Methyl benzoate is a natural product (floral volatile organic compound) that is currently used as a food flavoring ingredient. This compound has shown to be insecticidal in laboratory studies against agricultural and urban pests, including spotted wing drosophila Drosophila suzukii, brown marmorated stink bug Hyalomorpha halys, the diamondback moth Plutella xylostella, and the common bed bug Cimex lectularius, to name several insect taxa. In this study we topically treated adult Aedes aegypti females with methyl benzoate and analogs and determined their toxicities. We found that among adult females, 4 analogs—butyl benzoate, n-pentyl benzoate, vinyl benzoate, and methyl 3-methoxybenzoate—were more toxic than the parent compound, methyl benzoate.
Aedes aegypti (L.) is an anthropophilic mosquito species that is originally from Africa but found worldwide (Brown et al. 2011, 2014) and in many portions of the USA, where its range appears to be expanding (Kraemer et al. 2015, CDC 2018). This mosquito species has been incriminated as the primary disease vector of several viruses of human importance. For example, as of 2019 approximately a half million individuals were estimated to develop the severe form of dengue and require hospitalization annually, with 2.5% of those cases resulting in death (Shepard et al. 2016, Powell 2018, WHO 2019). Control of disease vectors has been primarily focused on chemical applications of insecticides. However, with the development of insecticide resistance and a limited pool of viable insecticide chemistries with only a few modes of action, chemical controls are failing (Liu 2015, WHO 2018, Kandel et al. 2019). Therefore, it is imperative that novel control chemistries be discovered to assist in the continued fight against disease vectors. One avenue of research that has seen an increase in popularity is with botanical compounds. Several studies have looked at botanical compounds, such as plant essential oils as synergists, to enhance the toxicity of current insecticides, and as insecticides themselves (Isman 2000, 2006; Tong and Bloomquist 2013; Dias and Moraes 2014; Gross et al. 2017; Norris et al. 2019). There are several advantages that botanical insecticides would have in terms of being successful insecticide candidates, and for total cost of development and registration. Reports have indicated that various botanical compounds have several different modes of action and molecular target sites than conventional pesticides do (Enan 2001, 2005; Parnas et al. 2009; Tong and Coats 2010; Tong et al. 2013).
Also, many botanical compounds are nontoxic to mammals, in addition to being volatile, reducing environmental retention times (Isman et al. 2011).
Methyl benzoate is a floral volatile organic compound that has been registered in the USA and European Union as a food-grade flavor additive (Feng and Zhang 2017). It, along with several analogs, has been shown to have insecticidal and repellent properties against several different species of insects. Topically, it has shown to be active against the red imported fire ant Solenopsis invicta (Buren), spotted wing drosophila Drosophila suzukii (Matsumura), the brown marmorated stink bug Hyalomorpha halys (Stål), the diamondback moth Plutella xylostella (L.), and the tobacco hornworm Manduca sexta (L.) (Feng and Zhang 2017, Zhang and Feng 2017). It has also been shown to have fumigant activity against several stored product pests, the red imported fire ant, and the common bed bug Cimex lectularius (L.) (Chen et al. 2019, Larson et al. 2020, Morrison et al. 2019). Since various species of insects are susceptible to methyl benzoate, it was our goal to determine the efficacy of methyl benzoate and several of its analogs against adult Ae. aegypti females.
MATERIALS AND METHODS
Insects and rearing
Aedes aegypti eggs were obtained from the USDA–ARS Center for Medical, Agricultural, and Veterinary Entomology, in Gainesville, FL. Larvae were reared within a Percival environmental chamber (Percival Scientific, Inc., Perry, IA) at 27°C, 70% RH, with a photoperiod of 12 h light and 12 h dark. Larvae were fed ground Tetramin® fish food (Spectrum Brand Pet, LLC, Blacksburg, VA). Upon emergence, adult mosquitoes were fed a 10% sucrose solution and maintained under the same conditions as were the larvae.
Methyl benzoate, acetophenone, butyl benzoate, hexyl benzoate, ethyl benzoate, methyl 2-methylbenzoate, propyl benzoate, vinyl benzoate, n-pentyl benzoate, and methyl 2-chlorobenzoate were purchased from Sigma-Aldrich (St. Louis, MO). Methyl 3-methoxybenzoate was purchased from TCI America (Portland, OR). Acetone (Honeywell Burdick and Jackson™, Fisher Scientific, Morristown, NJ) was used as the solvent for dilution for all concentrations of test compounds, and as a control.
Adult bioassay protocols
Adult Ae. aegypti females were anesthetized using ice, and 0.2 μl of compound (dissolved into acetone) was applied to the pronotum, using a handheld PB600-1 Hamilton repeating syringe (Hamilton Company, Reno, NV). For the determination of the median lethal dose (LD50) for each of the compounds, 5 different doses of each compound (that yielded 0–100% mortality) were applied to 10 mosquitoes each and replicated a minimum of 3 times. A solvent-only treatment was included in each replicate as a control. After dosing, mosquitoes were transferred to plastic containers covered with netting and were given a 10% sugar solution on cotton balls. Mortality was recorded 24 h after the treatment.
For all toxicity bioassays, control mortality (≥10%) was corrected using Abbott's formula (Abbott 1925). All LD50 values were calculated using probit analysis in PoloPlus software (LeOra Software Co., Petaluma, CA). The LD50 values were compared utilizing the formula for comparing lethal dose ratios found within Robertson et al. (2017). Regression analysis was performed in Microsoft Excel software (Microsoft, Redmond, WA).
The LD50 values for the compounds tested against adult female Ae. aegypti are given in Table 1. There was an approximate 14-fold change between the most effective compound and least effective compound screened. The most effective compound was butyl benzoate with an LD50 of 5.1 μg per adult female. n-Pentyl benzoate was the next most effective compound with an estimated LD50 of 7.34 μg per adult female. The LD50 of vinyl benzoate of 10.7 μg per adult female was not significantly different from n-pentyl benzoate. This is followed by methyl 3-methoxybenzoate, acetophenone, propyl benzoate, methyl 2-chlorobenzoate, methyl 2-methylbenzoate, methyl benzoate, hexyl benzoate, and ethyl benzoate with LD50 values of 14.7, 18.4, 19.2, 29.2, 36.4, 45.6, 50.9, and 71 μg per adult female, respectively. Figure 1 shows that there was no correlation (R2 = 0.1283) between alkyl chain length on the alcohol portion of the methyl benzoate analogs.
Aedes aegypti has been incriminated as the major transmission vector of several viruses that are responsible for yellow fever, dengue, chikungunya, and Zika infections in humans (Powell 2018). The estimated economic losses worldwide due to dengue, alone, is more than US$8 billion annually (Shepard et al. 2016). In the USA, common adulticides for mosquito control include organophosphate and pyrethroid insecticides (USEPA 2017), though reports of pyrethroid resistance in Ae. aegypti are emerging (Kandel et al. 2019). Methyl benzoate has been shown in laboratory tests to be toxic to several economically important agricultural pests, and to the common bed bug (Feng and Zhang 2017, Zhang and Feng 2017, Chen et al. 2019, Larson et al. 2020, Morrison et al. 2019). This current study demonstrates that methyl benzoate and its more active analogs may have a role in adult mosquito control. Feng and Zhang (2017) found that contact toxicity on developing and emerging spotted wing drosophila was negatively correlated with alkyl chain length, while Chen et al. (2019) reported that topical toxicity was positively correlated with alkyl chain length for the red imported fire ant. The current study found that there was no correlation between toxicity and the alkyl chain length on the alcohol. This could be the result of differences between the species of arthropods tested; however, further studies may be warranted to explore further alkyl chain relevance to toxicity.
While formulations would need to be explored and developed for use of these compounds within vector control, the incorporation of environmentally friendly active ingredients into a program directed at mosquito control would reduce our reliance on synthetic pesticides. Volatile organic compounds evidently can be used to modify vector behavior in plant-feeding insects (Aksenov et al. 2014), though the potential for use with medically important arthropods remains speculative (Hurd 2003, Lefèvre and Thomas 2008). It would be prudent to test these compounds further against the larval stages of mosquitoes as well as determine any behavioral effects they may have on the adults.
We thank members of the USDA–ARS Center for Medical, Agricultural, and Veterinary Entomology, in Gainesville, FL, for supplying the Ae. aegypti eggs that were hatched and reared to adults and used in this study. Mention of a proprietary product or company does not constitute an endorsement or a recommendation for its use by the USDA. The USDA is an equal opportunity employer.
Invasive Insect Biocontrol and Behavior Laboratory, USDA-ARS, Beltsville, MD 20705.
Department of Biology, Towson University, Towson, MD 21252.