Contact, fumigant and repellent activities of essential oils of cinnamon, lemon, and peppermint against larvae and adults of Musca domestica L.

Abstract

The house fly, Musca domestica L., is a common pest in domestic, medical, and veterinary settings. Traditional control methods involving the use of synthetic insecticides have led to resistance and harmed both the environment and human health. Hence, control alternatives are needed, and essential oils have shown contact insecticidal, fumigant, and repellent activities. The objective of this research was to assess, under laboratory conditions, the contact, fumigant, and repellent activities of essential oils of Citrus xlimon (L.) Osbeck, Cinnamomum zeylanicum Blume, and Mentha xpiperita L. against adults and larvae of houseflies. The major components in each essential oil were limonene (64.8%; C. limon), cinnamaldehyde (76.1%; C. zeylanicum), and menthol (74.92%; M. piperita). The highest contact toxicity was achieved by C. zeylanicum and M. piperita, both with a CL50 = 1.5 µl mL-1 water in larvae, and C. limon LC50 = 2.5 µ mL-1 water in adults. However, the treatments did not significantly differ. In the fumigant bioassay, C. zey/anicum in larvae (LC50 = 1.9 pL L-1 air) and adults (LC50 = 2.8 µ L-1 air) was the most toxic but was not significantly different from the other treatments. All the treatments resulted in repellent effects, but only the 10% (v/v) concentration resulted in 90% repelled insects. We concluded that C. limon, C. zeylanicum, and M. piperita are effective insecticides and repellents for M. domestica.

Headnote

Received: 3 September 2024; Accepted: 12 November 2024, doi:10.4067/50718-58392025000300318

ABSTRACT

The house fly, Musca domestica L., is a common pest in domestic, medical, and veterinary settings. Traditional control methods involving the use of synthetic insecticides have led to resistance and harmed both the environment and human health. Hence, control alternatives are needed, and essential oils have shown contact insecticidal, fumigant, and repellent activities. The objective of this research was to assess, under laboratory conditions, the contact, fumigant, and repellent activities of essential oils of Citrus xlimon (L.) Osbeck, Cinnamomum zeylanicum Blume, and Mentha xpiperita L. against adults and larvae of houseflies. The major components in each essential oil were limonene (64.8%; C. limon), cinnamaldehyde (76.1%; C. zeylanicum), and menthol (74.92%; M. piperita). The highest contact toxicity was achieved by C. zeylanicum and M. piperita, both with a CL50 = 1.5 µl mL-1 water in larvae, and C. limon LC50 = 2.5 µ mL-1 water in adults. However, the treatments did not significantly differ. In the fumigant bioassay, C. zey/anicum in larvae (LC50 = 1.9 pL L-1 air) and adults (LC50 = 2.8 µ L-1 air) was the most toxic but was not significantly different from the other treatments. All the treatments resulted in repellent effects, but only the 10% (v/v) concentration resulted in 90% repelled insects. We concluded that C. limon, C. zeylanicum, and M. piperita are effective insecticides and repellents for M. domestica.

Key words: Botanical insecticides, Cinnamomum zeylanicum, Citrus xlimon, essential oils, Mentha xpiperita, Musca domestica, terpenoids, urban pest.

INTRODUCTION

The house fly Musca domestica L. (Diptera: Muscidae) is a major domestic, medical, and veterinary pest that irritates, spoils food, and acts as a vector for many pathogenic organisms. This fly pest is commonly associated with animal facilities where it develops in feces and wet feed because manure and humidity provide a suitable environment for its development. A high density of flies causes stress to poultry workers and domestic animals and affects the economic value of their products. Historically, because of its rapid action, low cost, and effectiveness (Geden et al., 2021), house fly management has been directed toward adults using synthetic insecticides. However, this pest is notorious for its ability to develop behavioral and metabolic mechanisms to avoid and detoxify chemical insecticides. The Arthropod Pesticide Resistance Database (https://www.pesticideresistance.org/) records 463 cases of insecticide resistance worldwide, including practically all active compounds authorized for M. domestica control. Therefore, control alternatives, such as bioinsecticides based on plant essential oils, are needed.

Essential oils from plants and secondary metabolites from other plant tissues have high potential for use as insecticides. The volatile substances of essential oils are rich in monoterpenes that protect plants from herbivores and pathogens by blocking predation, deterring oviposition, inhibiting growth, and repelling or mimicking juvenile hormones (Mossa, 2016). The mode of action of essential oils has not been fully elucidated, but symptoms of poisoned insects are associated with a neurotoxic effect (Isman, 2020). First, some monoterpenes, even at lower concentrations, block octopamine receptors that control and modulate vital functions ranging from metabolism to behavior (Jankowska et al., 2018). Additionally, essential oils are considered weak inhibitors of acetylcholinesterase activity (Isman, 2020). Another proposed mechanism is positive allosteric modulation of GABA receptors (GABARs) (Jankowska et al., 2018). Previous studies of the biological activity of essential oils against M. domestica have shown that species such as Eucalyptus globulus Labill. (Rossi and Palacios, 2015), Lavandula angustifolia Mill. (Cossetin et al., 2018), Thymus spp. (Pavela et al., 2020; Xie et al., 2020), Mentha spp. (Benelli et al., 2018; Mohafrash et al., 2020), Ocimum basilicum L. (Senthoorraja et al., 2021) and Cymbopogon citratus (DC.) Stapf (Pinto et al., 2015) have shown insecticidal and repellent activity. To increase their efficacy, essential oils have been mixed with synthetic insecticides such as pyrethroids (Suwannayod et al., 2019) or chlorfenapyr (Yoon and Tak, 2022), and a possible synergistic effect has been identified. However, for many years, research into essential oils has focused on medicinal plants or native plants from reduced geographical areas, making their industrialization complex. An alternative is to formulate bioinsecticides with essential oils used in the cosmetic or food industry because are permanently available in the market or to extract essential oils from waste from wine or citrus industries for example. Thus, this research aimed to assess, under laboratory conditions, the contact, fumigant, and repellent activities of essential oils of Citrus xlimon (L.) Osbeck, Cinnamomum zeylanicum Blume, and Mentha xpiperita L. against adults and larvae of houseflies.

MATERIALS AND METHODS

Insects

Adult houseflies were collected from cattle-confined barns at the Facultad de Medicina Veterinaria of Universidad de Concepcion at Chillan, Chile, using a sweep net. Insects collected were moved to the laboratory and identified with the key of Carvalho and Mello-Patiu (2008) and reared under laboratory conditions in a PVC white mesh cage (30 x 30 x 30 cm) (BusoTh, China) at 25 + 5 °C, 65 + 2% relative humidity (RH), and a photoperiod of 16:8 h (L:D) in a bioclimatic chamber (Memnert Gmbh IPS 749, Schwabech, Germany). A Petri dish with a cotton swab soaked in 5 g milk powder was placed in each cage to feed the flies and oviposition sites. TWo grams of yeast was dissolved in 30 mL water and changed every 24 h, as suggested by Sinthusiri and Soonwera (2014). Eggs were transferred to the rearing medium, which consisted of strips of absorbent paper stacked in layers moistened with 2.0% milk and sugar solution, inside a container with a lid until larval emergence. In the barns where the insects were collected, the insecticides cypermethrin (Cyperkill Plus; Anasac S.A., Santiago, Chile) and thiamethoxam (Agita 10G; Elanco SpA, Santiago, Chile) were periodically sprayed. Therefore, before the bioassays were started, the susceptibility of the collected insects to commercial doses of cypermethrin and thiamethoxam was assessed exhibited a mortality of at least 90%.

Essential oils

The essential oils of lemon (Citrus xlimon (L.) Osbeck), cinnamon (Cinnamomum zeylanicum Blume), and peppermint (Mentha xpiperita L.) were acquired from Now essential oils (Bloomingdale, Illinois, USA) with 99% purity. These essential oils were selected because they are available on the market in large volumes because have gourmet and medicinal use. Chemical analysis was performed at the Laboratory of Natural Products, Department of Botany of Universidad de Concepcion at Concepcion, by using gas chromatography (GC) coupled with mass spectrometry (GC-MS) and high-performance gas chromatography-mass spectrometry (HPGC-MS; series || 5890, Hewlett Packard, Palo Alto, California, USA). Separation was achieved using a 5% poly diphenyl 95% dimethylsiloxane bonded phase column (i.d. 0.25 mm, length 30 m, film thickness 0.25 um). Operation conditions were as follow: Injector temperature, 250 °C; carrier gas (helium), flow rate 1 mL min? and split

injection with a split ratio 1:20. Mass spectrometry conditions were as follows: lonization voltage, 70 eV; emission current 40 mA; scan rate, 1 scan s·; source temperature 285 °С. Mass range was 35-300 Da. The oven temperature was 2 min isothermal at 60 °C and then increased to 210 °C, at the rate of 10 °C min", and to 260 °C, at rate of 10 °C min. Samples (1 pL) were dissolved with CH2Cl2 (1:100 v/v). The MS fragmentation pattern was checked with the retention time (RI), standards available in our laboratory, and by matching the MS data with the library NIST NBS54K or literature (Adams, 2007).

Contact activity

We used an acute topical bioassay to assess contact toxicity against adults and larvae. We determined the concentration range for each essential oil that caused 0% to 100% mortality. Then, three to four intermediate concentrations were evenly distributed among the original range of tested concentrations as suggested by Robertson et al. (2020) corresponding to 2.0, 5.0, 10, 15 and 20 pL oil mL in M. piperita, 1.0, 2.0, 5.0, 10 and 15 pL oil mL? in С. limon and 5.0, 10, 15, 20 and 25 pL oil mL· in С. zeylanicum. Groups of 50 M. domestica adults of 5-d-old and 20 third-instar larvae were anesthetized for 3 min with CO2 at 3 psi. Each sample was then treated on the pronotum with 1 pl oil solution in 1 mL acetone (99% purity) (Pavela et al. 2020) via a Burkard hand micro applicator (Burkard Manufacturing, Hertfordshire, UK). Each treatment had 10 replicates of 20 insects each, and the control was handled similarly to the insecticide treatment, except that they were treated with acetone. Mortality was assessed 24 h after treatment, and an insect was considered dead when there was no movement after prodding it with a dissection needle for 5 min. The maximum mortality in the control was 2.0%; hence, mortality in the treatments was adjusted using Abbott's formula (Abbott, 1925).

Fumigant activity

The fumigant toxicity of essential oils against larvae was assessed via the methodology of Xie et al. (2020). A filter paper impregnated with different concentrations of undiluted essential oils (12.50, 18.75, 25.00, 50.00, and 70.00 pL essential oil Lİ air) was pasted inside the lid of a 0.5 L plastic flask, while 20 third-instar larvae in a Petri dish (5 cm diameter) with strips of absorbent paper stacked in layers moistened with 2.0% milk and sugar solution were placed at the bottom. Each treatment had 10 replicates, and mortality was assessed at 72 h after treatment and adjusted with Abbott's formula (Abbott, 1925) because the control had a mortality of 3.0%.

In our previous adult bioassay, we evaluated the methods used by Zhang et al. (2017) and Xie et al. (2020) using filter paper, impregnated with essential oil. However, the flies were able to fly and settle on the impregnated surface, which could lead to an overestimation of fumigant activity due to potential contact toxicity. For this purpose, a special device was created (Figure 1). It consists of transparent plastic containers With a capacity of 0.5 L, each with a perforated lid. Two plastic caps without rubber stopper 10 mL vacuum blood tubes were securely attached with silicone. Additionally, a 1 mL Eppendorf tube was placed inside, with its tip cut and covered by a tulle mesh secured with an elastic band. This mesh contained a strip of filter paper that was impregnated with undiluted essential oil. In this way, the oil released its chemical compounds into the container, but the flies did not have contact with the impregnated surface even if they landed on the Eppendorf tube. Each treatment had 10 replicates with 25 adult flies, and mortality was assessed at 72 h after treatment and adjusted with Abbott's formula (Abbott, 1925) because the control contained 1.0% dead insects.

Repellency

This bioassay was performed via a double-choice method adapted from Haselton et al. (2015) (Figure 2). The device consisted of two plastic chambers of 500 mL, each containing a Petri dish with cotton impregnated with a solution of 5 g milk powder and 2 g yeast dissolved in 30 mL distilled water. One Petri dish was treated with essential oils at concentrations of 2.0%, 4.0%, 8.0%, and 10.0% (v/v), and the other dish without oil was the control. The treatment chambers were connected by transparent rubber tubes on both sides. One side was connected by rubber tubes 5 cm in diameter with a 1 L cage with 50 adult house flies, and the other side was connected with rubber tubes 0.5 cm in diameter with a flowmeter. The flowmeter regulates the air flow produced by an aquarium pump to 0.5 L min", purifies the air through a charcoal filter, and humidifies it before entering the treatment chambers. The cage that contained house flies had a tube covered with tulle fabric that allowed the airflow to escape but prevented insect escape. In previous tests, at 3 h, 100% of the insects selected an option; thus, at this time, the repellency was calculated by the number of insects that prefer the treated or control cage. Every treatment had four replicates of 50 insects each and the cages were washed with neutral washing soap, distilled water, 90% ethanol (v/v), and 90% acetone (v/v) between replicates, while the connections with the odor sources were alternated to minimize bias.

Statistical analysis

Data on contact and fumigant toxicity and repellency were analyzed via ANOVA and Tukey's test for comparison of means (P < 0.05) and via logarithmic regression with the Probit model (Finney, 1971) using the Proc Probit procedure of the Statistical Analysis System (SAS) software (SAS Institute, Cary, North Carolina, USA) to generate values for lethal concentration 50% (LCso) and 90% (LCs0) and the 90% repellency concentration (RCso) with their corresponding 95% confidence limits. The response was not considered significantly different when the confidence limits overlapped (Robertson et al., 2020).

RESULTS AND DISCUSSION

Chemical composition of essential oils

Among the three assessed essential oils, 98% of the chemical compounds were identified, and as expected, the predominant compounds were terpenoids (Table 1). The major components in each essential oil were limonene (64.8%; C. limon), cinnamaldehyde (76.1%; C. zeylanicum), and menthol (74.92%; C. piperita). Limonene is a monocyclic monoterpenoid found mainly in citrus oils. It is one of the main components of essential oils from orange (Citrus sinensis (L.) Osbeck), lemongrass (Cymbopogon citrates (D.C.) Stapf.), and Japanese pepper (Zanthoxylum piperitum D.C.), among others (Showler et al., 2019). This compound has been assessed singly and has insecticidal activity against M. domestica (Isman, 2020). Cinnamaldehyde is widely known as the characteristic compound of cinnamon spice and is obtained from the inner bark of several trees within the genus Cinnamomum. The most common species is C. zeylanicum, commonly referred to as true cinnamon, with a percentage of cinnamaldehyde of at least 80.0%. This compound has been widely used to impart a cinnamon flavor to edible products, cosmetics, and perfumes (Suriyagoda et al., 2021). Additionally, this compound has historically been known in traditional medicine for its bactericidal and antifungal activities (Shreaz et al., 2016) and ability to treat diseases such as diabetes (Zhu et al., 2017). With respect to insecticidal activity, da Silva et al. (2020) assessed this compound against M. domestica immature stages and reported larval and pupal mortality rates close to 70%. Menthol is the predominant monoterpene produced in the essential oil of maturing peppermint leaves during the filling of epidermal oil glands. This compound is used in confectionery, perfumery, liqueurs, cigarettes, nasal inhalers, and cough drop production. It is also used as a component of anesthetic, antiseptic, and gastric sedative drugs (Pergolizzi et al., 2018). With respect to insecticidal toxicity, Kumar et al. (2014a), using an isolated menthol in contact toxicity bioassays against larvae of M. domestica, obtained ап LCso of 0.02 ul cm? and registered 95.63% adult repellency. Zhang et al. (2017) assessed the fumigation toxicity of several monoterpenes against adults of M. domestica and reported that menthol and alcohol compounds, in general, had the strongest insecticidal effect, with a concentration of 1.38 pL L .

Contact activity

The highest toxicity against larvae was achieved with С. limon because a concentration of 15 ul oil mL? water resulted in 99% mortality. The essential oils of M. piperita resulted in maximum mortality of 87% at a concentration of 20 ul oil mL? water, and С. zeylanicum resulted in 91% mortality at a concentration of 25 ul ой ML? water (Figure 3). The lowest LCso was also obtained with С. limon (LCso = 3.14 ul oil mL? water), but the differences from those of M. piperita (LCso = 3.79 ul oil mL? water) and С. zeylanicum (LCso = 10.4 pl oil mL? water) were nonsignificant because the confidence limits overlapped (Robertson et al., 2020) (Table 2). The toxicity of the essential oil of M. piperita is consistent with that reported by Kumar et al. (2014b), who reported that the essential oils of M. piperita formulated as nanoparticles showed a larvae mortality of 100% and 93% under laboratory and field conditions respectively. With respect to cinnamon, Kékdener (2023) reported 100% larval mortality from a concentration of 1.0% essential oil (equivalent to 10 pL oil mL? water), which is a lower concentration than the essential oil used in this research. Furthermore, the effectiveness of essential oils of C. limon and M. piperita to larvae control makes it possible to formulate a less expensive bioinsecticide than C. zeylanicum, especially in countries that import this spice.

All treatments resulted in 90% mortality in the adult house fly bioassay, but С. zeylanicum presented the highest toxicity, with 97% dead insects in 20 pL oil mL? water and an LCso of 6.1 pL oil ML? water (Figure 3 and Table 2). Although С. limon mortality was 100% (LCso = 9.0 pL oil ML? water), this essential oil requires a 25 pL oil mL? water concentration. At the same concentration, M. piperita was 93% toxic (LCso = 13.3 pL oil mL? water). However, the overlap of confidence limits indicates nonsignificant difference between treatments. Considering the low distillation yields of essential oils and the lower concentration of C. zeylanicum required to reach 90% dead insects, it could be considered a better alternative for future commercial insecticide formulations. However, due to its high cost and demand by industries that use this compound as a flavoring, peppermint would be the best alternative since it can also be grown practically worldwide. The results agree with those of Boito et al. (2018), who reported that the essential oil of C. zeylanicum with concentrations of 10.0% (equivalent to 100 pL oil ML? water) and 5.0% (equivalent to 50 ul oil mL? water), formulated a nanoemulsion and obtained 100% control after 90 min of exposure. In the case of M. piperita Sinthusiri and Soonwera (2014), a concentration of 10% (v/v) (equivalent to 100 UL oil mL? water) essential oil resulted in 100% mortality at 24 h, which was almost four-fold higher than that required in this research to reach 90% dead insects (Table 2).

Fumigant activity

In the bioassay of fumigant activity against house fly adults, the three essential oils reached a mortality rate of 90%. Citrus limon and M. piperita, which had the highest assessed concentrations (70 uL L· air), resulted in 100% dead insects (Figure 4). However, when the LCso was analyzed, the lowest value was for M. piperita with 17.0 pL L· air, but there were nonsignificant differences from С. zeylanicum (LCso = 24.3 pL L air) and С. limon (LCso = 22.2 ul L· air) because the confidence limits overlapped (Robertson et al, 2020) (Table 3). Zhang et al. (2017) reported that the vapors of M. piperita contain high concentrations of menthol, which coincides with the phytochemical analysis of the essential oils used in this research (Table 1) and obtained a LCso of 4.28 pL Lİ air and concluded that menthol exhibited strong fumigation activity against M. domestica adults. Both values are lower than our results. In the case of C. zeylanicum essential oil, Khater and Geden (2019) reported 100% mortality of adult houseflies with a concentration of 0.6% (equivalent to 6 UL oil mL? air), which is less than the concentration of essential oil required in this research to obtain 90% mortality (Table 3). The most prominent previous studies on monoterpenes as fumigants for houseflies have used C. limon essential oil. Zhang et al. (2017) estimated an LCso of 3.22 ul LT air for limonene against adults of M. domestica, although the mortality rate was lower than 50%. The same trend was reported by Kumar et al. (2014a), who assessed the biological activity of several monoterpenes against houseflies and reported that limonene exhibited the poorest performance in all bioassays. Similar contact toxicity bioassay, C. limon and M. piperita are the best option for a bioinsecticide. In addition, the main monoterpenes of both essential oils, limonene and menthol respectively, can be obtained from the essential oil or synthetically formulated at low cost, allowing to produce a bioinsecticide and a synthetic insecticide with the same active compound.

In terms of larval toxicity, the highest toxicity was reached in M. piperita, with a mortality of 90.7% at 70 ul LT air. The essential oils of С. zeylanicum and С. limon at the same concentration resulted in 72.0% and 70.7% dead insects, respectively (Figure 4). Consistent with the mortality results, the lowest LCso was obtained with M. piperita (LCso = 20.4 ul Lİ air), but the LCso was not significantly different from that of С. limon (LCso = 34.0 pL Lİ air) because the confidence limits overlapped (Table 3). Although the essential oils of С. zeylanicum and С. limon showed similar toxicity, the higher mortality obtained with intermediate doses of С. limon resulted in confidence limits (22.4-60.6) that were wider than those of C. zeylanicum (39.1-47.2), resulting in overlapping values.

Research on fumigant activity against houseflies has focused mainly on adults rather than immature individuals. However, the results for M. piperita agreed with those of Kumar et al. (2014a), who assessed menthol, documenting an LCoo of 7.1 ul L air against housefly larvae at 48 h after exposure, which is less than the concentration of essential oil required in this research at the same toxicity level.

Repellency

In all treatments, adult houseflies mostly selected untreated milk rather than milk mixed with essential oils. As the concentration increased, the number of insects that preferred the untreated control also increased. All assessed concentrations of C. zeylanicum and M. piperita had repellent effects greater than 80%, without significant differences among the treatments (Table 4). The essential oil of C. limon at a concentration of 2.0% had a repellency of 76.2%, a value significantly lower than those obtained at 8.0% and 10.0%, but with no difference than those obtained at 4.0%. However, when comparing the RCoo values, the confidence limits of the three essential oils overlapped, so there were nonsignificant differences between them when repelling 90% of the population. The results for M. piperita agreed with those of Chauhan et al. (2018) who studied the repellency of four essential oils individually and mixed, reporting a repellency of 100% with M. piperita alone and mixed with Cymbopogon citratus (70:30). The repellent activity observed in M. piperita could be attributed to menthol, the major constituent of the oil (74.92%) (Table 1). Kumar et al. (2014a) reported that menthol alone had а repellency of 95.6%. In the case of С. zeylanicum, the results differ from Khater and Geden (2019), who assessed the repellency of essential oils of vetiver, cinnamon, lavender, and their blends and reported that none of the oils were repellent for adult house flies in olfactometer assays. In other fly species, Boito et al. (2018), with a concentration of 5.0%, reported 80% repellency of horn flies (Haematobia irritans L.; Diptera: Muscidae). In the case of С. xlimon essential oil, there is no evidence of repellency in M. domestica. However, with other citrus species, such as С. sinensis, Chauhan et al. (2018), using a concentration of 0.010 ul cm", obtained a repellency of 90% in M. domestica adults. Results with 8%-10%, make the possibility of developing a commercial repellent formulation, especially with essential oil of C. zeylanicum, very expensive. Therefore, growing M. piperita or using waste from the citrus industry could be a lower-cost alternative for a future commercial repellent.

CONCLUSIONS

The present study demonstrated the potential of lemon (Citrus xlimon), cinnamon (Cinnamomum zeylanicum), and peppermint (Mentha xpiperita) as effective insecticides and repellents for Musca domestica control. Although, the M. domestica stages showed differential susceptibility, because in contact toxicity the larva is more susceptible to M. piperita and C. zeylanicum and adult is to C. limon. While in fumigant toxicity C. zeylanicum was the most toxic to both stages. All essential oils were repellent but only the highest doses showed 90% effectiveness which can be expensive to formulate commercially a repellent. However, these findings must be validated under field conditions. Furthermore, in addition to their biological activity against houseflies, mint and lemon essential oils offer positive externalities. In the case of lemon, the essential oil and even limonene can be obtained from industry waste, which could be considered a circular economy. And although mint is a cultivated species, its use as a raw material for the production of an insecticide will increase its demand and consequently the interest of farmers in its cultivation.

Author contribution

Conceptualization: G.S., J.C.R., А.В. Methodology: G.S., T.V., M.R., G.O., G.C. Software: T.V., G.S., M.R., С.С. Validation: G.S,, G.C., G.O. Formal analysis: G.S. J.C.R., M.R., TV, А.К. Investigation: G.S., T.M., М.В. А.В. Resources: G.S., M.R. Data curation: TV, G.O. Writing-original draft: G.S., J.C.R., A.R., TV. Writing-review & editing: G.S., J.C.R., А.В. Visualization: TM., G.S. М.В. Supervision: G.S., M.R., G.C. All co-authors reviewed the final version and approved the manuscript before submission.

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