to deltamethrin. The lack of impact that we observed was in line with results obtained by a similar approach (ovitraps and back-pack aspirators) against Ae. aegypti in Puerto Rico, Jamaica and Venezuela  and in Panama .
Our results were disappointing because we had supposed that Ae. albopictus would be more vulnerable than Ae. aegypti because of its markedly exophilic behaviour . We suggest that the lack of efficacy was simply due to lack of interaction between aerosol particles and the mosquitoes: the mosquito favors resting sites, particularly vegetation, that are devoid of air movement but the insecticide particles depend on the nuances of air movement to deliver them to the mosquito. Indeed in nearly every country, Ae. albopictus is common in suburban and rural situations were important vegetation is available . In densely crowded urban areas which lack vegetation and outdoor breeding sites, Ae. albopictus may be rare or absent . Using a hot-wire anemometer, we observed at least a ten-fold reduction in air movement in the interior of bushes that were abundant in the study area and it was clear that there are many sites (e.g. the leeward side of trunks, branches, leaves, crevices and other hollow structures) where mosquitoes can shelter with minimal exposure to air currents.
Apart from the lack of delivery to resting sites, there are clearly other limitations to the efficacy of ULV, particularly in urban areas, where walls, buildings and other structures obstruct the drift of particles . In this context, the term “space spray” is appropriate because much of the aerosol drifts through open spaces, around or over obstacles. Moreover, mortality in a cage has little relation to mortality of resting or even free-flying mosquitoes, particularly at sites in vegetation where mosquitoes are likely to rest; in studies of the control of Ae. aegypti in Venezuela, mortality was more than 90% in caged mosquitoes set in the open but close to zero at typical indoor resting sites . Similarly, Mount et al.  reported 90% mortality of caged mosquitoes in an open field but 34–67% in vegetation and Andis et al.  observed 95.5% mortality in caged Ae. aegypti suspended in the open vs. 49% in more sheltered locations. Moreover, Bengoa et al.  evaluated the efficacy of the ULV truck-mounted vehicle and obtained nearly 100% mortality in caged mosquitoes of Ae. albopictus in an open area but stated that this impact would be lower in wild uncaged mosquitoes resting within vegetation.
Logistically, ULV should be the control method of choice: a single vehicle with a driver and an operator can cover 50–80 ha in about three hours, dependent on the layout of roads and ease of access . However, there is no documented evidence that ULV treatments have ever had a discernible impact on transmission of dengue or chikungunya anywhere in the world; this is not surprising, given our results.
By contrast, in our small test of thermal fog, the aerosol was applied at close quarters to the presumed resting sites, directed by the operator and boosted by the physical thrust from the exhaust energy of the machine. Therefore, hand-held thermal fogging was highly effective, eliminating between 61 and 95% of females and eggs after a single treatment directly applied in the vegetation surrounding the treated houses. These results are broadly in accordance with those of Britch et al.  who evaluated the efficiency of truck mounted ULV and thermal fogger and found that there is 100-fold greater chance that a droplet will come in contact with a mosquito in the sentinel cage in a thermal fog application versus a ULV application.
We conclude that in the event of outbreaks of disease, truck-mounted ULV is unlikely to have significant impact on transmission but that, despite being highly labor-intensive, thermal or ULV aerosols dispensed from portable sprayers are the method of choice. Clearly this is not practicable on any large scale but may be useful in the event of potential “hot-spots” of local transmission.
We would like to thank Thierry Baldet of IDRC/CRDI–Ottawa, Canada and Annelies Wilder-Smith of University of Umea, Umea, Sweden for useful discussions and advices for the work. Our thanks also to Xavier Allen of CIRAD, UMR15 CMAEE; INRA, Montpellier, France for providing blood for the mosquito rearing and Arnaud Cannet and Pascal Delaunay of Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, C3 M, Université de Nice-Sophia Antipolis for their technical help and all the staff of EID Méditerranée, Montpellier, France for their help in field trials.
The research was funded by the European Commission under the 7th Framework, Grant Agreement Number: 282589 .
Availability of data and materials
SCB, PR, CL and DF designed the experimental scheme, DR, MR and SCB performed the statistical analysis, SCB, MR and FC evaluated the insecticidal susceptibility in the laboratory, SCB, RB, MR, CT, BC and RT carried out the field collections and ULV/thermal fogger spraying, SCB, PR, DF, CL and DR drafted the manuscript. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
In France, authorization for insecticidal space sprays must be obtained at the national and local (prefectural) levels. At the national level we were granted authorization by the French Ministry of Social Affairs and Health for each of the two summers of the trials. These were listed as DGS/RI1/2013/182 of April 30th, 2013 and DGS/RI1/2014/136 of April 29th, 2014. (These update the guidelines related to implementation modalities of the anti-disseminating plan of chikungunya and dengue in Metropolitan France). At the local level, we were granted authorization by the Prefecture for the Department (County) of Alpes-Maritimes: decrees number 2013-339 of April 24th, 2013, and 2014-414 of May 22th, 2014. Furthermore, condominium boards as well as the residents of all the treated areas were duly informed at least one week before the insecticide space spraying. These rules govern the operations of EID Méditerranée, the sole mosquito-control agency authorized to respond to suspect cases of dengue and chikungunya in the French Mediterranean region. Our study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol of blood sampling from cattle was approved by the Committee on the Ethics Animal Experiments of the French Ministry of Higher Education and Research (permit number 12ANI01). Certificates of all the authorizations are available from the authors upon request.
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