TY - JOUR
AU - Da Silva, Aleksandro, S
AB - Abstract For the first time, the repellent and insecticidal effects of eucalypt essential oil (Eucalyptus globulus) in its free form and associated with different nanostructures (nanoemulsion and nanocapsules) were investigated against Musca domestica (Diptera: Muscidae) and Haemotobia irritans (Diptera: Muscidae) flies. Specimens of M. domestica were collected, separated into groups (n = 10), treated with aspersion of essential oil of eucalypt in its free (1, 5, and 10%) and nanostructured (nanoemulsion or nanoencapsulated at 1, 3, and 5%, respectively) forms. The determination of the insecticidal effect was performed by counting the number of dead flies at intervals of 30, 60, 120, 360 and 750 min after oil aspersion. E. globulus essential oil showed insecticidal effect against M. domestica at concentrations of 1 and 5%. Eucalypt essential oil (10%) caused 100% mortality of M. domestica after 750 min of the treatment. Nanocapsules (3 and 5%) showed efficacy by reducing the number of flies. On the other hand, nanoemulsion did not show insecticidal effect. The repellent action of E. globulus concentrations of 5% was tested against H. irritans on naturally infested cows. The repellent action against H. irritans was verified soon after pulverization. After 24 h, a significant reduction on horn flies’ population (83.33 and 66.66%) was observed using free and nanoencapsulated forms tested, respectively. E. globulus essential oil demonstrated insecticidal and repellent effects against M. domestica and H. irritans flies. Eucalyptus globulus, Haemotobia irritans, fly control Challenges regarding fly control are common in many countries throughout the world, since flies are important vectors for several pathogenic microorganisms such as viruses, bacteria, fungi, and parasites, causing reduction in animal productivity and higher production costs (Thyssen et al. 2004). The house fly (Musca domestica [Diptera: Muscidae]) is considered one of the main vectors disseminating pathogens to animals and man (Béjar et al. 2006). Another important fly related to several economic losses in animal production is the horn fly (Haemotobia irritans [Diptera: Muscidae]) due to its hematophagous habit, reflecting negatively in animal performance (Grisi et al. 2014). The indiscriminate use of insecticides is one of the main reasons for parasitic resistance to several drugs used for fly control (Scott et al. 2013). Also, it is important to emphasize that several pesticides remain in the environment for a long time, which may cause them to accumulate in food, eliciting a risk to ecosystem and human (Bianchin and Alves 2002, Gerage et al. 2017). In this sense, many researchers are testing new approaches to avoid environmental contamination and meat residues using more natural products (Clemente et al. 2007, 2010), as well as because resistance to natural products is low (Dayan et al. 2009). The eucalypt (Eucalyptus globulus) essential oil is a viable candidate (Chagas et al. 2001) to be tested. However, it presents a few concerning characteristics common to all essential oils: high concentration of volatile substances and increasing degradation when it is exposed to high temperatures. Is it possible to obtain positive results using essential oils of E. globulus against flies? Our doubts directed us to nanotechnology methodologies, since it is a well-known tool that can improve the therapeutic efficacy of essential oils and degradation of the active ingredients (Anton et al. 2008). This technology causes a new molecular organization with nanometric size, allowing a prolonged and controlled release of active principle that could be used for many drugs or essential oils (Misra et al. 2010). Therefore, the use of natural products using nanotechnology has drawn researcher’s attention, since it is able to increase the action of repellent products by inducing a better absorption of essential oil by the host (Vauthier and Bouchemal 2009). The nanoemulsion process has other advantages compared with nanocapsules that deserves to be mentioned, such as stability and immediate release that occurs due to the absence of polymeric wall. Thus, the oil content and its retention is influenced by the size and distribution of its drops (Anton et al. 2008). Based on these studies, we hypothesize that nanocapsules and nanoemulsions containing essential oil of eucalypt can potentiate the insecticidal and repellent effects of E. globules. Therefore, the aim of this study was to evaluate whether nanostructured forms of E. globulus essential oil could enhance its insecticidal effect against M. domestica in vitro, as well as repellent action to H. irritans using cattle as the animal experimental model. Materials and Methods Source and Characterization of E. globulus Essential Oil Eucalypt oil was purchased from Ferquima (São Paulo, Brazil) and its chemical composition was analyzed according to the methodology described by de Godoi et al. (2017) using a gas chromatograph Varian one Star 3400CX (CA) equipped with a flame ionization detector (GC-FID). The qualitative analysis of the compounds was performed by a Shimadzu QP2010 Plus gas chromatograph coupled to a mass spectrometer (GC/MS, Shimadzu Corporation, Kyoto, Japan). The analytes were identified based on the comparison of the mass spectra in the library available at the National Institute of Standards and Technology (NIST) and by comparing the calculated linear retention indices with those available in the scientific literature. The relative amount of each identified compound was obtained from the peak area obtained by the FID. The gas chromatograph analyses revealed that 1–8 cineol is the main component with 75.78% of the total essential oil composition (Fig. 1). Fig. 1. Open in new tabDownload slide Percentage of the main components present in the essential oil of Eucalyptus globulus used in this study. Fig. 1. Open in new tabDownload slide Percentage of the main components present in the essential oil of Eucalyptus globulus used in this study. To obtain the right concentrations for our study, the eucalypt oil was diluted in Triton for further dilution in distilled water at final concentrations of 1, 5, and 10%. These concentrations were based on a previous pilot study using M. domestica as the experimental model. Nanostructure Preparation and Characterization The nanoemulsion containing the E. globulus essential oil was prepared using the emulsification under high agitation with Ultra Turrax method (de Godoi et al. 2017). The oil phase was composed of 5% of eucalypt oil and 0.5 g of sorbitan monooleate (Span 80). The aqueous phase was composed of 0.5 g of polysorbate 80 (Tween 80) and 25 ml of ultrapure water. Both phases were solubilized separately using a magnetic stirrer during 15 min. Then, the oil phase was mixed with the aqueous phase under agitation (10,000 RPM) during 15 min and increased to 17,000 RPM during 1 h. The nanocapsules containing E. globulus essential oil were prepared according to the interfacial deposition of preformed polymer (Fessi et al. 1989). The oil phase was composed of 67 ml of acetone, 0.25 g of polycaprolactone (PLC), and 0.5 g of sorbitan monooleate (Span 80). This phase was maintained in a magnetic stirrer at 40ºC during 1 h, and later 1.25 g of eucalypt oil was added and agitated for 15 min. The aqueous phase was composed of 134 ml of ultrapure water and 0.5 g of polysorbate 80 (Tween 80) followed by solubilization in a magnetic stirrer without heating. Then, the oil phase was verted in the aqueous phase and maintained in agitation for 15 min. Later, the solution was rotaevapored to eliminate solvents and to adjust to the final volume (25 ml). Particle size and zeta potential were evaluated in diluted samples (500×) using Nano-ZS ZEN 3600 (Zetasizer, Malvern, England). The pH was assessed by the direct use of a potentiometer (Digimed DM-20, São Paulo, Brazil). Bioassays In Vitro Tests to M. domestica Aiming to evaluate the insecticidal effect of eucalypt essential oil against flies, the house fly (M. domestica) was used in our experimental model. Four hundred flies were captured using an adequate mesh and allocated in breeding cages with food (sugar) and water for 48 h. Later, 10 flies were allocated in test cages built as described by Klauck et al. (2014). Then, the tests were performed with a hand sprayer that proved to be effective to release microdroplets of approximately 100 µl per group of the tested solution. Three hundred (n = 300) flies were separated into four groups (n = 75 flies/group): control (CG), free oil (PoG), eucalypt essential oil nanoencapsulated (NcG), and eucalypt essential oil nanoemulsified (NeG). The treatments were performed in triplicate. The free oil was tested through the aspersion method at concentrations of 1, 5, and 10%, whereas the nanostructured forms were tested at concentrations of 1, 3, and 5%. Flies of the control group were pulverized with distilled water diluted in Triton (10%). The insecticidal effect was verified according to the number of dead flies after 30, 60, 120, 360, and 750 min postpulverization. At the end of the experiment, all flies were examined under a stereomicroscope and were confirmed to be M. domestica by morphological traits. Repellency Test for Horn Fly Repellency tests were conducted on crossbred cows (Holstein x Jersey) naturally infested by H. irritans. The experimental design was composed of three groups with three cows each: 1) control (pulverized with Triton diluted in water 10%); 2) pulverized with free eucalypt oil at 5%; and 3) pulverized with nanocapsules at 0.5%. Each animal was treated with the pulverized material (30 ml) on three distinct regions: head, neck, and dorsal region (cervical and thoracic). The animals were kept in the same environment, with free access to water, grass, and shade. The number of flies was counted right before the beginning of the treatments (9 a.m.) and 1, 3, 6, and 24 h posttreatment. For this visual counting, the right and left sides of each cow were photographed at the same moment (Digital Camera—Fujifilm, model Finepix S2980, 14 megapixels, São Paulo, Brazil). In the laboratory, these pictures were computer analyzed. The insecticidal effect was verified by counting the number of dead flies at specific times (30, 60, 120, 360, and 750 min). The repellent effect was verified by the amount of flies present in the animals at 1, 3, 6, and 24 h after the treatment. Statistical Analysis The data were firstly analyzed by descriptive statistics for contingency of information and description of data. The number of death flies (in vitro and repellency test) was tested for normality using the Shapiro–Wilk test (Shapiro and Wilk 1965). Analysis of variance (ANOVA) considered 30, 60, 120, 360, and 750 min for M. domestica and 1, 3, 6, and 24 h for H. irritans, followed by Tukey test (P < 0.05). Results Particle size (nm), polydispersity index, and zeta potential (mV) of the nanocapsules containing oil of E. globulus were as follows: 162.1 ± 1.25, 0.165 ± 0.01, and −14.3 ± 0.60, respectively; for nanoemulsion: 69 ± 0.71 nm, 0.20 ± 0.01, and −16.5 ± 0.65 and pH 4.68 ± 0.04, respectively. The eucalypt essential oil at concentrations of 1 and 5% caused fly mortality rate of 80% at the end of the experiment. However, at a concentration of 10%, the free eucalypt essential oil was fully effective (100% of effect) 750 min after treatment (Fig. 2A). The nanoemulsions containing essential oil of eucalypt did not exhibit insecticidal effect (P > 0.05; Fig. 2B). When nanocapsules containing essential oil of eucalypt at 1% were used, no insecticidal effect was observed. However, at 3 and 5% it showed 65 and 85% of efficacy, respectively, 750 min after treatment (Fig. 2C). Fig. 2. Open in new tabDownload slide Insecticidal effect of Eucalyptus globulus in its free (A) (1, 5, and 10%) and nanostructured form (B and C) (1, 3, and 5%) against Musca domestica. Number of dead flies was counted during the onset of the experiment and after 30, 60, 120, 360, and 750 min of the treatments. Data inside the same circle did not differ statistically (Tukey test; P > 0.05). Fig. 2. Open in new tabDownload slide Insecticidal effect of Eucalyptus globulus in its free (A) (1, 5, and 10%) and nanostructured form (B and C) (1, 3, and 5%) against Musca domestica. Number of dead flies was counted during the onset of the experiment and after 30, 60, 120, 360, and 750 min of the treatments. Data inside the same circle did not differ statistically (Tukey test; P > 0.05). There was a significant reduction on the number of H. irritans after the treatment with eucalypt essential oil (5% and its nanoencapsulated form (0.5%; Fig. 3). In addition, 24-h postpulverization, a reduction of 83.33 and 66.67% on the number of flies was observed, respectively. Fig. 3. Open in new tabDownload slide Percentage of Haematobia irritans alive after pulverization of free (5%) and nanoencapsulated (0.5%) oil of Eucalyptus globulus. The number of flies was counted on nine animals 1, 3, 6, and 24 h posttreatment application. The data inside the same circle did not differ statistically (Tukey test; P > 0.05). Fig. 3. Open in new tabDownload slide Percentage of Haematobia irritans alive after pulverization of free (5%) and nanoencapsulated (0.5%) oil of Eucalyptus globulus. The number of flies was counted on nine animals 1, 3, 6, and 24 h posttreatment application. The data inside the same circle did not differ statistically (Tukey test; P > 0.05). Discussion This is the first experimental study that demonstrates the insecticidal effect of E. globulus essential oil in its nanostructured form against M. domestica and H. irritans, whereas use of free eucalyptus essential oil was already reported against housefly (Kumar et al. 2012). We clearly observed that the nanocapsules had a similar effect compared with free oil against horn fly when using doses 10 times lower. Several studies demonstrated the potential effect of essential oils to control ectoparasites (Barker and Altman 2011, Ellse and Wall 2014). The insecticidal properties of some essential oils are linked with their capacity to penetrate the insects’ exoskeleton and cause changes in their physiological functions (Ozaki et al. 2003). In this sense, we expected that the nanostructured form would increase its insecticidal action; however, it did not occur. It is important to emphasize that the repellent effect of eucalypt essential oil against adult horn flies was not yet described in the scientific literature. Additionally, several other important essential oils are known by their potent insecticidal and repellent effects, such as Mentha arvensis, Mentha piperata, Mentha spicatae, and Cymbopogon spp. (Raja et al. 2001), as well as the use of Melaleuca alternifolia and Caraipa guianensis (Klauck et al. 2014). The E. globulus essential oil is composed of several terpenes such as the 1,8-cineol, α-pinene, p-cymene, and limonene that may have insecticidal and repellent effect. In this sense, Dias and Morais (2014) demonstrated that the insecticidal and repellent effect of citronella, Eucalyptus spp., and M. piperita essential oils was due to the presence of terpenes, such as the 1,8-cineol. Moreover, the 1,8-cineol, the main component of Lippia sidoides and E. globulus essential oils, might be linked with insecticidal and acaricidal activities against Tenebrio molitor and Rhipicephalus microplus (Chagas et al. 2001, Lima et al. 2011). According to Pauliquevis and Favero (2015), the terpenes act on octopaminergic sites of insects, inhibiting the octopamine, an essential neurotransmitter of the central nervous system of insects. According to Enan (2001), terpenes acts on octopaminergic sites of insects causing a blockage of octopamine receptor–binding sites, i.e., the action is linked to inhibition of octopamine receptors binding, as observed using eugenol and α-terpienol, terpenes that presented dose-dependent and species-dependent effects. Thus, the insecticidal and repellent effect of eucalypt essential oil may involve the effect on the octopaminergic sites of M. domestica and H. irritans. In this present study, the nanoemulsion did not exert insecticidal action on M. domestica. This may have occurred due to a too fast release, since the nanoemulsion is characterized by a rapid release of active principles when compared with other nanostructures, providing an immediately effect on the applied surface (Vauthier and Bouchemal 2009). On the other hand, the release of nanocapsules occurs slowly and continuously, justifying their insecticidal and repellent effects, as already described in tick (Pazinato et al. 2014). This condition validates our results, since the nanotechnology allows the use of low concentrations of the active principles without changes on their therapeutic efficacy. The nanotechnology also presented similar repellent effect when used in concentration 10 times lower. This result is similar to the one reported by Moorthi et al. (2015) against the common castor (Ergolis merione). According to Nair et al. (2016), increased therapeutic efficacy of many compounds when associated with nanotechnology is linked with some factors such as better solubilization, greater bioavailability, longer half-life, and higher protection of the active principle against enzymatic and hydrolytic degradation. In summary, the eucalypt oil in its free or nanoencapsulated form exhibited insecticidal effects against M. domestica, besides to its repellent effect on H. irritans. Additionally, nanotechnology was able to enhance the repellent effect using a concentration 10 times lower when compared with the free form. Therefore, nanocapsules containing essential oil of E. globulus can be considered an interesting option to prevent, as well as an alternative treatment to control flies associated with livestock production. Acknowledgments We thank CAPES and CNPq for their financial support in research in Brazil. 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TI - Low Dose of Nanocapsules Containing Eucalyptus Oil Has Beneficial Repellent Effect Against Horn Fly (Diptera: Muscidae)
JF - Journal of Economic Entomology
DO - 10.1093/jee/toy267
DA - 2018-12-14
UR - https://www.deepdyve.com/lp/oxford-university-press/low-dose-of-nanocapsules-containing-eucalyptus-oil-has-beneficial-BSdl15Gfwb
SP - 2983
VL - 111
IS - 6
DP - DeepDyve
ER -