Medical and Veterinary Entomology (2014) 28 (Suppl. 1), 33–39
Insecticidal and repellent effects of tea tree and andiroba oils on flies associated with livestock V. K L A U C K 1 , R. P A Z I N A T O 1 , L. M. S T E F A N I 1,2 , R. C. S A N T O S 3,4 , R. A. V A U C H E R 3 , M. D. B A L D I S S E R A 3 , R. R A F F I N 4 , A. B O L I G O N 5 , M. A T H A Y D E 5 , D. B A R E T T A 1 , G. M A C H A D O 6 and A. S. D A S I L V A 1 1
Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil, 2 Postgraduate Programme in Animal Science, UDESC, Lages, SC, Brazil, 3 Laboratory of Microbiology, Centro Universitário Franciscano (UNIFRA), Santa Maria, RS, Brazil, 4 Laboratory of Nanotechnology, UNIFRA, Santa Maria, RS, Brazil, 5 Department of Industrial Pharmacy, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil and 6 Laboratory of Veterinary Epidemiology (EPILAB), Faculdade de Veterinária (FAVET), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
Abstract. This study aimed to evaluate the insecticidal and repellent effects of tea tree, Melaleuca alternifolia (Myrtales: Myrtaceae), and andiroba, Carapa guianensis (Sapindales: Meliaceae), essential oils on two species of fly. For in vitro studies, free-living adult flies were captured and reared in the laboratory. To evaluate the insecticidal effects of the oils, adult flies of Haematobia irritans (L.) and Musca domestica L. (both: Diptera: Muscidae) were separated by species in test cages (n = 10 per group), and subsequently tested with oils at concentrations of 1.0% and 5.0% using a negative control to validate the test. Both oils showed insecticidal activity. Tea tree oil at a concentration of 5.0% was able to kill M. domestica with 100.0% efficacy after 12 h of exposure. However, the effectiveness of andiroba oil at a concentration of 5.0% was only 67.0%. The insecticidal efficacy (100.0%) of both oils against H. irritans was observed at both concentrations for up to 4 h. The repellency effects of the oils at concentrations of 5.0% were tested in vivo on Holstein cows naturally infested by H. irritans. Both oils demonstrated repellency at 24 h, when the numbers of flies on cows treated with tea tree and andiroba oil were 61.6% and 57.7%, respectively, lower than the number of flies on control animals. It is possible to conclude that these essential oils have insecticidal and repellent effects against the species of fly used in this study. Key words. Andiroba, essential oils, flies, insects, tea tree.
Introduction Flies are considered important vectors of disease, carrying pathogenic microorganisms such as viruses, bacteria, fungi and parasites (Axtell & Arends, 1990; Béjar et al., 2006). Houseflies (Musca domestica) may disseminate pathogens, contaminating food and surfaces on which they land (Béjar et al., 2006). In addition, many flies have direct influence on animal production, such as the horn fly (Haematobia irritans), stable fly [Stomoxys calcitrans (Diptera: Muscidae)], and flies that cause
myiasis [Cochliomyia hominivorax and Chrysomya spp. (both: Diptera: Calliphoridae)]. The horn fly is a blood-sucking insect which causes great economic losses in the cattle industry (Campbell et al., 2001) as a result of its painful bite and subsequent decreases in animal food intake (Palavesam et al., 2012) and production (Guimarães, 1984). Fly control is usually accomplished by the use of chemicals, which, when used incorrectly, can lead to environmental contamination, as well as to fly resistance (Barros, 2001; Freitas, 2008). The use of herbal medicine on conventional farms
Correspondence: Aleksandro S. Da Silva, Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Rua Beloni Trombeta Zanin, 680-E, Bairro Santo Antônio, Chapecó, SC CEP 89815-630, Brazil. Tel.: + 55 49 3330 9400; Fax: + 55 49 3330 9401; E-mail: [email protected] © 2014 The Royal Entomological Society
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has been suggested as a strategy to extend the life of neurotoxic insecticides. Recently, researchers demonstrated that tea tree and lavender essential oils can be clinically useful in louse control (Bovicola ocellatus) when used as part of a grooming routine, and suggested that with further development these might form the basis of an easy-to-apply component of a louse management programme for donkeys (Ellse et al., 2013). In addition, these natural products are less toxic to the animal and the environment (Vieira & Cavalcante, 1999). In this search for approaches to insect control that are economical and less harmful to the environment, many researchers have turned their attention to more natural and biodegradable products for pest control (Chagas, 2004). Some essential oils are already used for the treatment and control of flies, such as citronella oil (Chagas et al., 2002; Cárcamo et al., 2007) and neem oil (Deleito & Borja, 2008; Pissinati et al., 2009). Other essential oils, such as those of melaleuca and andiroba, have shown medicinal antiparasitic properties (Miot, 2004; Vieira et al., 2004; Castro et al., 2005). Carapa guianensis (andiroba) belongs to the botanical family Meliaceae and is a large tree found throughout the Amazon basin (Boufleuer, 2004). Andiroba oil has repellent and larvicidal effects against mosquitoes (Miot, 2004; Emerick, 2005) and adult bees (Santos et al., 2012). Melaleuca alternifolia (tea tree) belongs to the family Myrtaceae, derived originally from Australia (Vieira et al., 2004). Tea tree essential oil has been shown to have bactericidal and antifungal properties against various pathogens (Castro et al., 2005). The chemical composition of the oil from the leaves of M. alternifolia is well known for being rich in terpinen-4-ol, which is thought to be largely responsible for its medicinal properties (Vieira et al., 2004). Therefore, the aim of this study was to evaluate the insecticidal and repellent effects of andiroba and tea tree oils against flies. Materials and methods Plant material Andiroba oil (C. guianensis) RF3150 was purchased from Beraca Sabará Químicos e Ingredientes S/A (São Paulo, SP, Brazil). Tea tree oil (M. alternifolia) was purchased from Chemical Importer Delaware Ltda (São Paulo, SP, Brazil). To obtain the desired concentrations, both essential oils were diluted in a non-ionic surfactant triton solution (Chagas et al., 2003; Farias et al., 2007). First, essential oils were diluted in triton solution (50.0% v/v) and manually homogenized for 5 min. Then, the solution (oil and triton) was diluted with distilled water to obtain concentrations of 1.0% and 5.0% to be used in the study. The concentrations used in this study were selected after a pilot study had tested five concentrations (0.5%, 1.0%, 3.0%, 5.0% and 10.0%) against M. domestica. Oil characterization: andiroba and tea tree Gas chromatography (GC) analyses were carried out using an Agilent 6890N GC-FID system (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.), equipped with a DB-5 capillary column (30 mm by 0.25 mm internal diameter; film thickness of
0.25 mm) connected to a flame ionization detector (FID). The injector and detector temperatures were set at 280 ∘ C. Helium was used as the carrier gas at a flow rate of 1.3 mL/min. The thermal program escalated from 50 ∘ C to 300 ∘ C at a rate of 5 ∘ C/min. Two replicates of oil samples were processed in the same way. Relative component concentration was calculated based on GC peak areas without using correction factors. The injection volume of the C. guianensis (andiroba) and M. alternifolia (tea tree) essential oils was 1 μL (Boligon et al., 2013). Gas chromatography mass spectroscopy (GC-MS) analyses were performed using an Agilent Technologies AutoSystem XL GC-MS system operating in the EI mode at 70 eV, equipped with a split/splitless injector (250 ∘ C). The transfer line temperature was 280 ∘ C. Helium was used as the carrier gas (1.5 mL/min). The capillary columns used were an HP 5MS (30 m by 0.25 mm internal diameter; film thickness of 0.25 mm) and an HP Innowax (30 m by 0.32 mm internal diameter; film thickness of 0.50 mm). The temperature program was identical to that used for the GC analyses. The injected volume of each essential oil was 1 μL. Component identification in both oils was performed on the basis of the retention index (RI), determined with reference to the homologous series of n-alkanes, C7 –C30 , under identical experimental conditions, and compared with a search of the mass spectra libraries [National Institute of Standards and Technology (NIST) and Wiley Registry of Mass Spectral Data] and with the mass spectra literature (Adams, 1995). The relative amounts of individual components were calculated based on the GC peak area (FID response). Fly acquisition and essential oils bioassays In vitro insecticidal effect against Musca domestica. Adult houseflies were captured with the aid of a net from a property in the city of Chapecó, Santa Catarina in southern Brazil. After capture, 252 flies were housed in cages and fed with sugar and water up to the test date (48 h). Each cage (20 × 20 × 30 cm) was built using a 2-L plastic bottle with an iron frame covered by a thin nylon mesh at one end. At the other end of the plastic bottle was a screw cap. Through this opening (2.1 cm in diameter) flies were introduced and sprayed. For this, we used a hand-sprayer able to release microdroplets of the essential oil into the cage (Fig. 1). A new hand-sprayer was used for each repetition and treatment (i.e. hand-sprayers were not reused). A total of 180 M. domestica flies were separated into groups of 10 insects per group to test andiroba and tea tree oils by spraying (100 μL/group) each oil at concentrations of 1.0% and 5.0%. A control group was sprayed with distilled water mixed with 5.0% triton. All tests were performed in triplicate. Insecticidal effects were verified by observing the numbers of dead flies at predetermined times of 1 h, 2 h, 3 h, 6 h, 9 h, 12 h and 24 h after oil exposure. Tests with H. irritans In vitro test. In vitro tests with horn flies were performed as previously described for M. domestica. Horn fly capture
© 2014 The Royal Entomological Society, Medical and Veterinary Entomology, 28 (Suppl. 1), 33–39
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Table 1. Qualitative and quantitative analyses of Carapa guianensis (andiroba) and Melaleuca alternifolia (tea tree) essential oils.
Fig. 1. Equipment used to test the toxicity of essential oils in vitro against flies in the laboratory. A hand-sprayer (1) was used to spray the oil solution as microdroplets into cages containing flies (2), at a spray volume per cage of 100 μL. Each repetition and treatment used a new cage.
was performed using a net at a dairy farm with a high level of infestation. Tests with tea tree and andiroba oils against H. irritans were performed simultaneously, and the results for each oil were compared with those for the control group (water + triton). In vivo tests. In vivo tests were performed on a dairy farm in Chapecó (SC, Brazil). On this property Holstein cows were naturally highly infested by H. irritans with approximately similar number of flies per cow. Three groups of five animals each with similar coats were sprayed using hand-sprayers containing, respectively, tea tree oil at 5.0% (Group A), andiroba oil at 5.0% (Group B) and triton diluted in water (control group, Group C). Each animal was treated with 10 mL of solution by spraying on the head, neck and dorsal region of the body (for a total of 30 mL/animal). During testing, all animals were placed in the same environment and given free access to water, shade and pasture. All H. irritans located on the animals were counted before (at 09.00 hours) and after treatments at 1 h, 2 h, 3 h, 6 h, 9 h, 24 h, 48 h and 72 h. To count the flies, two observers photographed both sides (left and right) of each cow at the same time using high-resolution digital cameras (Nikon Coolpix P510, 16.1 megapixels; Nikon Corp., Tokyo, Japan) and made a visual count of all flies. Later, in the laboratory, with the aid of computers, the photographic images (two per animal) were carefully analysed and counts were checked against those obtained in the field.
Components
Andiroba oil
Tea tree oil
Camphene 𝛼-pinene Octanone p-Cymene Limonene Linalool Menthol 𝛼-terpineol Carvacrol 𝛼-cubebene 𝛽-elemene 𝛽-caryophyllene 𝛽-humulene 𝛼-humulene Germacrene-D Bicyclogermacrene Germacrene-A 𝛾-cadinene 𝛼-cadidene Germacrene-B Caryophyllene oxide Cedrol Eudesmol 𝛼-cadinol Sabinene 𝛼-terpinene 1.8-Cineole 𝛾-terpinene Terpinen-4-ol Terpinolene Aromadendrene Ledene 𝛿-cadinene Globulol Viridiflorol Total
0.87% 3.28% 0.25% 0.13% 1.67% 0.79% 0.53% 2.31% 1.34% 0.41% 4.57% 5.08% 2.47% 23.81% 12.07% 17.46% 2.58% 0.73% 1.13% 8.83% 1.85% 3.19% 0.32% 1.47% – – – – – – – – – – – 97.14%
– 3.51% – 2.27% 1.39% – – 2.43% – – – – – – – – – – – – – – – – 0.46% 9.85% 6.03% 20.15% 41.98% 4.17% 1.04% 0.80% 0.63% 0.97% 0.18% 95.86%
and H. irritans in vitro, and 1 h, 2 h, 3 h, 6 h, 9 h, 24 h, 48 h and 72 h for H. irritans in vivo) and Tukey’s honestly significant differences (HSD) test was used in post hoc analysis. A P-value of < 0.05 was considered to indicate a significant difference. The whole statistical process was carried out using R Version 2.15.2 (R Foundation for Statistical Computing, Vienna, Austria).
Statistical analysis Results The experimental data were first analysed using descriptive statistics for contingency of the information and for further assumptions. The numbers of dead flies (in vitro and in vivo) were tested for normality of variance using the Shapiro–Wilk test (Shapiro & Wilk, 1965), and for skewedness, kurtosis and homogeneity using Levene’s test (Box, 1953). The counts of all cages were transformed by [Log10 (x + 1)] before variance analysis. Analysis of variance (anova) was considered for each time-point (1 h, 2 h, 3 h, 6 h, 9 h, 12 h and 24 h for M. domestica
Oil components Qualitative and quantitative analyses of C. guianensis (andiroba) and M. alternifolia (tea tree) essential oils are shown in Table 1. The three main components of andiroba oil were 𝛼-humulene, bicyclogermacrene and germacrene-D (53.34%). The two main components of tea tree oil were terpinen-4-ol and 𝛾-terpinene (62.13%).
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(A)
(B) Fig. 2. Insecticidal effects of (A) tea tree and (B) andiroba oils at concentrations of 1.0% and 5.0% against Musca domestica. Numbers of dead flies were counted at baseline and at 1 h, 2 h, 3 h, 4 h, 9 h, 12 h and 24 h post-oil exposure. Data within the same circle are not statistically different (Tukey’s honestly significant differences test, P > 0.05).
Essential oils: insecticidal effect
Discussion
Tea tree oil at 1.0% had no insecticidal effect on M. domestica (F = 3.24, d.f. = 7, P > 0.05). However, at the concentration of 5.0% it was able to kill and was 100.0% effective after 12 h of oil exposure (F = 210.1, d.f. = 2, P < 0.05) (Fig. 2A). Andiroba oil was effective at both concentrations tested (1.0% and 5.0%); its insecticidal effect against houseflies formed a dose-dependent curve (F = 53.1, d.f. = 7, P < 0.05) (Fig. 2B) showing statistically similar results at almost all the time-points observed for both concentrations. However, the effectiveness of andiroba oil at 5.0% was 67.0% lower than that of tea tree oil against M. domestica. Insecticidal effects of tea tree oil (Fig. 3A) (F = 41.3, d.f. = 7, P < 0.05) and andiroba (Fig. 3B) (F = 25.6, d.f. = 7, P < 0.05) were also observed at both concentrations against H. irritans. Fly death (100.0%) was observed at 4 h post-treatment (Fig. 3). A significant reduction in the number of H. irritans was observed on cows sprayed with both essential oils compared with animals in the control group (F = 30.04, d.f. = 2, P < 0.05) (Fig. 3C). The effect lasted for up to 24 h, but by 48 h after application no difference in fly numbers could be detected among the groups (F = 2.12, d.f. = 2, P > 0.05).
According to the literature, there is a growing body of evidence indicating the potential value of essential oils as control agents against a range of arthropod ectoparasites, although it is unclear whether these effects derive from neurotoxicity or mechanical suffocation (Ellse & Wall, 2013). The study of oil toxicity is subject to many difficulties, mainly because of the wide variation in the chemical composition of oils. Nevertheless, the use of essential oils in the control of veterinary ectoparasites holds considerable potential and research in this area is still at an early stage. The data presented here show the insecticidal effects of oils of both andiroba and tea tree against H. irritans and M. domestica. However, the mortality caused by the andiroba oil was lower than that caused by the tea tree oil. Similar results were reported by Farias et al. (2009), who conducted in vitro tests against third-instar M. domestica larvae using andiroba oil and noted 80.0% larval mortality. Cárcamo et al. (2007) used citronella oil at 1.0% and found it to cause 58.7% mortality against larvae of Lucilia sericata (Diptera: Calliphoridae). Researchers explain that the insecticidal effects of these essential oils is derived from their penetration of insect tissues and subsequent modification of some physiological functions (Ozaki et al., 2003).
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(A)
(B)
(C) Fig. 3. The percentage of Haematobia irritans alive at various time-points after exposure to (A) tea tree and (B) andiroba oils at concentrations of 1.0% and 5.0%. (C) Number of H. irritans counted on the bodies of 15 cattle treated with 5.0% tea tree oil, 5.0% andiroba oil or a control (water and triton) solution at baseline and at 1 h, 2 h, 3 h, 6 h, 9 h, 24 h, 48 h and 72 h post-oil exposure. Data within the same circle are not statistically different (Tukey honestly significant differences test, P > 0.05).
Studies on the repellent and insecticidal effects of andiroba and tea tree essential oils in adult H. irritans have not been described until now. However, other oils have been shown to have effect against this fly. Agnolin (2009) showed the efficiency of citronella oil on H. irritans and presented results similar to those of the present study for in vitro tests, in which treatments
with 5.0% oil gave 100.0% mortality. In vivo repellency tests using these oils showed a decrease in the number of flies found on treated animals. These results probably derived from the insecticidal and repellent effects of both tested oils (Ozaki et al., 2003; Coitinho et al., 2006; Farias et al., 2009). The insecticidal and repellent activities against mosquitoes and flies
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of oils of Mentha arvensis, Mentha piperita, Mentha spicata and Cymbopogon nardus have been reported previously (Raja et al., 2001). The insecticidal effect of tea tree oil can be related to its major components of terpinen-4-ol and 𝛾-terpinene. Terpinen-4-ol was tested against larvae of the oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae), and found to have a toxic effect (Ma & Zhang, 2004), which may also have occurred in the current study. The other component is 𝛾-terpinene, which has also been shown to have insecticidal effects (Abbassy et al., 2009). The literature contains no specific studies of the three major components present in andiroba oil (𝛼-humulene, bicyclogermacrene and germacrene-D), which may be effective against flies. However, these components are present in other plants with proven insecticidal activities (François et al., 2009; Dell’Agli et al., 2012). It is important to note that other components are present in these oils in lower concentrations and may also be responsible for both insecticidal and repellent activities. Therefore, further studies of isolated components will be conducted in the future by our research group. In summary, the current study concludes that essential oils of tea tree and andiroba have insecticidal effects against flies (M. domestica and H. irritans), as well as repellent effects against H. irritans. The horn fly was more susceptible to both concentrations of the oils than was the housefly.
Author’s declaration of interests No competing interests have been declared.
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Insecticidal action of oils on flies François, T., Michel, J.D.P., Lambert, S.M., Ndifor, F., Vyry, W.N.A., Henri, A.Z.P. & Chantal, M. (2009) Comparative essential oils composition and insecticidal effect of different tissues of Piper capense L., Piper guineense Schum. et Thonn., Piper nigrum L. and Piper umbellatum L. grown in Cameroon. African Journal of Biotechnology, 8, 424–431. Freitas, S.R.Q. (2008) Bioatividade de extratos aquosos de Eucalyptus Sp. L’hér. (Myrtaceae) E Melia Azedarach L. (Meliaceae) sobre Musca domestica L. (Diptera, Muscidae). Dissertação apresentada ao Programa de Pós-Graduação em Parasitologia da Universidade Federal de Pelotas, Pelotas. http://www.dominiopublico.gov. br/pesquisa/DetalheObraForm.do?select_action=&co_obra=125537 [Accessed 17 June 2013]. Guimarães, J.H. (1984) Mosca dos estábulos – uma importante praga do gado. Agroquímica Ciba: Geigy, 23, 10–14. Ma, Z.Q. & Zhang, X. (2004) Insecticidal activity of terpinen-4-ol against larvae of the oriental armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae). Acta Entomologica Sinica, 47, 329–333. Miot, H.A. (2004) Comparative study of the topical effectiveness of the andiroba oil (Carapa guianensis) and DEET 50% as repellent for Aedes sp. Revista do Instituto de Medicina Tropical de São Paulo, 45, 253–256. Ozaki, M., Takahara, T., Kawahara, Y. et al. (2003) Perception of noxious compounds by contact chemoreceptors of the blowfly, Phormia regina: putative role of an odorant binding protein. Chemical Senses, 28, 349–359.
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Palavesam, A., Guerrero, F.D., Heekin, A.M. et al. (2012) Pyrosequencing-based analysis of the microbiome associated with the horn fly, Haematobia irritans. Microbiome, 7, 8–12. Pissinati, A., Mikami, A.Y., Marques, C.R.G. et al. (2009) Use of neem and kaolin on nymphs of whitefly in cabbage. Revista Brasileira de Agroecologia, 4, 1487–1490. Raja, N., Albert, S., Ignacimuthu, S. & Dorn, S. (2001) Effect of volatile oils in protecting stored Vigna unguiculata (L.) Walpers against Callosobruchus maculatus (F.) (Coleóptera: Bruchidae) infestation. Journal of Stored Products Research, 37, 127–132. Santos, R.C.V., Alves, C.F.S., Schneider, T. et al. (2012) Antimicrobial activity of Amazonian oils against Paenibacillus species. Journal of Invertebrate Pathology, 109, 265–268. Shapiro, S.S. & Wilk, M.B. (1965) An analysis of variance test for normality (complete samples). Biometrika, 52, 591–611. Vieira, L.S. & Cavalcante, A.C.R. (1999) Resistência anti-helmíntica em rebanhos caprinos no Estado do Ceará. Pesquisa Veterinaria Brasileira, 19, 99–103. Vieira, T.R., Barbosa, L.C.A., Maltha, C.R.A., Paula, V.F. & Nascimento, E.A. (2004) Constituintes químicos de Melaleuca alternifolia (Myrtaceae). Quimica Nova, 27, 536–539. Accepted 19 December 2013
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Insecticidal and repellent effects of tea tree and andiroba oils on flies associated with livestock V. K L A U C K 1 , R. P A Z I N A T O 1 , L. M. S T E F A N I 1,2 , R. C. S A N T O S 3,4 , R. A. V A U C H E R 3 , M. D. B A L D I S S E R A 3 , R. R A F F I N 4 , A. B O L I G O N 5 , M. A T H A Y D E 5 , D. B A R E T T A 1 , G. M A C H A D O 6 and A. S. D A S I L V A 1 1
Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil, 2 Postgraduate Programme in Animal Science, UDESC, Lages, SC, Brazil, 3 Laboratory of Microbiology, Centro Universitário Franciscano (UNIFRA), Santa Maria, RS, Brazil, 4 Laboratory of Nanotechnology, UNIFRA, Santa Maria, RS, Brazil, 5 Department of Industrial Pharmacy, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil and 6 Laboratory of Veterinary Epidemiology (EPILAB), Faculdade de Veterinária (FAVET), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
Abstract. This study aimed to evaluate the insecticidal and repellent effects of tea tree, Melaleuca alternifolia (Myrtales: Myrtaceae), and andiroba, Carapa guianensis (Sapindales: Meliaceae), essential oils on two species of fly. For in vitro studies, free-living adult flies were captured and reared in the laboratory. To evaluate the insecticidal effects of the oils, adult flies of Haematobia irritans (L.) and Musca domestica L. (both: Diptera: Muscidae) were separated by species in test cages (n = 10 per group), and subsequently tested with oils at concentrations of 1.0% and 5.0% using a negative control to validate the test. Both oils showed insecticidal activity. Tea tree oil at a concentration of 5.0% was able to kill M. domestica with 100.0% efficacy after 12 h of exposure. However, the effectiveness of andiroba oil at a concentration of 5.0% was only 67.0%. The insecticidal efficacy (100.0%) of both oils against H. irritans was observed at both concentrations for up to 4 h. The repellency effects of the oils at concentrations of 5.0% were tested in vivo on Holstein cows naturally infested by H. irritans. Both oils demonstrated repellency at 24 h, when the numbers of flies on cows treated with tea tree and andiroba oil were 61.6% and 57.7%, respectively, lower than the number of flies on control animals. It is possible to conclude that these essential oils have insecticidal and repellent effects against the species of fly used in this study. Key words. Andiroba, essential oils, flies, insects, tea tree.
Introduction Flies are considered important vectors of disease, carrying pathogenic microorganisms such as viruses, bacteria, fungi and parasites (Axtell & Arends, 1990; Béjar et al., 2006). Houseflies (Musca domestica) may disseminate pathogens, contaminating food and surfaces on which they land (Béjar et al., 2006). In addition, many flies have direct influence on animal production, such as the horn fly (Haematobia irritans), stable fly [Stomoxys calcitrans (Diptera: Muscidae)], and flies that cause
myiasis [Cochliomyia hominivorax and Chrysomya spp. (both: Diptera: Calliphoridae)]. The horn fly is a blood-sucking insect which causes great economic losses in the cattle industry (Campbell et al., 2001) as a result of its painful bite and subsequent decreases in animal food intake (Palavesam et al., 2012) and production (Guimarães, 1984). Fly control is usually accomplished by the use of chemicals, which, when used incorrectly, can lead to environmental contamination, as well as to fly resistance (Barros, 2001; Freitas, 2008). The use of herbal medicine on conventional farms
Correspondence: Aleksandro S. Da Silva, Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Rua Beloni Trombeta Zanin, 680-E, Bairro Santo Antônio, Chapecó, SC CEP 89815-630, Brazil. Tel.: + 55 49 3330 9400; Fax: + 55 49 3330 9401; E-mail: [email protected] © 2014 The Royal Entomological Society
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has been suggested as a strategy to extend the life of neurotoxic insecticides. Recently, researchers demonstrated that tea tree and lavender essential oils can be clinically useful in louse control (Bovicola ocellatus) when used as part of a grooming routine, and suggested that with further development these might form the basis of an easy-to-apply component of a louse management programme for donkeys (Ellse et al., 2013). In addition, these natural products are less toxic to the animal and the environment (Vieira & Cavalcante, 1999). In this search for approaches to insect control that are economical and less harmful to the environment, many researchers have turned their attention to more natural and biodegradable products for pest control (Chagas, 2004). Some essential oils are already used for the treatment and control of flies, such as citronella oil (Chagas et al., 2002; Cárcamo et al., 2007) and neem oil (Deleito & Borja, 2008; Pissinati et al., 2009). Other essential oils, such as those of melaleuca and andiroba, have shown medicinal antiparasitic properties (Miot, 2004; Vieira et al., 2004; Castro et al., 2005). Carapa guianensis (andiroba) belongs to the botanical family Meliaceae and is a large tree found throughout the Amazon basin (Boufleuer, 2004). Andiroba oil has repellent and larvicidal effects against mosquitoes (Miot, 2004; Emerick, 2005) and adult bees (Santos et al., 2012). Melaleuca alternifolia (tea tree) belongs to the family Myrtaceae, derived originally from Australia (Vieira et al., 2004). Tea tree essential oil has been shown to have bactericidal and antifungal properties against various pathogens (Castro et al., 2005). The chemical composition of the oil from the leaves of M. alternifolia is well known for being rich in terpinen-4-ol, which is thought to be largely responsible for its medicinal properties (Vieira et al., 2004). Therefore, the aim of this study was to evaluate the insecticidal and repellent effects of andiroba and tea tree oils against flies. Materials and methods Plant material Andiroba oil (C. guianensis) RF3150 was purchased from Beraca Sabará Químicos e Ingredientes S/A (São Paulo, SP, Brazil). Tea tree oil (M. alternifolia) was purchased from Chemical Importer Delaware Ltda (São Paulo, SP, Brazil). To obtain the desired concentrations, both essential oils were diluted in a non-ionic surfactant triton solution (Chagas et al., 2003; Farias et al., 2007). First, essential oils were diluted in triton solution (50.0% v/v) and manually homogenized for 5 min. Then, the solution (oil and triton) was diluted with distilled water to obtain concentrations of 1.0% and 5.0% to be used in the study. The concentrations used in this study were selected after a pilot study had tested five concentrations (0.5%, 1.0%, 3.0%, 5.0% and 10.0%) against M. domestica. Oil characterization: andiroba and tea tree Gas chromatography (GC) analyses were carried out using an Agilent 6890N GC-FID system (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.), equipped with a DB-5 capillary column (30 mm by 0.25 mm internal diameter; film thickness of
0.25 mm) connected to a flame ionization detector (FID). The injector and detector temperatures were set at 280 ∘ C. Helium was used as the carrier gas at a flow rate of 1.3 mL/min. The thermal program escalated from 50 ∘ C to 300 ∘ C at a rate of 5 ∘ C/min. Two replicates of oil samples were processed in the same way. Relative component concentration was calculated based on GC peak areas without using correction factors. The injection volume of the C. guianensis (andiroba) and M. alternifolia (tea tree) essential oils was 1 μL (Boligon et al., 2013). Gas chromatography mass spectroscopy (GC-MS) analyses were performed using an Agilent Technologies AutoSystem XL GC-MS system operating in the EI mode at 70 eV, equipped with a split/splitless injector (250 ∘ C). The transfer line temperature was 280 ∘ C. Helium was used as the carrier gas (1.5 mL/min). The capillary columns used were an HP 5MS (30 m by 0.25 mm internal diameter; film thickness of 0.25 mm) and an HP Innowax (30 m by 0.32 mm internal diameter; film thickness of 0.50 mm). The temperature program was identical to that used for the GC analyses. The injected volume of each essential oil was 1 μL. Component identification in both oils was performed on the basis of the retention index (RI), determined with reference to the homologous series of n-alkanes, C7 –C30 , under identical experimental conditions, and compared with a search of the mass spectra libraries [National Institute of Standards and Technology (NIST) and Wiley Registry of Mass Spectral Data] and with the mass spectra literature (Adams, 1995). The relative amounts of individual components were calculated based on the GC peak area (FID response). Fly acquisition and essential oils bioassays In vitro insecticidal effect against Musca domestica. Adult houseflies were captured with the aid of a net from a property in the city of Chapecó, Santa Catarina in southern Brazil. After capture, 252 flies were housed in cages and fed with sugar and water up to the test date (48 h). Each cage (20 × 20 × 30 cm) was built using a 2-L plastic bottle with an iron frame covered by a thin nylon mesh at one end. At the other end of the plastic bottle was a screw cap. Through this opening (2.1 cm in diameter) flies were introduced and sprayed. For this, we used a hand-sprayer able to release microdroplets of the essential oil into the cage (Fig. 1). A new hand-sprayer was used for each repetition and treatment (i.e. hand-sprayers were not reused). A total of 180 M. domestica flies were separated into groups of 10 insects per group to test andiroba and tea tree oils by spraying (100 μL/group) each oil at concentrations of 1.0% and 5.0%. A control group was sprayed with distilled water mixed with 5.0% triton. All tests were performed in triplicate. Insecticidal effects were verified by observing the numbers of dead flies at predetermined times of 1 h, 2 h, 3 h, 6 h, 9 h, 12 h and 24 h after oil exposure. Tests with H. irritans In vitro test. In vitro tests with horn flies were performed as previously described for M. domestica. Horn fly capture
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Table 1. Qualitative and quantitative analyses of Carapa guianensis (andiroba) and Melaleuca alternifolia (tea tree) essential oils.
Fig. 1. Equipment used to test the toxicity of essential oils in vitro against flies in the laboratory. A hand-sprayer (1) was used to spray the oil solution as microdroplets into cages containing flies (2), at a spray volume per cage of 100 μL. Each repetition and treatment used a new cage.
was performed using a net at a dairy farm with a high level of infestation. Tests with tea tree and andiroba oils against H. irritans were performed simultaneously, and the results for each oil were compared with those for the control group (water + triton). In vivo tests. In vivo tests were performed on a dairy farm in Chapecó (SC, Brazil). On this property Holstein cows were naturally highly infested by H. irritans with approximately similar number of flies per cow. Three groups of five animals each with similar coats were sprayed using hand-sprayers containing, respectively, tea tree oil at 5.0% (Group A), andiroba oil at 5.0% (Group B) and triton diluted in water (control group, Group C). Each animal was treated with 10 mL of solution by spraying on the head, neck and dorsal region of the body (for a total of 30 mL/animal). During testing, all animals were placed in the same environment and given free access to water, shade and pasture. All H. irritans located on the animals were counted before (at 09.00 hours) and after treatments at 1 h, 2 h, 3 h, 6 h, 9 h, 24 h, 48 h and 72 h. To count the flies, two observers photographed both sides (left and right) of each cow at the same time using high-resolution digital cameras (Nikon Coolpix P510, 16.1 megapixels; Nikon Corp., Tokyo, Japan) and made a visual count of all flies. Later, in the laboratory, with the aid of computers, the photographic images (two per animal) were carefully analysed and counts were checked against those obtained in the field.
Components
Andiroba oil
Tea tree oil
Camphene 𝛼-pinene Octanone p-Cymene Limonene Linalool Menthol 𝛼-terpineol Carvacrol 𝛼-cubebene 𝛽-elemene 𝛽-caryophyllene 𝛽-humulene 𝛼-humulene Germacrene-D Bicyclogermacrene Germacrene-A 𝛾-cadinene 𝛼-cadidene Germacrene-B Caryophyllene oxide Cedrol Eudesmol 𝛼-cadinol Sabinene 𝛼-terpinene 1.8-Cineole 𝛾-terpinene Terpinen-4-ol Terpinolene Aromadendrene Ledene 𝛿-cadinene Globulol Viridiflorol Total
0.87% 3.28% 0.25% 0.13% 1.67% 0.79% 0.53% 2.31% 1.34% 0.41% 4.57% 5.08% 2.47% 23.81% 12.07% 17.46% 2.58% 0.73% 1.13% 8.83% 1.85% 3.19% 0.32% 1.47% – – – – – – – – – – – 97.14%
– 3.51% – 2.27% 1.39% – – 2.43% – – – – – – – – – – – – – – – – 0.46% 9.85% 6.03% 20.15% 41.98% 4.17% 1.04% 0.80% 0.63% 0.97% 0.18% 95.86%
and H. irritans in vitro, and 1 h, 2 h, 3 h, 6 h, 9 h, 24 h, 48 h and 72 h for H. irritans in vivo) and Tukey’s honestly significant differences (HSD) test was used in post hoc analysis. A P-value of < 0.05 was considered to indicate a significant difference. The whole statistical process was carried out using R Version 2.15.2 (R Foundation for Statistical Computing, Vienna, Austria).
Statistical analysis Results The experimental data were first analysed using descriptive statistics for contingency of the information and for further assumptions. The numbers of dead flies (in vitro and in vivo) were tested for normality of variance using the Shapiro–Wilk test (Shapiro & Wilk, 1965), and for skewedness, kurtosis and homogeneity using Levene’s test (Box, 1953). The counts of all cages were transformed by [Log10 (x + 1)] before variance analysis. Analysis of variance (anova) was considered for each time-point (1 h, 2 h, 3 h, 6 h, 9 h, 12 h and 24 h for M. domestica
Oil components Qualitative and quantitative analyses of C. guianensis (andiroba) and M. alternifolia (tea tree) essential oils are shown in Table 1. The three main components of andiroba oil were 𝛼-humulene, bicyclogermacrene and germacrene-D (53.34%). The two main components of tea tree oil were terpinen-4-ol and 𝛾-terpinene (62.13%).
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(A)
(B) Fig. 2. Insecticidal effects of (A) tea tree and (B) andiroba oils at concentrations of 1.0% and 5.0% against Musca domestica. Numbers of dead flies were counted at baseline and at 1 h, 2 h, 3 h, 4 h, 9 h, 12 h and 24 h post-oil exposure. Data within the same circle are not statistically different (Tukey’s honestly significant differences test, P > 0.05).
Essential oils: insecticidal effect
Discussion
Tea tree oil at 1.0% had no insecticidal effect on M. domestica (F = 3.24, d.f. = 7, P > 0.05). However, at the concentration of 5.0% it was able to kill and was 100.0% effective after 12 h of oil exposure (F = 210.1, d.f. = 2, P < 0.05) (Fig. 2A). Andiroba oil was effective at both concentrations tested (1.0% and 5.0%); its insecticidal effect against houseflies formed a dose-dependent curve (F = 53.1, d.f. = 7, P < 0.05) (Fig. 2B) showing statistically similar results at almost all the time-points observed for both concentrations. However, the effectiveness of andiroba oil at 5.0% was 67.0% lower than that of tea tree oil against M. domestica. Insecticidal effects of tea tree oil (Fig. 3A) (F = 41.3, d.f. = 7, P < 0.05) and andiroba (Fig. 3B) (F = 25.6, d.f. = 7, P < 0.05) were also observed at both concentrations against H. irritans. Fly death (100.0%) was observed at 4 h post-treatment (Fig. 3). A significant reduction in the number of H. irritans was observed on cows sprayed with both essential oils compared with animals in the control group (F = 30.04, d.f. = 2, P < 0.05) (Fig. 3C). The effect lasted for up to 24 h, but by 48 h after application no difference in fly numbers could be detected among the groups (F = 2.12, d.f. = 2, P > 0.05).
According to the literature, there is a growing body of evidence indicating the potential value of essential oils as control agents against a range of arthropod ectoparasites, although it is unclear whether these effects derive from neurotoxicity or mechanical suffocation (Ellse & Wall, 2013). The study of oil toxicity is subject to many difficulties, mainly because of the wide variation in the chemical composition of oils. Nevertheless, the use of essential oils in the control of veterinary ectoparasites holds considerable potential and research in this area is still at an early stage. The data presented here show the insecticidal effects of oils of both andiroba and tea tree against H. irritans and M. domestica. However, the mortality caused by the andiroba oil was lower than that caused by the tea tree oil. Similar results were reported by Farias et al. (2009), who conducted in vitro tests against third-instar M. domestica larvae using andiroba oil and noted 80.0% larval mortality. Cárcamo et al. (2007) used citronella oil at 1.0% and found it to cause 58.7% mortality against larvae of Lucilia sericata (Diptera: Calliphoridae). Researchers explain that the insecticidal effects of these essential oils is derived from their penetration of insect tissues and subsequent modification of some physiological functions (Ozaki et al., 2003).
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(A)
(B)
(C) Fig. 3. The percentage of Haematobia irritans alive at various time-points after exposure to (A) tea tree and (B) andiroba oils at concentrations of 1.0% and 5.0%. (C) Number of H. irritans counted on the bodies of 15 cattle treated with 5.0% tea tree oil, 5.0% andiroba oil or a control (water and triton) solution at baseline and at 1 h, 2 h, 3 h, 6 h, 9 h, 24 h, 48 h and 72 h post-oil exposure. Data within the same circle are not statistically different (Tukey honestly significant differences test, P > 0.05).
Studies on the repellent and insecticidal effects of andiroba and tea tree essential oils in adult H. irritans have not been described until now. However, other oils have been shown to have effect against this fly. Agnolin (2009) showed the efficiency of citronella oil on H. irritans and presented results similar to those of the present study for in vitro tests, in which treatments
with 5.0% oil gave 100.0% mortality. In vivo repellency tests using these oils showed a decrease in the number of flies found on treated animals. These results probably derived from the insecticidal and repellent effects of both tested oils (Ozaki et al., 2003; Coitinho et al., 2006; Farias et al., 2009). The insecticidal and repellent activities against mosquitoes and flies
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of oils of Mentha arvensis, Mentha piperita, Mentha spicata and Cymbopogon nardus have been reported previously (Raja et al., 2001). The insecticidal effect of tea tree oil can be related to its major components of terpinen-4-ol and 𝛾-terpinene. Terpinen-4-ol was tested against larvae of the oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae), and found to have a toxic effect (Ma & Zhang, 2004), which may also have occurred in the current study. The other component is 𝛾-terpinene, which has also been shown to have insecticidal effects (Abbassy et al., 2009). The literature contains no specific studies of the three major components present in andiroba oil (𝛼-humulene, bicyclogermacrene and germacrene-D), which may be effective against flies. However, these components are present in other plants with proven insecticidal activities (François et al., 2009; Dell’Agli et al., 2012). It is important to note that other components are present in these oils in lower concentrations and may also be responsible for both insecticidal and repellent activities. Therefore, further studies of isolated components will be conducted in the future by our research group. In summary, the current study concludes that essential oils of tea tree and andiroba have insecticidal effects against flies (M. domestica and H. irritans), as well as repellent effects against H. irritans. The horn fly was more susceptible to both concentrations of the oils than was the housefly.
Author’s declaration of interests No competing interests have been declared.
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