Susceptibility of Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say to 19 Pesticides with Different Modes of Action

Abstract

The mosquito Aedes aegypti L. (Diptera: Culicidae) transmits viral pathogens of humans, including yellow fever (Gillett and Ross 1955, Philip 1962, Soper 1967, Aitken et al. 1977) and dengue (Mattingly 1967, Rudnick 1967, Coleman and McLean 1973, Degallier et al. 1988), both of which can cause severe human morbidity and mortality. The mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is the vector of the filarial parasite Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), which causes bancroftian filariasis in human (Sabatinelli et al. 1994, Samuel et al. 2004). Cx. quinquefasciatus Say is also a vector of West Nile virus (Godsey et al. 2005), Japanese encephalitis virus (Nitatpattana et al. 2005), and Saint Louis encephalitis virus (Jones et al. 2002). In North America, the common malaria mosquito Anopheles quadrimaculatus Say (Diptera: Culicidae) is a vector for human malaria (Box et al. 1953, Micks and Mc 1953).

The primary approach used for mosquito control has mainly relied on pesticides. However, very few types of pesticides are currently registered for mosquito control. Furthermore, many mosquito species have developed resistance to various classes of pesticides (Su and Mulla 2004, Tia et al. 2006, Xu et al. 2006), creating an urgent need to seek and identify new effective pesticides to control these important disease vectors. To search for pesticides that are effective as mosquito adulticides, we selectively chose 19 pesticides (Table 1) from the Insecticide Resistance Action Committee (IRAC) Mode of Action (MoA) classification list (http://www.irac-online.org/documents/IRAC%20MoA%20Classification%20v5_3.pdf), with each pesticide representing a different category of pesticide and evaluated their activities against three species of mosquitoes— Aedes aegypti, Cx. quinquefasciatus, and An. quadrimaculatus. Our results revealed that these three mosquitoes had different susceptibilities to various pesticides, showing the need to select the most efficacious compounds for the least susceptible mosquito species to achieve successful mosquito control.

Table 1

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Modes of action of the 19 selected pesticides used in the study

Table 1

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Modes of action of the 19 selected pesticides used in the study

Materials and Methods

Mosquitoes and Pesticides.

All three species of mosquitoes were reared in the insectary of the Mosquito and Fly Research Unit at Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), USDA-ARS. Ae. aegypti and An. quadrimaculatus have been established in the insectary since 1952 from Orlando, FL, strains. Cx. quinquefasciatus has been established in the insectary since 1995 from a Gainesville, FL, strain. Female adults were used for all experiments because only this sex takes blood meals and is of concern to the general public. Mosquitoes were reared using standard procedures (Reinert et al. 1997, McCall and Eaton 2001, Pridgeon et al. 2007). Briefly, collected eggs were hatched in a flask, and the hatched larvae were held overnight in the flask and transferred to a plastic tray containing distilled water. Larval diet was added to each tray. Mosquitoes were reared in an environmental chamber set with a temperature profile representing a simulated summer day regimen (ranging from 22 to 30°C) and 80% RH. Incandescent lighting was set to a crepuscular profile with a photoperiod of 14 h:10 h (L:D), including 2 h of simulated dawn and 2 h of simulated dusk. Adults were held in a screened cage and provided 10% sucrose ad libitum. Bovine blood in 1% heparin that had been placed in a pig intestine and warmed to 37°C was provided to adults twice a week. Eggs were hatched, and larvae were reared in containers as described above. Nineteen pesticides with different modes of action were selected (Table 1). All pesticides were purchased in technical grade from Chem Service (West Chester, PA).

Adult Bioassays and Data Analysis.

To determine precisely the activity of each pesticide against female mosquitoes, each chemical was serially diluted in acetone and topically applied to individual mosquitoes. Before pesticide application, 5- to 7-d-old females were briefly anesthetized for 30 s with carbon dioxide and placed on a 4°C chill table (BioQuip Products, Rancho Dominguez, CA). A droplet of 0.5 μl of pesticide solution was applied to the dorsal thorax using a 700 series syringe and a PB 600 repeating dispenser (Hamilton, Reno, NV). Six concentrations providing a range of 0–100% of mortality were used on 25–30 females per concentration. Tests were replicated three times. Control treatments with 0.5 μl of acetone alone gave control mortality rates of <10%. After treatment, mosquitoes were kept in plastic cups and supplied with 10% sucrose solution for 24 h before mortality was recorded. Temperature and humidity were maintained at 26°C and 80% RH, respectively. Every bioassay was conducted at 27°C and 80% RH and replicated three times. Bioassay data were analyzed using PoloPlus probit and logit analysis software (LeOra Software, Petaluma, CA). Control mortality was corrected using Abbott’s formula. χ² goodness-of-fit tests were performed, and LD₅₀/LD₉₅ values were calculated using PoloPlus program.

Results and Discussion

To determine the susceptibility of Ae. aegypti to the 19 selected pesticides, topical application bioassays were performed. The bioassay results are summarized in Table 2. Our results revealed that, among the 19 pesticides tested, fipronil, a gamma amino butyric acid (GABA)-gated chloride channel antagonist, was the most toxic pesticide against Ae. aegypti, with an LD₅₀ value as low as 4.6 × 10^–7μg/mg of mosquito (Table 2). The order of the next most toxic pesticides against Ae. aegypti was as follows: permethrin, a sodium channel modulator (LD₅₀ = 4.9 × 10^–5μg/mg) > abamectin, a chloride channel activator (LD₅₀ = 4.6 × 10^–4μg/mg) > diazinon, an acetylcholinesterase inhibitor representing organophosphates (LD₅₀ = 6.7 × 10^–4μg/mg) > imidacloprid, a nicotinic acetylcholine receptor antagonist (LD₅₀ = 7.7 × 10^–4μg/mg) > spinosad, a nicotinic acetylcholine receptor agonist (LD₅₀ = 8.8 × 10^–4μg/mg) > carbaryl, an acetylcholinesterase inhibitor representing carbamates (LD₅₀ = 9.5 × 10^-4μg/mg) > indoxacarb, a voltage dependent sodium channel blocker (LD₅₀ = 1.5 × 10^-3μg/mg) > chlorfenapyr, an uncoupler of oxidative phosphorylation through disruption of H⁺ gradient (LD₅₀ = 1.9 × 10^–3μg/mg) > pyridaben, a mitochondrial complex I electron transport inhibitor (LD₅₀ = 3 × 10^–3μg/mg; Table 2). Our results also revealed that the least toxic pesticide against Ae. aegypti was bifenazate, a neuron inhibitor currently registered as miticide with unknown mode of action, with an LD₅₀ value as high as 1.49 μg/mg. The activity order of the next least toxic pesticides tested were dicofol, a registered miticide with unknown mode of action (LD₅₀ = 0.48 μg/mg) < amitraz, an insecticide and acaricide to control red spider mites and control bollworms acting as an octopaminergic agonist (LD₅₀ = 0.41 μg/mg) < propargite, a registered miticide acting as an inhibitor of oxidative phosphorylation and ATP synthase (LD₅₀ = 0.24 μg/mg) < hydramethylnon, a mitochondrial complex II electron transport inhibitor (LD₅₀ = 0.2 μg/mg) < cyhexatin, a miticide acting as an inhibitor of oxidative phosphorylation and ATP synthase (LD₅₀ = 5.6 × 10^–2μg/mg) < diafenthiuron, an inhibitor of oxidative phosphorylation and ATP synthase (LD₅₀ = 4.8 × 10^-2μg/mg) < DNOC (dinitro-o-cresol), a pesticide registered for killing locusts and spider mites acting as an uncoupler of oxidative phosphorylation through disruption of H⁺ gradient (LD₅₀ = 2.5 × 10^–2μg/mg) < azocyclotin, a miticide acting as an inhibitor of oxidative phosphorylation and ATP synthase (LD₅₀ = 8.8 × 10^–3μg/mg) (Table 2).

Table 2

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Toxicities of 19 pesticides against female adults of Ae. aegypti × topical application

Table 2

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Toxicities of 19 pesticides against female adults of Ae. aegypti × topical application

To study whether different mosquito species have various susceptibilities to the 19 selected pesticides, topical application bioassays were performed against female adults of Cx. quinquefasciatus and An. quadrimaculatus. The bioassay results are presented in Tables 3 and 4. Among the 19 pesticides tested, fipronil, the most toxic pesticide against Ae. aegypti, was also the most toxic pesticide against Cx. quinquefasciatus and Anopheles quadrimaculatus, with an LD₅₀ value of 3.3 × 10^–7 and 6.8 × 10^–5μg/mg, respectively (Tables 3 and 4). However, to our surprise, An. quadrimaculatus was the least susceptible species to fipronil, with 206-fold higher LD₅₀ value than Cx. quinquefasciatus and 148-fold higher LD₅₀ value than Ae. aegypti. This could be simply because of species variability. An alternative explanation is that the An. quadrimaculatus strain might have previous exposure to pesticides with similar modes of action as fipronil (i.e., GABA-gated chloride channel antagonist). The second most toxic pesticide tested against all three mosquito species was permethrin. However, the three species showed different susceptibilities against permethrin, with Cx. quinquefasciatus as the least susceptible species, whereas Ae. aegypti was the most susceptible species (Table 5). Three relatively new pesticides (spinosad, imidacloprid, and abamectin) showed slightly lower activities against all three mosquito species than permethrin, with activities <20-fold lower than permethrin (Table 5). However, when LD₅₀ values were compared, Cx. quinquefasciatus was the least susceptible species to the three pesticides (Table 5). Furthermore, Cx. quinquefasciatus also showed the least susceptibility to six other pesticides tested (carbaryl, diazinon, permethrin, chlorfennapyr, azocyclotin, and DNOC; Table 5). The relatively low susceptibility of Cx. quinquefasciatus to nine pesticides tested (DNOC, azocyclotin, chlorfenapyr, carbaryl, spinosad, imidacloprid, diazinon, abamectin, and permethrin) may be simply caused by natural species-specific tolerance to the nine pesticides. Different susceptibility of various mosquito species to pesticides has been previously reported (Pampiglione et al. 1985, Campos and Andrade 2003, Somboon et al. 2003). For example, it has been reported that, when female mosquitoes engorged blood from mice injected subcutaneously 12 h previously with avamectin MK-933 at 82 mg (AI)/kg, mortality rates after 36 h were 100% for An. stephensi, >60% for Ae. aegypti, and >50% for Cx. quiquefasciatus (Pampiglione et al. 1985). Similarly, our results also showed that Cx. quiquefasciatus was the least susceptible species to abamectin with the highest LD₅₀ value, followed by Ae. aegypti and An. quadrimaculatus (Table 5), although we used a different bioassay method.

Table 3

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Toxicities of 19 pesticides against female adults of Cx. quinquefasciatus by topical application

Table 3

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Toxicities of 19 pesticides against female adults of Cx. quinquefasciatus by topical application

Table 4

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Toxicities of nineteen pesticides against female adults of An. quadrimaculatus by topical application

Table 4

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Toxicities of nineteen pesticides against female adults of An. quadrimaculatus by topical application

Table 5

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Toxicity comparison of the 19 selected pesticides against Ae. aegypti, Cx. quinquefasciatus, and An. quadrimaculatus

Table 5

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Toxicity comparison of the 19 selected pesticides against Ae. aegypti, Cx. quinquefasciatus, and An. quadrimaculatus

Although the three mosquito species showed different susceptibility to certain pesticides, they also showed similar susceptibility to some other pesticides. For example, DNOC, a registered pesticide used agriculturally as a larvicide, ovicide, and pesticide against locusts and other insects, had very similar activity against Ae. aegypti, Cx. quinquefasciatus, and An. quadrimaculatus, with LD₅₀ values of 2.5 × 10^–2, 3.5 × 10^–2, and 3.5 × 10^–2μg/mg, respectively. Another example was bifenazate, the active ingredient in acramite to control mites on a variety of fruit crops. The LD₅₀ values of bifenzate against Ae. aegypti, Cx. quinquefasciatus, and An. quadrimaculatus were 1.49, 1.6, and 2.46 μg/mg, respectively. These results suggest that the three species of mosquito tested had no previous exposure to either bifenzate or DNOC.

On the basis of 24-h LD₅₀ values, the most toxic pesticide tested was fipronil and the least toxic pesticide tested was bifenzate. Our results revealed that the three mosquito species had very similar susceptibility to relatively new pesticides such as DNOC and bifenzate. However, the three mosquito species also showed various susceptibilities to some pesticides such as fipronil and permethrin. Therefore, it is evident that the evaluation and selection of the most efficacious compound for the least susceptible mosquito species is an important step for effective mosquito control. Based on activity, fipronil seems to be the best compound of the 19 chemicals tested for successful mosquito control. However, fipronil is a broad-specturm pesticide that is also very toxic to aquatic nontargets (Overmyer et al. 2007). Therefore, it is not likely that fipronil will be approved as arial sprays. Permethrin, one of the pyrethroids currently registered for mosquito control, is the second highest active compound against all three mosquito species. Therefore, unless field strains have developed resistance, pyrethroids are still highly recommended for mosquito control. Of the 19 pesticides tested, 5 (carbaryl, spinosad, imidacloprid, diazinon, and abamectin) showed slightly lower activity than permethrin (<20-fold). Carbary and diazion are both currently registered as effective arial sprays for mosquito control. Therefore, they are recommended as alternative mosquito control compounds unless resistance has been reported. Abamectin, a relatively new pesticide not currently registered for mosquito control, is a natural fermentation product of soil bacterium Streptomyces avermitilis. Because abamectin showed only slightly lower activity than permethrin (<10-fold), we propose that abamectin is a compound worthy of pursuing as a mosquito adulticide. Imidacloprid, another relatively new pesticide, showed slightly lower activity than permethrin (<20-fold) against the three mosquito species tested. However, use of imidacloprid is highly controversial because it is believed to be responsible for high losses in bees. Therefore, its registration as a mosquito adulticide is not likely. Spinosad (spinosyn A and spinosyn D), a new chemical class of pesticides that are registered by the EPA to control a variety of insects, also showed slightly lower activity than permethrin (<20-fold). Because the active ingredient of spinosad is derived from a naturally occurring soil dwelling bacterium Saccharopolyspora spinosa and spinosad has very low impact to mammals, the environment, birds and predatory beneficials, we propose that spinosad is also worthy of pursuing as a mosquito adulticide.

In summary, we evaluated the potency of 19 pesticides with different modes of action against adult Ae. aegypti, Cx. quinquefasciatus Say, and An. quadrimaculatus Say. Our results revealed that different species of mosquitoes had different susceptibility to different pesticides, showing the need to select the most efficacious compound for the least susceptible mosquito species to achieve successful mosquito control.

Acknowledgements

We thank Drs. S. M. Valles (USDA-ARS) and G. B. White (University of Florida) for critical reviews of the manuscript; and M. H. Brown, H. Furlong, G. Allen, N. Newlon, and L. Jefferson (USDA-ARS) for support on mosquitoes. This study was supported by a Deployed War-Fighter Protection Research Program Grant funded by the U.S. Department of Defense through the Armed Forces Pest Management Board.

References Cited